Engineered lysin-derived peptide as potent antibacterial active against c. acnes and s. aureus

Abstract

The present invention provides engineered peptides, compositions, and methods useful for killing, reducing and / or inhibiting colonization of bacteria, and for the prophylactic and therapeutic amelioration and treatment of bacteria, including Cutibacterium and Staphylococcus bacterial strains, particularly C. acnes and S. aureus, including pathogenic and antibiotic-resistant bacteria, and related conditions, including chronic inflammatory skin conditions and acne vulgaris. The invention provides compositions incorporating engineered peptide(s) and variants derived from a lysin polypeptide and to methods and treatments utilizing the engineered peptides and compositions.

Description

ENGINEERED LYSIN-DERIVED PEPTIDE AS POTENT ANTIBACTERIAL ACTIVE AGAINST C. ACNES AND S. AUREUSFIELD OF THE INVENTION

[0001] The present invention relates to engineered peptides, compositions, and methods useful for killing, reducing and / or inhibiting colonization of bacteria, and for the prophylactic and therapeutic amelioration and treatment of bacteria, including Cutibacterium and Staphylococcus bacterial strains, particularly C. acnes and A aureus, including pathogenic and antibiotic-resistant bacteria, and related conditions, including chronic inflammatory skin conditions and acne vulgaris. The invention relates to compositions incorporating engineered peptide(s) and variants derived from a lysin polypeptide and to methods and treatments utilizing the engineered peptides and compositions.SEQUENCE LISTING

[0002] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on March 23, 2026, is entitled “1119-86 PCT_ST26.xml”, and is 69,001 bytes in size.BACKGROUND OF THE INVENTION

[0003] A major problem in medicine has been the development of drugresistant bacteria as more antibiotics are used for a wide variety of illnesses and other conditions. The use of more antibiotics and the number of bacteria showing resistance has prompted longer treatment times. Furthermore, broad, nonspecific antibiotics, some of which have detrimental effects on the patient, are being used more frequently. A related problem with this increased use is that many antibiotics do not penetrate mucus linings easily. Additionally, the number of people allergic to antibiotics appears to be increasing. Accordingly, there is a commercial need for new antibacterial approaches, especially those that operate via new modalities or provide new means to kill pathogenic bacteria.

[0004] Novel antimicrobial therapy approaches include enzyme-based antibiotics (“enzybiotics”) such as bacteriophage lysins. Phages use these lysins to digest the cell wall of their bacterial hosts, releasing viral progeny through hypotonic lysis. A similar outcome results when purified, recombinant lysins are addedexternally to bacteria. The high lethal activity of lysins against pathogens makes them attractive candidates for development as therapeutics. Bacteriophage lysins were initially proposed for eradicating the nasopharyngeal carriage of pathogenic streptococci (Loeffler, J. M. et al (2001) Science 294: 2170-2172; Nelson, D. et al (2001) Proc Natl Acad Sci USA 98:4107-4112). Lysins have been shown to demonstrate a high lethal activity against numerous Gram-positive pathogens (especially the bacterium from which they were cloned), raising the possibility of their development as therapeutics (Fischetti, V.A. (2008) Curr Opinion Microbiol 11:393-400; Nelson, D. L. et al (2001) Proc Natl Acad Sci USA 98:4107-4112).

[0005] Acne vulgaris is a common skin problem, affecting most adolescents in the United States, and in some cases, may persist into adulthood. Acne is a chronic inflammatory disease of the skin pilosebaceous unit that produces sebum to lubricate both the skin and hair, and acts as a natural barrier against external environmental factors. While the pathogenesis of acne is multifactorial, dysbiosis of the skin microbiome is an important factor, and the bacterium Cutibacterium acnes (C. acnes, formerly Propionibacterium acnes) plays a key role. Mechanistically, C. acnes contribute to the inflammatory process that is typical of acne vulgaris, and therefore, the elimination of C. acnes is part of current therapeutic protocols (Zaenglein AL (2018) N Engl J Med 379:1343-1352; Williams HC et al (2012) Lancet 379:361-72; Hauk L (2017) Am Fam Physician 95:740-741). In some cases, other skin microbiome members, such as staphylococci, including Staphylococcus aureus (S'. aureus), can contribute to skin inflammation (Dessinioti C, Katsambas A (2024) Dermatol Ther (Heidelb) 14:31-44). Moreover, S'. aureus is one of several microbes that can contribute to the skin microbiome imbalances described in acne vulgaris (Totte JE et al (2016) Eur J Clin Microbiol Infect Dis 35:1069-77).

[0006] C. acnes is a lipophilic Gram-positive bacterium. While C. acnes grow ideally in anaerobic lipid rich conditions, it is also an aerotolerant bacterium that can detoxify oxygen and, therefore, can be sustained on the surface of the skin. Although considered a commensal, C. acnes involvement in various infections - including bone and prosthesis, spinal disk, eyes after cataract surgery, central nervous system catheters, and others - led to its emergence as an opportunistic pathogen (Mayslich C, Grange PA, Dupin N (2021) Microorganisms 9).

[0007] Current protocols for the treatment of acne vulgaris include the use of antibiotics to eradicate C. acnes, thereby mitigating the typical inflammatory process in acne lesions (Dessinioti C, Katsambas A (2024) Dermatol Ther (Heidelb) 14:31-44; Hauk L (2017) Am Fam Physician 95:740-741). These antibiotics are often combined with topical benzoyl peroxide or retinoids to further mitigate inflammation. Due to concerns about the development of antibiotic resistance, current acne treatment guidelinesrecommend limiting antibiotic use to a duration of up to 3 months. However, in clinical practice, the length of antibiotic treatment is often significantly longer, and the prevalence of C. acnes strains that are resistant to various antibiotics is increasing (Coates P et al (2002) Br J Dermatol 146: 840-8; Dessinioti C, Katsambas A (2022) Yale J Biol Med 95:429-443). Taken together, the need for treatment alternatives is evident.

[0008] Lysins are phage-encoded enzymes used by the phage to degrade the bacterial cell wall and promote hypotonic lysis, thereby releasing progeny virions from the phage-infected cells. When delivered externally as a purified recombinant protein, lysins may be used as efficient antimicrobials to rapidly lyse the target bacteria. Native lysins that kill gram-positive (G+) pathogens degrade the peptidoglycan while lysins directed to gram-negative (G-) bacteria must initially disrupt the outer membrane to subsequently access and degrade the peptidoglycan. Because of this dual action, most native G- lysins structurally comprise a single globular peptidoglycan-degrading catalytic domain with a C-terminal cationic region responsible for destabilizing the outer membrane (Ghose C, Euler CW (2020) Antibiotics (Basel) 9).

[0009] Currently, there is a lack of effective lysins and treatments other than antobiotics for therapeutic use against C. acnes, particularly exhibiting significant (greater than 3 log) colony forming units (CFU) reduction. Furthermore, no lysin-derived peptide has been shown to be active against C. acnes. It is evident from the deficiencies and problems associated with current traditional antibacterial agents that there still exists a need in the art for additional specific bacterial agents, including directed against C. acnes and S. aureus, particularly without high risks of acquired resistance.

[0010] The citation of references herein shall not be construed as an admission that such is prior art to the present invention.SUMMARY OF THE INVENTION

[0011] In a general aspect, the invention provides an engineered or variant peptide. In particular, the engineered peptide is derived from a lysin polypeptide sequence. The engineered or variant peptide is an anti-bacterial peptide and is capable of killing one or more bacteria, including distinct species of bacteria, including certain gram-positive and gram-negative bacteria. Notably, the original lysin polypeptide is active against gram-negative bacteria. The engineered peptide is effective to kill both gram-negative and gram¬ positive bacteria. The engineered peptide is active against and capable of killing both Cutibacterium and Staphylococcus species bacteria. Particularly susceptible bacteria are Cutibacterium acnes and Staphylococcus aureus, both of which are associated with and contribute to chronic inflammatory disease of the skin, particularly acne and acne vulgaris. The original lysin polypeptide has low bactericidal activityagainst C. acnes, therefore in an aspect of the invention the engineered peptide is more active and effective against bacteria.

[0012] The engineered peptide sequence provided herein is a variant derived from an amino acid sequence of the lysin PlyPi01 (Genbank MBQ0073608) which is a lysin of the Prevotella species of bacteria, particularly Prevotella intermedia. The C-terminal cationic region of the PlyPiOl lysin, corresponding to amino acids 102-132 of the 141 amino acid lysin, denoted PiPOl, was used as a basis for generating engineered variant peptides. The full lysin PlyPiOl exhibits low activity against C. acnes. Also, the C- terminal peptide PiPOl exhibits low activity against C. acnes.

[0013] In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:1, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the sequence KAKAPRAEIYAQFNK. WVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO: 1, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria.

[0014] In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. In accordance with an aspect of the invention, the PiPOl sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6) was modified by strategically introducing cationic amino acids, particularly lysine (K) and / or arginine (R) residues, at the N and / or C-terminal end(s), to improve its antibacterial activity.

[0015] In an aspect an engineered peptide is provided, wherein one or more lyine (K) and / or arginine (R) amino acid is added at the N-terminus and / or C-terminus. In an aspect an engineered peptide is provided,wherein one or more lysine (K) and / or arginine (R) and / or histidine (H) amino acids is / are added at the N- terminus and / or C-terminus. In an aspect a poly amino acid, particularly of two or more amino acids, particularly of three or more amino acids is added at the N-terminus and / or C-terminus. In an aspect, a poly amino acid consisting of 2, 3, 4, 5, 6, 7, or 8 cationic amino acids is covalently attached to the N-terminus and / or the C-terminus of the PiPOl sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6). In an aspect, a poly amino acid is covalently attached to each of the N-terminus and the C- terminus of the PiPOl sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6). In an aspect, the poly amino acid attached to the N-terminus is the same or different from the poly amino acid attached to the C-terminus. In an aspect, such poly amino acid may be a poly-arginine, a poly-lysine, or a poly-histidine, or a combination thereof. A poly amino acid may be, for example, KK (SEQ ID NO: 16), KKK (SEQ ID NO: 17), KKKK (SEQ ID NO: 18), KKKKK (SEQ ID NO: 19), RR (SEQ ID NO: 20), RRR (SEQ ID NO: 21 ), RRRR (SEQ ID NO: 22), RRRRR (SEQ ID NO: 23), HH (SEQ ID NO: 24), HHH (SEQ ID NO: 25), HHHH (SEQ ID NO: 26), HHHHH (SEQ ID NO: 27), RK (SEQ ID NO: 28), KR (SEQ ID NO: 29), RH (SEQ ID NO: 30), HR (SEQ ID NO: 31), KH (SEQ ID NO: 32), HK (SEQ ID NO: 33), RKR (SEQ ID NO: 34), KRK (SEQ ID NO: 35), HRK (SEQ ID NO: 36), RKH (SEQ ID NO: 37), RRKK (SEQ ID NO: 38), RKRK (SEQ ID NO: 39), KKRR (SEQ ID NO: 40), KRKR (SEQ ID NO: 41), HRHR (SEQ ID NO: 42), RHHRK (SEQ ID NO: 43), etc.

[0016] In particular aspects, the engineered peptide is selected from KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRK (SEQ ID NO:2), KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRKK (SEQ ID NO:3), KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:4), RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:1), and RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO:5). In an aspect, the engineered peptide is not SEQ ID NO: 1.

[0017] In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the sequence RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:1) or an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:1 with one or more cationic amino acids at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the sequence RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ IDNO: 1 ) or an amino acid sequence having at least 90% identity to SEQ ID NO:1 that has one or more cationic amino acids at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria.

[0018] In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the sequence RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO: 5) or an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:5 that has one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. In an aspect, the engineered peptide with at least about 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 5 which has one or more cationic amino acids added at the N- terminus and / or C-terminus is not SEQ ID NO: 1. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the sequence RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO:5) or an amino acid sequence having at least 90% identity to SEQ ID NO:5 and having one or more cationic amino acids at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. In an aspect, the engineered peptide with an amino acid sequence having at least 90% identity to SEQ ID NO:5 and that has one or more cationic amino acids at the N-terminus and / or C-terminus is not SEQ ID NO:1.

[0019] In one particular aspect, the engineered peptide consists of RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:1). In one particular aspect, the engineered peptide consists of RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO:5).

[0020] In alternative embodiments of the invention, the engineered peptide comprises or consists of a consensus sequence selected from the following sequences:X1KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRX2 (SEQ ID NO: 11 )wherein Xi is one or more natural or unnatural cationic amino acid, and wherein X2 is one or more natural or unnatural cationic amino acid.X1KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRX2 (SEQ ID NO:12)wherein X1 is 1, 2 or 3 natural or unnatural cationic amino acids, and wherein X2 is 1, 2 or 3 natural or unnatural cationic amino acid.X1KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRX2 (SEQ ID NO: 13)wherein X, is one or more lysine (K) or arginine (R), and wherein X2 is one or more lysine (K) or arginine (R).X1X2AX3APX4AEIYAQFNX5WVYAGGX6X7LSGLVX8X9X10X11 (SEQ ID NO:14)wherein Xj is one or more lysine (K) or arginine (R); X2 is one or more lysine (K) or arginine (R); X3 is one or more lysine (K) or arginine (R); X4 is one or more lysine (K) or arginine (R); X5 is one or more lysine (K) or arginine (R); Xg is one or more lysine (K) or arginine (R); X? is one or more lysine (K) or arginine (R); Xs is one or more lysine (K) or arginine (R); X? is one or more lysine (K) or arginine (R); X10 is one or more lysine (K) or arginine (R); Xi 1 is one or more lysine (K) or arginine (R).X1X2AX3APX4AEIYAQFNX5WVYAGGX6X7LSGLVX8X9X10X11 (SEQ ID NO:15)wherein Xi is one or more lysine (K) or arginine (R); X2 is lysine (K) or arginine (R); X3 is lysine (K) or arginine (R); X4 is lysine (K) or arginine (R); Xs is lysine (K) or arginine (R); Xe is lysine (K) or arginine (R); X7 is lysine (K) or arginine (R); Xs is lysine (K) or arginine (R); X9is lysine (K) or arginine (R); X10 is lysine (K) or arginine (R); X11 is one or more lysine (K) or arginine (R). In an embodiment, the engineered peptide of SEQ ID NO: 11, 12, 13, 14 and / or 15 is not SEQ ID NO: 1.

[0021] In an aspect, an engineered peptide comprises or consists of an amino acid sequence with at least about 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 6 that has at least two cationic amino acid residues at the N-terminus and / or C-terminus, wherein the total number of amino acids in the amino acid sequence of the engineered peptide is 33, 34, 35, 36, 37, 38, 39, 40 or 41 amino acids. In an embodiment, the engineered peptide comprises or consists of SEQ ID NO: 6 modified with at least two cationic amino acid residues at the N-terminus and / or C-terminus, wherein the total number of amino acids in the amino acid sequence of the engineered peptide is 33, 34, 35, 36, 37, 38, 39, 40 or 41 amino acids. In an embodiment, the at least two cationic amino acid residues are positioned at the N-terminus and at the C-terminus. In an embodiment, the at least two cationic amino acid residues is 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In an embodiment, the at least two cationic amino acid residues is 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In an embodiment, the at least two cationic amino acid residues is a poly amino acid as described above. In an embodiment, the engineered peptide is not SEQ ID NO: 1.

[0022] In an aspect, an engineered peptide comprises or consists of an amino acid sequence with at least about 85%, 90%, or 95% sequence identity to SEQ ID NO: I with retention of at least two cationic amino acid residues at the N-terminus and / or C-terminus, wherein the total number of amino acids in the aminoacid sequence of the engineered peptide is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 amino acids. In an embodiment, the at least two cationic amino acid residues are positioned at the N-terminus and at the C- terminus. In an embodiment, the at least two cationic amino acid residues is 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In an embodiment, the at least two cationic amino acid residues is a poly amino acid as described above. In an embodiment, the engineered peptide comprises bactericidial activity in an in vitro assay using C. acnes. In an embodiment, the engineered peptide is not SEQ ID NO: 1.

[0023] In an aspect, an engineered peptide comprises or consists of an amino acid sequence with at least about 85%, 90%, or 95% sequence identity to SEQ ID NO: 5 with retention of at least one cationic amino acid residue at the N-terminus and / or C -terminus, wherein the total number of amino acids in the amino acid sequence of the engineered peptide is 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 amino acids. In an embodiment, the at least one cationic amino acid residue is positioned at the N-terminus or at the C-terminus. In an embodiment, the at least one cationic amino acid residue is 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In an embodiment, the at least one cationic amino acid residue is 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In an embodiment, the at least one cationic amino acid residues is a poly amino acid as described above. In an embodiment, the engineered peptide is not SEQ ID NO:1.

[0024] In a particular embodiment, the engineered peptide is capable of killing gram-positive and / or gram¬ negative bacteria.

[0025] In some embodiments, the engineered peptide is capable of killing pathogenic and / or antibiotic¬ resistant bacteria.

[0026] In other embodiments, the engineered peptide is capable of killing bacteria, including C. acnes, in the presence of one or more of retinoid, benzoyl peroxide, or salicylic acid. In other embodiments, the engineered peptide is capable of killing bacteria, including C. acnes and S', aureus, in the presence of one or more of retinoid, benzoyl peroxide, or salicylic acid.

[0027] In other embodiments, the engineered peptide is capable of killing bacteria in the presence of surfactant. In other embodiments, the engineered peptide is capable of killing bacteria in oral / saliva conditions.

[0028] In an embodiment, the engineered peptide is capable of killing Cutibacterium and Staphylococcus bacterial strains. In an embodiment, the engineered peptide effectively kills C. acnes and S. aureus. In an embodiment, the engineered peptide inhibits colonization of C. acnes and S. aureus bacteria. In an embodiment, the engineered peptide is capable of killing S’, aureus and S. epidermidis bacteria. In an embodiment, the engineered peptide is capable of killing gram positive and gram negative bacteria. In anembodiment, the engineered peptide is capable of killing gram positive and gram negative bacteria capable of colonizing skin. In an embodiment, the engineered peptide is capable of killing Prevotella bacteria. In an embodiment, the engineered peptide is capable of killing P. intermedia. In an embodiment, the engineered peptide is capable of killing gram negative Acinetobacter, Escherichia, and Pseudomonas bacteria. In an embodiment, the engineered peptide is capable of killing A. baumannii, E. coli, and P aeruginosa bacteria.

[0029] The invention provides compositions, including therapeutic and pharmaceutical compositions comprising one or more engineered lysin-derived peptide of the invention.

[0030] In aspects, a composition is provided comprising the engineered peptide. In an aspect, a composition is provided comprising one or more engineered peptide. In an aspect, a composition is provided comprising the peptides of SEQ ID NO: 1 and SEQ ID NO:5. In an aspect, a composition is provided comprising one or more engineered peptide, wherein the engineered peptide is not SEQ ID NO: 1.

[0031] In one aspect, the composition is an antimicrobial composition further comprising a pharmaceutically acceptable carrier.

[0032] In an aspect, the composition further comprises another antibacterial agent. In an aspect, the composition further comprises one or more other antibacterial agent. In some aspects, the composition further comprises one or more antibiotic. In some aspects, the composition further comprises one or more other antibacterial peptide.

[0033] In some embodiments, the composition further comprises one or more of retinoid, benzoyl peroxide, or salicylic acid.

[0034] In some aspects, a retinoid is a derivative of vitamin A or is related to vitamin A. In aspects, retinoid may be selected from retinol, retinal, retinoic acid (tretinoin), isotretinoin, alitretinoin, etretinate, acitretin, adapalene, bexarotene, tazarotene and trifarotene.

[0035] In other aspects, the composition is suitable for topical administration and effective to kill one or more type of bacteria.

[0036] In aspects, the engineered peptide and compositions thereof retain high bactericidal activity against C. acnes at NaCl concentrations up to 50 mM. In aspects, C. acnes is reduced to CFU / ml below the level of detection (LOD) at NaCl concentrations up to 50 mM. In an aspect, the peptide P157 (SEQ ID NO:5) is capable of reducing C. acnes to CFU / ml below the level of detection (LOD) at NaCl concentrations up to 50 mM. In aspects, the engineered peptide and compositions thereof reduce C. acnes to CFU / ml below the level of detection (LOD) at 5 pg / ml. In aspects, the engineered peptide and compositions thereof reduce C.acnes strain HSSB to CFU / ml below the level of detection (LOD) at 5 pg / ml. In aspects, the engineered peptide and compositions thereof reduce C. acnes strain HSSB, HSSC and HSSE to CFU / ml below' the level of detection (LOD) at 5 pg / ml. In an aspect, the peptide P157 and compositions thereof reduce C. acnes strain HSSB to CFU / ml below the level of detection (LOD) at 5 pg / ml. In an aspect, the engineered peptide and compositions thereof retain high bactericidal activity against C. acnes at temperatures of from 15°C to 40°C. In an aspect, the peptide P157 and compositions thereof reduce C. acnes to CFU / ml below the level of detection (LOD) at temperatures of from 15°C to 40°C.

[0037] The invention provides a pharmaceutical composition for killing gram-positive or gram-negative bacteria comprising the engineered peptide in an amount effective to kill the gram-positive or gram¬ negative bacteria.

[0038] The invention provides, in another aspect, a cosmetic composition comprising one or more of the engineered peptides disclosed herein. The cosmetic composition may comprise the engineered peptide in an amount effective to kill the gram-positive and / or gram-negative bacteria. The cosmetic composition may include one or more cosmetic ingredients. Cosmetic ingredients may include, for example, a blend of water, emulsifiers, preservatives, thickeners, moisturizers, colors, and / or fragrances designed to cleanse, beautify, and / or alter the skin's appearance. Other exemplary cosmetic ingredients include glycerin, hyaluronic acid, retinol, niacinamide, and parabens, which are used for hydration, anti-aging, and / or preservation.

[0039] In another aspect, the invention includes a composition suitable for application to the skin. In aspects, such a composition is suitable for topical application. In aspects, the composition for topical application may be a topical anti-infective. In aspects, the composition is a cream, aqueous gel, or other suitable formulation for topical application or cosmetic use. The composition may include other components frequently added to or present in cosmetics or makeup. The composition may include dyes or skin tints.

[0040] In another embodiment, nucleic acid capable of encoding the engineered peptide is provided.

[0041] In some embodiments, the said nucleic acid is operatively linked to an expression control sequence. In an embodiment, a unicellular host transformed with the nucleic acid is provided.

[0042] In aspects, the invention provides a method of killing bacteria or reducing a population of bacteria comprising the step of contacting the bacteria with an amount of one or more engineered peptide, or wdth a composition comprising one or more engineered peptide, effective to kill the bacteria.

[0043] In an aspect of the method, the bacteria is gram negative or gram positive.

[0044] In an aspect of the method, the bacteria is Cutibacterium or Staphylococcus. In one such aspect, the bacteria is C. acnes or S. aureus.

[0045] In an aspect of the method, the bacteria is Cutibacterium, Staphylococcus, Prevotella, Acinetobacter, Escherichia, and Pseudomonas bacteria.

[0046] In an aspect of the method, the bacteria is an antibiotic-resistant bacteria.

[0047] In an aspect of the method, the bacteria is methicillin-resistant Staphylococcus aureus (MRSA), vancomycin intermediate-sensitivity Staphylococcus aureus (VISA), or vancomycin resistant Staphylococcus aureus (VRSA).

[0048] In another aspect, a method is provided for treating acne vulgaris in a human comprising administering to a human having acne an effective amount of one or more engineered peptide, or a composition comprising one or more engineered peptide, whereby the acne vulgaris is reduced or controlled.

[0049] In aspects, methods are provided wherein the number of C. acnes and / or S. aureus bacteria associated with the acne is reduced.

[0050] In aspects, methods are provided wherein the composition further comprises one or more of retinoid, benzoyl peroxide, salicylic acid, or an antibiotic.

[0051] In aspects, methods are provided for reducing the number of bacteria on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing the number of Cutibacterium, Staphylococcus or Prevotella bacteria on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing the number of C. acnes and / or S. aureus bacteria on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs.

[0052] In aspects, methods are provided for reducing the number of Cutibacterium, Staphylococcus, Prevotella, Acinetobacter, Escherichia, and / or Pseudomonas bacteria on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs.

[0053] In aspects, methods are provided for reducing a bacterial infection on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing the number of Cutibacterium, Staphylococcus or Prevotella bacteria in a bacterial infection on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing thenumber of C. acnes and / or S. aureus bacteria in a bacterial infection on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs.

[0054] In other aspects, methods are provided for reducing the size of a site of bacterial growth on skin, such as a red, warm, painful or swollen area, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing the number of Cutibacterium, Staphylococcus or Prevotella bacteria associated with the site of bacterial growth on skin. In aspects, methods are provided for reducing the number of C. acnes and / or 5. aureus bacteria associated with the site of bacterial growth. In aspects, methods are provided for reducing the number of C. acnes and / or S', aureus and / or S. epidermis bacteria associated with the site of bacterial growth.

[0055] In some aspects, the bacterial growth on skin is associated with infections or conditions such as impetigo, cellulitis, or abscesses. In some aspects, the bacterial growth on skin is associated with infections or conditions such as folliculitis or rosacia.

[0056] A method for treating or controlling contaminations of or infections by one or more bacteria of Cutibacterium, Staphylococcus or Prevotella bacteria, comprising administering an effective amount of one or more engineered lysin-derived peptide, or with a composition of one or more engineered lysin-derived peptide.

[0057] In an aspect of the method, the bacteria is C. acnes or S. aureus. In an aspect of the method, the bacteria is P. intermedia.

[0058] In an aspect of the above methods of killing gram positive bacteria, the methods are performed in vitro or ex vivo so as to sterilize or decontaminate a solution, material or device, particularly intended for use by or in a human.

[0059] In accordance with any of the methods of the invention, the composition thereof may further comprise a carrier, including a pharmaceutically acceptable carrier, additive or diluent. In accordance with any of the methods of the invention, the composition thereof may further comprise a suitable vehicle for delivery of the polypeptide to a site of infection. In accordance with any of the methods of the invention, the composition thereof may further comprise one or more antibiotic. In accordance with any of the methods of the invention, the composition thereof may further comprise one or more agent effective against bacteria or the condition associated with bacteria that are the target of killing by the engineered peptide(s).

[0060] Other objects and advantages will become apparent to those skilled in the art from a review of the following description which proceeds with reference to the following illustrative drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIGURE 1. Predicted structure of the parental lysin-derived peptide PiPOl. AlphaFold was used to compare the predicted structure of the lysin-derived peptide PiPOl to that of the lysin-derived peptides P307 and PaPl-2. AH three peptides are predicted to adapt comparable helix-loop-helix hairpin structural motifs. For comparison, the alpha-helical structure of the human AMP LL-37 is also shown. Blue, positively charged amino acids; Red, negatively charged amino acids.

[0062] FIGURE 2. Bactericidal activity of five modified peptides derived from the P. intermedia PlyPiOl lysin on various strains of C. acnes. The amino acid sequence pertaining to the cationic C- terminal region of PlyPiOl (aa 102-132) was isolated and strategically engineered with varying cationic aa modifications. A total of five peptide derivatives (Pl 1, Pi ll, P16, P156, and P157) were assayed for bactericidal activity against seven clinical isolates of C. acnes using a dose-response killing assay. Following 1 hour treatment in 20 mM Tris, pH 7.2, at 37°C, bacterial viability was assessed via serial dilution and plating. The LOD was 200 CFU / mL. Error bars represent the ±SEM from duplicate experiments.

[0063] FIGURE 3. Pll, Pill, P16, P156 and P157 antibacterial activity against MSSA and MRSA strains of 5. aureus. (A) MSSA strain §325, (B) MRSA strain USA 300 and (C) MRSA strain USA 400 were grown under aerobic conditions to mid-log phase. A dose-response killing assay was performed to assess the bactericidal activity of Pl l, Pi ll, P16, P156, and P157 against each S. aureus strain in 20 mM Tris, pH 7.2, for 1 hour at 37°C. (D) The MRSA strain USA 300 was grown under anaerobic conditions to mid-log phase and then treated with Pl 56 at 25 pg / ml in 20 mM Tris, pH 7.2, for 1 hour at 37°C. Bacterial viability was quantified via serial dilution and plating. The LOD (dashed lines) was 10 CFU / mL. Error bars represent the ±SEM from triplicate experiments.

[0064] FIGURE 4. Bactericidal properties of P156 under various relevant skin conditions. A) The killing kinetics of Pl 56 (at 25 pg / mL) against C. acnes strain ATCC 6919 (in 20 mM Tris, pH 7.2, at 37°C) was assessed over 30 min at various time points by measuring bacterial viability. (B-D) The bactericidal activity of P156 (at 25 pg / mL) quantitated under various values of (B) temperature (15°C-40°C), (C) pH (20 mM MES: pH 5.5-6.5; 20 mM NaPO4: pH 6.5-7.0; 20 mM Tris, pH 7.0-8.0) or (D) NaCl concentrations (0-200 mM). The LOD (dashed lines) was 10 CFU / mL. Error bars represent the ±SEM from triplicate experiments.

[0065] FIGURE 5. Pl 56 cytotoxicity towards eukaryotic cells. Human red blood cells (hRBCs) from healthy donors were incubated for 4 hours at 37°C in 5% CO2with either PBS (negative control, blue square), 0.1% Triton X-100 (positive control, black dot), P156 (green triangle), or LL37 (a human antimicrobial peptide used as a control, red triangle). For the peptide treatments, a concentration gradient ranging from 0.5 to 256 pg / mL in PBS was applied. After the incubation, intact. hRBCs were removed, and the relative concentration of hemoglobin released into the supernatant was quantified by measuring the absorbance at 405 nm.

[0066] FIGURE 6. Effect of retinoids on P156 bactericidal activity. Retinoic acid at various concentrations (0.01%, 0.03%, 0.05%, 0.1%) was incubated with MRSA USA300 either alone or in combination with Pl 56 (25 pg / mL) during a 1-hour killing assay in Tris, pH 7.2, at 37°C. Bacterial viability was assessed by serial dilution and plating. The LOD (dashed lines) was 10 CFU / mL. Error bars represent the ±SEM from triplicate experiments.

[0067] FIGURE 7. Antibacterial activity of lysin PlyPiOl and peptide PiPOl against C. acnes. The antibacterial activity of the PlyPiOl lysin was compared to its isolated C -terminal cationic peptide, PiPOl. Bactericidal activity was measured against C. acnes strains ATCC 6919 (left) and HSS F (right) in 20 mM Tris, pH7.2, for I h at 37°C. Bacterial viability was assessed via serial dilution and plating. The limit of detection (LOD, dashed lines) was 10 CFU / mL. Bactericidal activity is defined as >3-log CFU reduction with respect to the untreated control. Error bars represent the ±SEM from triplicate experiments. 9-values were calculated using an unpaired / -test between each peptide or lysin dose and the untreated group. * <0.05 and a <3-log CFU reduction, **p<0.05 and a>3-log CFU reduction.

[0068] FIGURE 8. Bacterial killing of P156 against Staphylococcus epidermidis. The S', epidermidis strains ATCC 12228 and ATCC 35984 were grown to mid-log phase in aerobic conditions and then treated with Pl 56 (25 pg / mL). Bacterial viability was quantified via serial dilution and plating. The LOD (dashed lines) was 10 CFU / mL.

[0069] FIGURE 9. Bactericidal properties of P157 under various relevant skin conditions. (A) The killing kinetics of Pl 57 (25 pg / mL) against C. acnes strain ATCC 6919 was assessed over 30 min at various time points in 20 mM Tris, pH 7.2, at 37°C. (B-D) The bactericidal activity of Pl 57 (25 pg / mL) was quantitated at several (B) temperatures ( 15-40°C), (C) pH values (20 mM MES, pH 5.5-6.5; 20 mM NaPO4, pH 6.5-7.0; 20 mM Tris, pH 7.0-8.0) and (D) NaCl concentrations (0-300 mM). Bacterial viability' was quantitated by serial dilution and plating. The limit of detection was 10 CFU / mL. Error bars correspond to the ± standard error of the mean from triplicate biological replicates.

[0070] FIGURE 10. P157 antibacterial activity against S. epidermidis. The bactericidal activity of Pl 57 (25 pg / mL) was measured against S. epidermidis strains ATCC 12228 and ATCC 35984 in 20 mM Tris, pH 7,2, for 1 h at 37°C. Bacterial viability was quantitated via serial dilution and plating. The limit of detection was 10 CFU / mL. Error bars correspond to the ± standard error of the mean of triplicate biological replicates.

[0071] FIGURE 11. P157 cytotoxicity towards eukaryotic cells. hRBCs from healthy donors were incubated for 4 h at 37CC in 5% CO2with either PBS (negative control, blue square), 0.1% Triton X-100 (positive control, black dot), Pl 57 (green triangle), or LL37 (a human antimicrobial peptide used as a control, red triangle). For the peptide treatments, a concentration gradient ranging from 0.5-256 pg / mL in PBS was applied. After the incubation, intact hRBCs were removed, and the relative concentration of hemoglobin released into the supernatant was quantified by measuring the absorbance at 405 nm. Data shown represent one of three independent experiments with similar results.DETAILED DESCRIPTION

[0072] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art.

[0073] Therefore, if appearing herein, the following terms shall have the definitions as provided and set out below and in this section.

[0074] The terms "engineered lysin-derived peptide”, “engineered peptide”, “engineered variant peptide”, “engineered polypeptide”, “PiPOl derived peptide”, “PiPOl variant peptide” and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and the profile of activities set forth herein and in the Claims, including wherein the peptide comprises a sequence engineered or derived from SEQ ID NO:6, or from a sequence having % identity to SEQ ID NO:6. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts.

[0075] A "lytic enzyme" includes any bacterial cell wall lytic enzyme that kills one or more bacteria under suitable conditions and during a relevant time period. Examples of lytic enzymes include, withoutlimitation, various amidase cell wall lytic enzymes, A lytic enzyme is capable of specifically cleaving bonds that are present in the peptidoglycan of bacterial cells to disrupt the bacterial cell wail.

[0076] " A variant sequence lytic enzyme" includes a lytic enzyme characterized by a polypeptide sequence that is different from that of a lytic enzyme, but retains functional activity.

[0077] " Percent amino acid sequence identity" with respect to the peptide sequences identified is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the original lytic enzyme sequence or lytic enzyme fragment sequence, after aligning the sequences in the same reading frame and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In aspects, percent identity may not consider any conservative substitutions as part of the sequence identity.

[0078] " Percent nucleic acid sequence identity" with respect to the peptide sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the original lytic enzyme sequence or lytic enzyme fragment sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0079] To determine the percent identity of two nucleotide or amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first nucleotide sequence). The nucleotides or amino acids at corresponding nucleotide or amino acid positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity^# of identical positions / total # of positions.times.100).

[0080] The determination of percent identity between two sequences may be accomplished using a mathematical algorithm, A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al., Proc, Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST program which may be used to identify sequences having the desired identity to nucleotide sequences ofthe invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized as described in Altschui et al., Nucleic Acids Res, 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) may be used. See the programs provided by National Center for Biotechnology Information, National Library' of Medicine, National Institutes of Health. In one embodiment, parameters for sequence comparison may be set at W=12. Parameters may also be varied(e.g., W=5 or W=20). The value " W" determines how many continuous nucleotides must be identical for the program to identify two sequences as containing regions of identity.

[0081] " Polypeptide" includes a polymer molecule comprised of multiple amino acids joined in a linear manner. A polypeptide can, in some embodiments, correspond to molecules encoded by a polynucleotide sequence which is naturally occurring. The polypeptide may include conservative substitutions where the naturally occurring amino acid is replaced by one having similar properties, where such conservative substitutions do not alter the function of the polypeptide.

[0082] A "chimeric protein" or "fusion protein" comprises all or (preferably a biologically active) part of a polypeptide of the invention operably linked to a heterologous polypeptide. Chimeric proteins or peptides are produced, for example, by combining two or more proteins having two or more active sites. In a chimeric or fusion protein, a first polypeptide may be covalently attached to an entity which may provide additional function or enhance the use or application of the first polypeptide(s), including for instance a tag, label, targeting moiety or ligand, a cell binding or cell recognizing motif or agent, an antibacterial agent, an antibody, an antibiotic. Exemplary labels include a radioactive label, such as the isotopes3H,14C,32P,35S,36C1,5’Cr,57Co,58Co,59Fe,90Y,125I,131I, and186Re. The label may be an enzyme, and detection of the labeled lysin polypeptide may be accomplished by any of the presently utilized or accepted colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art. Chimeric protein and peptides can act independently on the same or different molecules, and hence have a potential to treat two or more different bacterial infections at the same time. Chimeric proteins and peptides also may be used to treat a bacterial infection by targeting or killing the bacteria via more than one mechanism or target, thus potentially providing more rapid or effective (or synergistic) killing from a single molecule or chimeric peptide.

[0083] A chimeric protein or fusion protein includes wherein a first protein or polypeptide of interest is combined with another distinct heterologous protein or peptide. A chimeric protein or fusion protein includes wherein a first protein or polypeptide of interest is combined with a targeting protein or targeting sequence which may direct the first protein or polypeptide to a particular cell type, a particular cell receptor, or a tissue or region of the body of an animal for instance. A chimeric protein or fusion protein includes wherein a first protein or polypeptide of interest is combined with a targeting protein or targeting sequence which may direct the first protein outside of the ceil of expression, such as to be expressed or located systemically in an animal, or to the blood or local tissues in the animal. A chimeric protein includes wherein a first protein is combined with a label, tag or enzyme. A tag orlabel or enzyme may be a functional molecule. A tag or label may be an epitope. A tag or label may be a detectable molecule, protein or other entity. A tag or label may be a fluorescent molecule, a radioactive molecule, etc. Suitable fluorescent molecules are known and available in the art. A fluorescent molecule may be a green fluorescent protein (GFP) for example.

[0084] A chimeric or fusion protein may include wherein the engineered peptide is combined or linked with another or alternative antibacterial peptide, or another cationic peptide. The antibacterial peptide may include a peptide antibiotic. Multiple engineered peptides of the invention may be linked or fused. One or more engineered peptides provided herein may be linked, fused, covalently or non-covalently attached to a polyamino acid, or a polymer for targeting or stabilization, such as polyethylene glycol, poly histidine, etc.

[0085] A "heterologous" region of a DNA construct or peptide construct is an identifiable segment of DNA within a larger DNA molecule or peptide within a larger peptide molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally- occurring mutational events do not give rise to a heterologous region of DNA or peptide as defined herein.

[0086] The term "operably linked" means that the polypeptide of the disclosure and the / a heterologous polypeptide are fused in-frame. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the polypeptide of the disclosure. Chimeric proteins may be produced enzymatically by chemical synthesis, or by recombinant DNA technology. One example of a useful fusion protein is a GST fusion protein in which the polypeptide of the disclosure is fused to the C-terminus of a GST sequence. Such a chimeric protein can facilitate the purification of a recombinant polypeptide of the disclosure.

[0087] In another embodiment, the chimeric protein or peptide contains a heterologous signal sequence at its N-terminus. For example, the native signal sequence of a polypeptide of the disclosure can be removed and replaced with a signal sequence from another protein.

[0088] The fusion protein may combine the engineered peptide herein with a protein or polypeptide of having a different capability, or providing an additional capability' or added character to the engineered peptide. The fusion protein may be an immunoglobulin fusion protein in which all or part of a polypeptide of the disclosure is fused to sequences derived from a member of the immunoglobulin protein family. Theimmunoglobulin may be an antibody, for example an antibody directed to a surface protein or epitope of a susceptible or target bacteria. The chimeric or fusion protein may include a heterologous sequence, molecule, or entity that serves to stabilize, target, or protect the engineered peptide. In aspects, the heterologous sequence, molecule, or entity can be a lipophilic sequence, a pegylation moiety, a liposome, an N-terminal an / or C-terminal blocking or stabilizing sequence or molecule, or a cell receptor or cell recognition sequence, a concatemerization sequence or molecule serving to concatemerize the engineered peptide, etc. Chimeric and fusion proteins and peptides of the disclosure can be produced by standard recombinant DNA techniques.

[0089] The fusion or chimeric gene / construct can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary' overhangs between two consecutive gene fragments which subsequently can be annealed and reamplified to generate a chimeric gene sequence. Moreover, many expression vectors are commercially available that already encode a fusion moiety (i.e., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety’ is linked in-frame to the polypeptide of the invention.

[0090] A signal sequence of a polypeptide can facilitate transmembrane movement of the protein and peptides and peptide fragments of the disclosure to and from mucous membranes, as well as by facilitating secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. A nucleic acid sequence encoding a signal sequence of the disclosure can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from an eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art-recognized methods. Alternatively, the signal sequence can be linked to a protein of interest using a sequence which facilitates purification, such as with a GST domain.

[0091] The term “cationic polymer(s)” refers to long-chain, synthetic or natural macromolecules possessing a net positive charge, such as those used to attract and neutralize negatively charged particles, contaminants, or surfaces.

[0092] Cationic polymers can be used in drug, agent, peptide or therapeutic delivery, and can be used as agents, such as for transfection or delivery, based on their ability to bind or associate with negatively charged entities, molecules or targets. Cationic polymers have recognized encapsulation efficacy, enhanced bioavailability, low toxicity' and improved release profile and can confer these qualities and characteristics by attachment, fusion or linkage to a drug, agent, peptide or therapeutic compound. Cationic polymers can be protein in nature, such as amino acids, and polysaccharide in nature. Examples of cationic polymers include gelatin, chitosan, cellulose, dextran, poly-lysine, poly(2-N, N-dimethylaminoethylmethacrylate), poly(ethyleneeimine), poly(amidoamine). Cationic lipopeptides, such as based on palmitoyl or myristoyl chains, can have applications for antibacterial and preservative applications. In particular aspects, the cationic polymer is positively charged at neutral pH, or under physiological conditions, including at the natural pH of external skin surfaces,

[0093] The term “cationic amino acid(s)” refers to cationic or positively charged amino acids that are protonated at physiological pH, neutral pH or at about pH 6-8 or 7. Cationic amino acids can include natural and synthetic or unnatural amino acids. Natural cationic amino acids include arginine, lysine, histidine. Synthetic cationic amino acids include modified, non-proteinogenic, or engineered amino acids designed to carry a permanent or inducible positive charge, with examples including derivatives of arginine (e.g., b 2-amino-3-guariidinopr0i>ionic acid (Gpf). l-2-amino-4-guanidinobutyric acid (Gbt), and homoarginine) and lysine (e.g., ornithine, 2,4-diaminobutyric acid, and 2,3-diaminopropionic acid. Positively charged (cationic) unnatural amino acids (uAAs) are non-canonical amino acids containing basic functional groups (e.g., amine derivatives, guanidinium derivatives) that remain protonated at physiological pH. Used extensively in peptide design, they enhance stability, binding affinity, and antimicrobial activity. Examples include homoarginine, N-methylarginine, 4-guanidinophenylalanine, and ornithine derivatives. Other examples include 4-guanidinophenylalanine or 4-guanidinomethylphenylalanine, N-methylarginine orother N-alkylated basic amino acids.

[0094] The present invention also pertains to other variants of the engineered peptides and polypeptides of the invention. Such variants may have an altered amino acid sequence. Variants can be generated by mutagenesis, i.e., discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limitedfunction. Treatment of a subject with a variant having a subset of the biological activities of the original form of the protein can have fewer side effects in a subject relative to treatment with the original form of the protein.

[0095] The amino acid residues described herein are preferred to be in the " L" isomeric form. However, residues in the " D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. It should be noted that all amino-acid residue sequences are represented herein in accepted single letter code designations and by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.

[0096] Mutations can be made in the amino acid sequences, or in the nucleic acid sequences encoding the engineered peptides and polypeptides herein, including in the base sequence SEQ ID NO: 6, or in SEQ ID NO: 1, or in any of SEQ ID NOs:2-5, such that a particular codon is changed to a codon which codes for a different amino acid, an amino acid is substituted for another amino acid, or one or more amino acids are deleted. In a particular aspect, mutations are made in the amino acid sequence, or in the nucleic acid sequence encoding the engineered peptide of the base sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6). In a particular aspect, mutations are made in the amino acid sequence, or in the nucleic acid sequence encoding the engineered peptide of the RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:1). In a particular aspect, mutations are made in the amino acid sequence, or in the nucleic acid sequence encoding the engineered peptide of sequence RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO: 5). Such a mutation is generally made by making the fewest amino acid or nucleotide changes possible. Such a mutation can be made in order to retain the charge or character of an amino acid or necessary activity of an amino acid, but increase its stability or resistance to proteases or to destabilization under altered pH or temperature or other relevant conditions. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an aminoacid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change can be more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or characteristics of the resulting peptide.

[0097] In an aspect, mutations are made in the amino acid sequence, or in the nucleic acid sequence encoding the engineered peptide of SEQ ID NO: 6, SEQ ID NO: 1, or SEQ ID NO:5, wherein the amino acid tryptophan (W) is retained or unchanged. In an aspect, mutations are made in the amino acid sequence, or in the nucleic acid sequence encoding the engineered peptide of SEQ ID NO: 6, SEQ ID NO: 1, or SEQ ID NO:5, wherein the amino acid tryptophan (W) is retained or unchanged, and further wherein the glutamine (Q) is retained or is replaced with a glutamic acid (E). In an aspect, mutations are made in the amino acid sequence, or in the nucleic acid sequence encoding the engineered peptide of SEQ ID NO: 6, SEQ ID NO:1, or SEQ ID NO:5, wherein the amino acid tryptophan (W) is retained or unchanged and further wherein the tyrosine (Y) is retained or unchanged.

[0098] The following is one example of various groupings of amino acids:Amino acids with nonpolar R groupsAlanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, MethionineAmino acids with uncharged polar R groupsGlycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, GlutamineAmino acids with charged polar R groups (negatively charged at Ph 6.0)Aspartic acid, Glutamic acidBasic amino acids (positively charged at pH 6.0)Lysine, Arginine, Histidine (at pH 6.0)

[0099] Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine[000100] Another grouping may be according to molecular weight (i.e., size of R groups).[000101] Particularly preferred substitutions are:- Lys for Arg and vice versa such that a positive charge may be maintained;- GIu for Asp and vice versa such that a negative charge may be maintained;- Ser for Thr such that a free -OH can be maintained; and- Gin for Asn such that a free NH2 can be maintained.[000102] Exemplary and preferred conservative amino acid substitutions include: glutamine (Q) for glutamic acid (E) and vice versa; leucine (L) for valine (V) and vice versa; serine (S) for threonine (T) and vice versa; isoleucine (I) for valine (V) and vice versa; lysine (K) for glutamine (Q) and vice versa; isoleucine (I) for methionine (M) and vice versa; serine (S) for asparagine (N) and vice versa; leucine (L) for methionine (M) and vice versa; lysine (K) for glutamic acid (E) and vice versa; alanine (A) for serine (S) and vice versa; tyrosine (Y) for phenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid (D) and vice versa; leucine (L) for isoleucine (I) and vice versa; lysine (K) for arginine (R) and vice versa.[000103] Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces [3-turns in the protein's structure.[000104] The term “specific” may be used to refer to the situation in which one member of a specific binding pair will not show significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.[000105] The term “comprise” generally used in the sense of include, that is to say permitting the presence of one or more features or components.[000106] The term “consisting essentially of’ refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product. In the case of the peptide of the invention hereof, those of skill in the art will appreciate that minor modifications to the N- or C- terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.[000107] The term “isolated” refers to the state in which the polypeptide(s) of the invention, or nucleic acid encoding such polypeptides w ill be, in accordance with the present invention. Polypeptides and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Polypeptides and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the polypeptides will normally be mixed withpolymers or mucoadhesives or other carriers, or will be mixed with pharmaceutically acceptable carriers or diluents, when used in diagnosis or therapy.[000108] Nucleic acids capable of encoding the engineered peptide(s) / polypeptide(s) of the invention are provided herein and constitute an aspect of the invention.[000109] A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo i.e., capable of replication under its own control.[000110] A "vector" is a repiicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.[000111] A " DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3’ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).[000112] An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.[000113] A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g,, mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.[000114] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.[000115] A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3’ terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary toinitiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain " TATA" boxes and " CAT" boxes. Prokaryotic promoters contain Shine- Dalgamo sequences in addition to the -10 and -35 consensus sequences.[000116] An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.[000117] A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety' of proteins native to prokaryotes and eukaryotes.[000118] The term "oligonucleotide," as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.[000119] The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single¬ stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method.[000120] The primers herein are selected to be "substantially" complementary' to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5’ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively,non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.[000121] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.[000122] A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single ceil or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.[000123] Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.[000124] In preferred embodiments of the present disclosure, stringent conditions may be defined as those under which DNA molecules with more than 25% sequence variation (also termed "mismatch") will not hybridize. In a more preferred embodiment, stringent conditions are those under which DNA molecules with more than 15% mismatch will not hybridize, and more preferably still, stringent conditions are those under which DNA sequences with more than 10% mismatch will not hybridize. Preferably, stringent conditions are those under which DNA sequences with more than 6% mismatch will not hybridize.[000125] The degeneracy of the genetic code further widens the scope of the embodiments as it enables major variations in the nucleotide sequence of a DNA molecule while maintaining the amino acid sequence of the encoded protein. For example, a representative amino acid residue is alanine. This may be encoded in the cDNA by the nucleotide codon triplet GCT. Because of the degeneracy of the genetic code, threeother nucleotide codon triplets-GCT, GCC and GCA-also code for alanine. Thus, the nucleotide sequence of the gene could be changed at this position to any of these three codons without affecting the amino acid composition of the encoded protein or the characteristics of the protein. The genetic code and variations in nucleotide codons for particular amino acids are well known to the skilled artisan. Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from the cDNA molecules disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences. DNA sequences which do not hybridize under stringent conditions to the cDNA sequences disclosed by virtue of sequence variation based on the degeneracy of the genetic code are herein comprehended by this disclosure.[000126] Thus, it should be appreciated that also within the scope of the present invention are DNA sequences encoding an engineered peptide of the present invention, but which are degenerate thereto. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that certain codons can be used interchangeably to code for each specific amino acid.[000127] One skilled in the art will recognize that the DNA mutagenesis techniques known in the art can produce a wide variety of DNA molecules that code for an engineered peptide of the invention yet that maintain the essential characteristics of the peptides described and provided herein. Such derivatives include those with variations in amino acid sequence including minor deletions, additions and substitutions.[000128] Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions may be made so as to generate no significant effect on the protein characteristics or when it is desired to finely modulate the characteristics of the protein. Amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions are described above and will be recognized by one of skill in the art.[000129] Substantial changes in function or immunological identity may be made by selecting substitutions that are less conservative, for example by selecting residues that differ more significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which: (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.[000130] The effects of these amino acid substitutions or deletions or additions may be assessed for derivatives or variants of the polypeptide(s) by analyzing the ability of the derivative or variant proteins to lyse or kill susceptible bacteria,[000131] Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes, if not already part of the DNA sequence, the provision of an initiation codon, AT G, in the correct reading frame upstream of the DNA sequence. A wide variety of host / expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors are known and available in the art. Also, any of a wide variety of expression control sequences — sequences that control the expression of a DNA sequence operatively linked to it — may be used in these vectors to express the DNA sequences of this invention. A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.[000132] A wide range of antimicrobial peptides is secreted in plants and animals to challenge attack by foreign viruses, bacteria or fungi (Boman, H. G. (2003) J. Intern. Med. 254 (3): 197-215). These form part of the innate immune response to infection, which is short term and fast acting relative to humoral immunity. These peptides are heterogeneous in length, sequence and structure, but most are small, cationic and amphipathic (Zasloff, M. (2002) Nature 415(6870):389-395). Antimicrobial peptides have been considered as prospective antibiotics agents because their effect is rapid, broad spectrum and indifferent to resistance to standard antibiotics such as penicillins (Fischetti, V. A. (2003) Ann. N. Y. Acad. Sci. 987:207-214; Hancock, R. E, (1999) Drugs 57(4):469-473). Various antimicrobial peptides have been studied in order to understand the relationship between the structural features of the peptides and their antimicrobial activity, for the purpose of designing a new generation of antibiotics. While the external cell wall may be the initial target, evidence suggests that antimicrobial peptides act by lysing bacterial membranes. Cells become permeable following exposure to peptides, and their membrane potential is correspondingly reduced.[000133] Protamines or polycationic amino acid peptides containing combinations of one or more recurring units of cationic amino acids, such as arginine (R), tryptophan (W), lysine (K), even synthetic polyarginine, polytryptophan, polylysine, have been shown to be capable of killing microbial cells. These peptides cross the plasma membrane to facilitate uptake of various biopolymers or small molecules (Mitchell DJ et al (2002) J Peptide Res 56(5):318-325).[000134] In contrast to antibiotics, pathogens are unlikely to develop resistance to antimicrobial peptides, including engineered lysin-derived peptides of the disclosure, due to their rapid action on bacterial membrane. Lytic peptides have been evaluated for antibiotic resistance and shown not to lead to resistance. This is confimed by treating susceptible or target bacteria in vitro with different concentrations of one or more of the engineered lysin-derived peptides, and observing and evaluating for potential resistant mutants.[000135] The success of antimicrobial peptides thus far has been limited, largely due to the requirement that they be present in a fairly high concentration to achieve killing. This high concentration can exert a potentially cytotoxic effect on human erythrocytes as well as other cells and tissues for example. The high concentrations are due, in part to the susceptibility of antimicrobial peptides to native proteases in an animal or otherwise produced and present at the site of therapeutic target.[000136] Peptides for use in the present disclosure may include synthetic, recombinant or peptidomimetic entities. The peptides may be monomers, polymers, multimers, dendrimers, concatamers of various forms known or contemplated in the art, and may be so modified or multimerized so as to improve activity, specificity or stability'. For instance, and not by way of limitation, several strategies have been pursued in efforts to increase the effectiveness of antimicrobial peptides including dendrimers and altered amino acids (Tam, J. P. et al (2002) Eur J Biochem 269 (3): 923-932; Janiszewska, J. et al (2003) Bioorg Med Chem Lett 13 (21 ):3711-3713; Ghadiri et al. (2004) Nature 369(6478):301 -304; DeGrado et al (2003) Protein Science 12(4)1647-665; Tew et al. (2002) PNAS 99(8)15110-5114; Janiszewska, J et al (2003) Bioorg Med Chem Lett 13 (21); 3711-3713). U. S. Patent No. 5,229,490 to Tam discloses a particular polymeric construction formed by the binding of multiple antigens to a dendritic core or backbone.[000137] In an aspect of the disclosure, the engineered lysin-derived peptides of the disclosure may be attached to another molecule or may be labeled, including labeled with a detectable label. The label may include or may be selected from radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. The engineered peptide can also be labeled with a radioactive element or with an enzyme. Theradioactive label can be detected by any of the currently available counting procedures, The isotope may be selected from3H,14C,32P,35S,36C1,51Cr,57Co,58Co,59Fe,90Y,125I,131I, and186Re. Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme may be conjugated to the engineered lysin-derived peptide by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, B-glucuronidase, B-D-glucosidase, B-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.[000138] In an aspect of the disclosure, the engineered lysin-derived peptides of the disclosure may be covalently attached to another molecule or may be a fusion protein. Thus, conjugates or fusion proteins of the present disclosure, wherein the peptide of the present disclosure, or one or more peptide(s) o f the present disclosure are conjugated or attached to other molecules or agents further include, but are not limited to peptides conjugated to a cell or pathogen targeting agent or sequence, membrane binding agent or sequence, lipophilic agent or sequence, toxin, immunomodulator, cytokine, cytotoxic agent, one or more anti-bacterial agent or drug.[000139] In a general aspect, the invention provides an engineered or variant peptide, derived from a lysin polypeptide sequence. In particular, the engineered or variant peptide is an anti-bacterial peptide and is capable of killing one or more bacteria, including distinct species of bacteria, including gram-positive and gram-negative bacteria. In an aspect, the engineered peptide is capable and effective to kill bacteria species which are not effectively killed by the original umodified lysin polypeptide sequence. In an aspect of the invention, the engineered peptide is capable and effective to kill gram-positive bacteria, which are not effectively killed by the original unmodified lysin polypeptide. In aspects, the engineered peptide is capable and effective to kill various gram-negative and various gram-positive bacteria.[000140] The engineered peptide is active against and capable of killing both Cutibacterium and Staphylococcus species bacteria. Particularly susceptible bacteria are Cutibacterium acnes and Staphylococcus aureus, both of which are associated with and contribute to chronic inflammatory disease of the skin, particularly acne and acne vulgaris, hi aspects of the invention, the engineered peptide is active against and capable of killing Prevotella species bacteria. In an aspect of the invention, the engineered peptide is active against Staphylococcus aureus, Staphylococcus epidermidis, and antibiotic resistant Staphylococcus aureus. In some embodiments, the engineered peptide is capable of killing pathogenic and antibiotic-resistant bacteria.[000141] The engineered peptide sequence provided herein is a variant derived from an amino acid sequence of the lysin PlyPiOl (Genbank MBQ0073608) which is a lysin of the Prevotella species of bacteria, particularly Prevotella intermedia. The C-terminal cationic region of the PlyPiOl lysin, corresponding to amino acids 102-132 of the 141 amino acid lysin, denoted PiPOl, was used as a basis for generating engineered variant peptides. The full lysin PlyPiOl exhibits low activity against C. acnes. Also, the C-terminal peptide PiPOl exhibits low activity against C. acnes. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the PiPOl sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6) and having one or more cationic amino acids, or a cationic polymer, added at the N-terminus and / or C-terminus, wherein the peptide is effective to kili bacteria. An engineered lysin-derived peptide comprising the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having one or more cationic amino acids or a cationic polymer added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO: 1, wherein the peptide is effective to kill bacteria.[000142] In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the PiPOl sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6) and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. An engineered lysin-derived peptide comprising the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:1, wherein the peptide is effective to kill bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:5, wherein the peptide is effective to kill bacteria.[000143] In aspects, the engineered peptide comprises the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N-terminusand / or C-terminus, wherein the peptide length is 32-40 amino acids, particularly at least 33 amino acids, particularly 33-35 amino acids, particularly 32, 33, 34 or 35 amino acids. In aspects, the engineered peptide comprises the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having two or more cationic amino acids added at the N-terminus and / or C-terminus. In aspects, the engineered peptide comprises the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having two or more cationic amino acids added at the N-terminus and C-terminus. In aspects, the engineered peptide comprises the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having one or two cationic amino acids added at the N-terminus and one or two cationic amino acids added at the C-terminus. In aspects, the engineered peptide comprises the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having up to three cationic amino acids added at the N-terminus and one or two cationic amino acids added at the C-terminus. In aspects, the engineered peptide comprises the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having up to three cationic amino acids added at the N-terminus and up to two cationic amino acids added at the C-tenninus. In aspects, the engineered peptide comprises the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having two cationic amino acids added at the N-terminus and one or two cationic amino acids added at the C-tenninus. In aspects, the engineered peptide comprises the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having two cationic amino acids added at the N-terminus and two cationic amino acids added at the C-terminus.[000144] In an aspect of the present invention, an engineered lysin-derived peptide is provided comprising the PiPOl sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill gram-positive and gram-negative bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:1, wherein the peptide is effective to kill gram-positive and gram-negative bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:5, wherein the peptide is effective to kill gram-positive and gram-negative bacteria. In an aspect of the present invention, an engineered lysin-derived peptide is provided comprising the PiPOl sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N- terminus and / or C-terminus, wherein the peptide is effective to kill Cutibacterium and Staphylococcus species bacteria and also Prevotella species of bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill Cutibacterium and Staphylococcus species bacteria and also Prevotella, Acinetobacter, Escherichia, and Pseudomonas species of bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:1, wherein the peptide is effective to kill Cutibacterium and Staphylococcus species bacteria and also Prevotella, Acinetobacter, Escherichia, and Pseudomonas species of bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO: 1, wherein the peptide is effective to kill Cutibacterium and Staphylococcus species bacteria and also Prevotella species of bacteria. In accordance with the present invention, an engineered lysin- derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:1, wherein the peptide is effective to kill Cutibacterium and Staphylococcus species bacteria and also Prevotella, Acinetobacter, Escherichia, and Pseudomonas species of bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:5, wherein the peptide is effective to kill Cutibacterium and Staphylococcus species bacteria and also Prevotella species of bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:5, wherein the peptide is effective to kill Cutibacterium and Staphylococcus species bacteria and also Prevotella, Acinetobacter, Escherichia, and Pseudomonas speciesof bacteria. In particular, susceptible and / or killed bacteria include C. acnes, S. aureus, S. epidermidis, Prevotella intermedia.[000145] In an aspect of the present invention, an engineered lysin-derived peptide is provided comprising the PiPOl sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill Cutibacterium and Staphylococcus species bacteria. In accordance with the present invention, an engineered lysin-derived peptide is provided comprising an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:1, wherein the peptide is effective to kill Cutibacterium and Staphylococcus species bacteria.[000146] In accordance with the present invention, an engineered lysin-derived peptide is provided comprising the PiPOl sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria. In an aspect, an engineered lysin-derived peptide is provided comprising the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6, and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria.[000147] In accordance with the invention, the PiPO 1 sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6) was modified by strategically introducing cationic amino acids, particularly introducing lysine (K) and / or arginine (R) residues at the N and / or C-terminal end(s), to improve its antibacterial activity. The following peptide sequences are provided:Peptide P11: KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRK (SEQ ID NO:2)Peptide P111: KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRKK (SEQ ID NO:3)Peptide P16: KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:4)Peptide P156: RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:1)Peptide P157: RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO:5)[000148] In another aspect, the following peptide is provided:KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRRK (SEQ ID NO: 10).[000149] In aspects of the invention, an engineered lysin-derived peptide is provided comprising the sequence RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:1), or an amino acidsequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO: 1. In some aspects, an engineered lysin-derived peptide is provided comprising the sequence RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO: 1 ), or an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO: 1 and having one or more cationic amino acids added at the N-terminus and / or C -terminus, wherein the peptide is effective to kill bacteria.[000150] In aspects of the invention, an engineered lysin-derived peptide is provided comprising the sequence RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO: 5), or an amino acid sequence having at least 80% identity, 85% identity', 90% identity or 95% identity to SEQ ID NO:5. In some aspects, an engineered lysin-derived peptide is provided comprising the sequence RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO:5), or an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:5 and having one or more cationic amino acids at the N-terminus and / or C -terminus, wherein the peptide is effective to kill bacteria. In some aspects, an engineered lysin-derived peptide is provided comprising the sequence RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO:5), or an amino acid sequence having at least 80% identity, 85% identity, 90% identity or 95% identity to SEQ ID NO:5 and having one or more cationic amino acids added at the N-terminus and / or C -terminus, wherein the peptide is effective to kill bacteria. In an aspect, the engineered peptide is not SEQ ID NO: 1.[000151] In aspects of the invention, the engineered peptide(s) are effective to kill gram-positive and gram-negative bacteria. In aspects of the invention, the engineered peptide(s) are effective to kill Cutibacterium and Staphylococcus species bacteria and also Prevotella, Acinetobacter, Escherichia, and Pseudomonas species of bacteria. In aspects of the invention, the engineered peptide(s) are effective to kill Cutibacterium, Staphylococcus, and Prevotella species bacteria. In aspects of the invention, the engineered peptide(s) are effective to kill Cutibacterium and Staphylococcus species bacteria. In aspects of the invention, the engineered peptide(s) are effective to kill Cutibacterium and Staphylococcus species bacteria. Particularly susceptible bacteria are Cutibacterium acnes and Staphylococcus aureus. In aspects the engineered peptide(s) are effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus. In an aspect, the engineered peptide(s) are effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus in skin. In an aspect, the engineered peptide(s) are effective to kill or decolonize Cutibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis in skin. In aspects, the engineered peptide(s) are effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureusassociated with acne vulgaris and / or acne skin disorders in humans. In aspects, the Pl 56 engineered peptide(s) are effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus associated with acne vulgaris and / or acne skin disorders in humans. In aspects, the Pl 57 engineered peptide(s) are effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus associated with acne vulgaris and / or acne skin disorders in hurnans.[000152] In an aspect, the engineered peptide denoted Pl 56 RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:1) is effective to kill Cutibacterium and Staphylococcus species bacteria. Particularly susceptible bacteria are. Cutibacterium acnes and Staphylococcus aureus. In aspects the Pl 56 engineered peptide (SEQ ID NO:1) is effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus. In an aspect, the Pl 56 engineered peptide (SEQ ID NO: 1) is effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus in the presence of one or more of retinoids or benzoyl peroxide. In an aspect, the P156 engineered peptide (SEQ ID NO:1) is effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus in skin. In aspects, the Pl 56 engineered peptide (SEQ ID NO:1) is effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus associated with acne vulgaris and / or acne skin disorders in humans.[000153] In an aspect, the engineered peptide denoted Pl 57 RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO:5) is effective to kill Cutibacterium and Staphylococcus species bacteria. Particularly susceptible bacteria are Cutibacterium acnes and Staphylococcus aureus. In aspects, the Pl 57 engineered peptide (SEQ ID NO:5) is effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus. In an aspect, the P157 engineered peptide (SEQ ID NO:5) is effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus in the presence of one or more of retinoids or benzoyl peroxide. In an aspect, the Pl 57 engineered peptide (SEQ ID NO:5) is effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus in or on skin. In aspects, the P157 engineered peptide (SEQ ID NO:5) is effective to kill or decolonize Cutibacterium acnes and Staphylococcus aureus associated with acne vulgaris and / or acne skin disorders in humans.[000154] In other embodiments, the engineered peptide is capable of killing bacteria, including C. acnes, in the presence of one or more of retinoid, benzoyl peroxide, or salicylic acid. In other embodiments, the engineered peptide is capable of killing bacteria, including C. acnes and S. aureus, in the presence of one or more of retinoid, benzoyl peroxide, or salicylic acid.[000155] In other embodiments, the engineered peptide is capable of killing bacteria in the presence of surfactant. In other embodiments, the engineered peptide is capable of killing bacteria in oral / saliva conditions. In other embodiments, the engineered peptide is capable of killing bacteria in the presence of one or more antibiotic.[000156] In other embodiments, the engineered peptide is capable of killing bacteria, including C. acnes, in the presence of one or more cosmetic ingredients. The engineered peptide is capable of killing bacteria, including C. acnes, in the presence of one or more cosmetic ingredients selected from emulsifiers, preservatives, thickeners, moisturizers, colors, and fragrances. Exemplary ingredients include glycerin, hyaluronic acid, retinol, niacinamide, and parabens. In an aspect, the engineered peptide is capable of killing bacteria, including C. acnes, in the presence of one or more cosmetic ingredient selected from retinoid, benzoyl peroxide, and salicylic acid. In other embodiments, the engineered peptide is capable of killing bacteria, including C. acnes and S. aureus, in the presence of one or more cosmetic ingredient. In other embodiments, the engineered peptide is capable of killing bacteria, including C. acnes, S. aureus, and S. epidermidis in the presence of one or more cosmetic ingredient.[000157] In aspects of the invention, the engineered peptide is not toxic to cells. In aspects of the invention, the engineered peptide is not toxic to native human cells. In aspects of the invention, the engineered peptide is not toxic to human red blood cells.[000158] Cutibacterium acnes (Propionibacterium acnes) is a relatively slow-growing,typically aerotolerant anaerobic, gram-positive bacterium linked to the skin condition of acne. It can also cause and / or is associated with eye conditions such as chronic blepharitis, and is associated with ophthalmic complications and conditions, such as endophthalmitis, particularly following intraocular surgery. C. acnes may also cause comeal ulcers. C. acnes has been found in herniated discs. The propionic acid which it secretes creates micro-fractures of the surrounding bone. C. acnes can be found in bronchoalveolar lavage of approximately 70% of patients with sarcoidosis and is associated with disease activity. C. acnes is often considered an opportunistic pathogen, causing a range of postoperative and device-related infections, notably including surgical infections, post-neurosurgical infections, infected joint prostheses (especially shoulder), neurosurgical shunt infections and endocarditis in patients with prosthetic heart valves.[000159] The bacteria species is largely commensal and part of the skin flora present on most healthy adult humans' skin. It also can be found throughout the gastrointestinal tract. Cutibacteriumacnes bacteria predominantly live deep within follicles and pores, although they are also found on thesurface of healthy skin. The cellular damage, metabolic byproducts and bacterial debris produced by the rapid growth of C. acnes in follicles can trigger inflammation. This inflammation can lead to the symptoms associated with some common skin disorders, such as folliculitis and acne vulgaris. Acne vulgaris is a chronic inflammatory disease of the pilosebaceous unit, which includes the hair follicle, hair shaft, and sebaceous gland.[000160J The damage caused by C. acnes and the associated inflammation make the affected tissue more susceptible to colonization by opportunistic bacteria, such as Staphylococcus aureus. Some studies show healthy pores are colonized by C. acnes, while unhealthy ones then include the nonporeresident Staphylococcus epidermidis, amongst other bacterial contaminants. In addition to contributing to skin inflammation and acne lesions, an imbalance in these bacteria may also impair the skin's ability' to heal and regenerate, leading to prolonged and more severe acne outbreaks. This disruption can also affect the skin's overall microbiome diversity, potentially increasing susceptibility to other skin conditions such as eczema or rosacea.[000161] Cutibacterium acnes bacteria are susceptible to a wide range of antimicrobial molecules, from both pharmaceutical and natural sources. The antibiotics most frequently used to combat / kill C. acnes and treat acne vulgaris are erythromycin, clindamycin, doxycycline, and minocycline. Several other families of antibiotics are also active against C. acnes bacteria, including quinolones, cephalosporins, pleuromutilins, penicillins, and sulfonamides. C. acnes bacteria are susceptible to many types of antimicrobial chemicals found in over-the-counter antibacterial products, including benzoyl peroxide, triclosan, chloroxylenol, and chlorhexidine gluconate.[000162] Therapeutic or pharmaceutical compositions comprising the engineered lysin-derived peptides of the invention are provided in accordance with the invention, as well as related methods of use and methods of manufacture. Therapeutic or pharmaceutical compositions may comprise one or more engineered lysin-derived peptide(s), optionally combined with other components such as a carrier, vehicle, polypeptide, polynucleotide, one or more antibiotics or suitable excipients, carriers or vehicles.[000163] In aspects, a composition is provided comprising the engineered peptide. In an aspect, a composition is provided comprising one or more engineered peptide. In one aspect, the composition is an antimicrobial composition further comprising a pharmaceutically acceptable carrier.[000164] In some aspects, the composition further comprises one or more antibiotic.[000165] In embodiments, the compositions further comprise one or more agent used for treatment or amelioration of acne or a skin disorder or inflammation, including any such disorder associated with acne.In some embodiments, the composition further comprises one or more of retinoid, benzoyl peroxide, or salicylic acid.[000166] In some aspects, a retinoid is a derivative of vitamin A or is related to vitamin A. In aspects, retinoid may be selected from retinol, retinal, retinoic acid (tretinoin), isotretinoin, alitretinoin, etretinate, acitretin, adapalene, bexarotene, tazarotene and trifarotene.[000167] In other aspects, the composition is suitable for topical administration and effective to kill one or more type of bacteria.[000168] The invention provides a pharmaceutical composition for killing gram-positive or gramnegative bacteria comprising the engineered peptide in an amount effective to kill the gram-positive or gram-negative bacteria.[000169] The invention provides therapeutic compositions or pharmaceutical compositions of the engineered peptide(s) of the invention, including Pl 56 (SEQ ID NO: 1 ) or variants thereof (such as variants having 90% sequence identity to SEQ ID NO: 1), for use in treating, reducing or controlling contamination and / or infections by gram positive bacteria, particularly including C. acnes and £ aureus, including in contamination or infection of or via an external surface such as skin. The invention provides therapeutic compositions or pharmaceutical compositions of the engineered peptide(s) of the invention, including Pl 57 (SEQ ID NO:5) or variants thereof (such as variants having 90% sequence identity to SEQ ID NO:5), for use in treating, reducing or controlling contamination and / or infections by gram positive bacteria, particularly including C. acnes and £ aureus, including in contamination or infection of or via an external surface such as skin. Compositions are thereby contemplated and provided for topical or dermatological applications and general administration to the exterior, including the skin or other external surface. Compositions of the engineered peptide or of one or more engineered peptide(s) are provided herein for use in the killing, alleviation, decolonization, prophylaxis or treatment of gram-positive bacteria, including bacterial infections or related conditions, particularly of Cutibacterium or Staphylococcus, including C. acnes or S. aureus or 5. epidermidis, and antibiotic resistant bacteria, such as antibiotic-resistant £ aureus.[000170] The pharmaceutical composition can also include one or more engineered peptides, including variants thereof, produced by chemical synthesis or DNA recombinant techniques. In particular, engineered peptide(s) or variants thereof can be produced by amino acid substitution, deletion, truncation, chimerization, shuffling, or combinations thereof. The pharmaceutical composition may contain a combination of one or more engineered peptide, or a combination of one or more engineered peptide andone or more variant engineered peptide. The pharmaceutical composition may also contain addition of one or more complementary agent, and a pharmaceutically acceptable carrier or diluent.[000171] The pharmaceutical composition can contain a complementary agent, including one or more antimicrobial agent and / or one or more conventional antibiotics. In order to accelerate treatment of the infection, the therapeutic agent may further include at least one complementary' agent which can also potentiate the bactericidal activity of the lytic enzyme. Antimicrobials act largely by interfering with the structure or function of a bacterial cell by inhibition of cell wall synthesis, inhibition of cell-membrane function and / or inhibition of metabolic functions, including protein and DNA synthesis. Antibiotics can be subgrouped broadly into those affecting cell wall peptidoglycan biosynthesis and those affecting DNA or protein synthesis in gram positive bacteria. Cell wall synthesis inhibitors, including penicillin and antibiotics like it, disrupt the rigid outer cell wall so that the relatively unsupported cell swells and eventually ruptures. Antibiotics affecting cell wall peptidoglycan biosynthesis include: Glycopeptides, which inhibit peptidoglycan synthesis by preventing the incorporation of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) peptide subunits into the peptidoglycan matrix. Available glycopeptides include vancomycin and teicoplanin.; Penicillins, which act by inhibiting the formation of peptidoglycan cross-links. The functional group of penicillins, the p-lactam moiety, binds and inhibits DD-transpeptidase that links the peptidoglycan molecules in bacteria. Hydrolytic enzymes continue to break down the cell wall, causing cytolysis or death due to osmotic pressure. Common penicillins include oxacillin, ampicillin and cloxacillin; and Polypeptides, which interfere with the dephosphorylation of the Css-isoprenyl pyrophosphate, a molecule that carries peptidoglycan building-blocks outside of the plasma membrane. A cell wall-impacting polypeptide is bacitracin.[000172] The complementary agent may be an antibiotic, such as erythromycin, clarithromycin, azithromycin, roxithromycin, other members of the macrolide family, penicilins, cephalosporins, and any combinations thereof in amounts which are effective to synergistically enhance the therapeutic effect of the lytic enzyme. Virtually any other antibiotic may be used with the engineered peptide(s). Similarly, other lytic enzymes or ly sin peptides may be included in the carrier to treat other bacterial infections. Antibiotic supplements may be used in virtually all uses of the engineered peptide(s)when treating different diseases. The pharmaceutical composition can also contain a peptide or a peptide fragment of at least one lytic protein, one holin protein, or at least one holin and one lytic protein, which lytic and holin proteins are each derived from the same or different bacteria species, with an optional addition of a complementary agents, and a suitable carrier or diluent.[000173] The invention provides cosmetic compositions comprising the engineered peptide. The cosmetic composition may comprise the engineered peptide in an amount effective to kill the gram-positive or gram-negative bacteria. The cosmetic composition may include one or more cosmetic ingredient. Cosmetic ingredients may include a blend of water, emulsifiers, preservatives, thickeners, moisturizers, colors, and / or fragrances designed to cleanse, beautify, and / or alter the skin's appearance. Exemplary ingredients include glycerin, hyaluronic acid, retinol, niacinamide, and parabens. Ingredient categories and examples include: (i) Moisturizers / Humectants - Glycerin, hyaluronic acid, propylene glycol, and urea attract moisture to the skin; (ii) Emollients - Petrolatum, shea butter, oils, and silicones (dimethicone) soften skin and prevent water loss; (iii) Surfactants - Sodium lauryl sulfate (SLS) and cocamidopropyl betaine are used in cleansers to remove dirt and oil; (iv) Preservatives - Phenoxyethanol and parabens extend shelf life and prevent microbial growth; (v) Thickeners - Xanthan gum, carbomer, and cetyl alcohol improve product consistency; (vi) Active Ingredients - Retinol (anti-aging), Salicylic Acid (acne), and Alpha Hydroxy Acids (exfoliation); (vii) Colorants / Pigments - Iron oxides, mica, and titanium dioxide.[000174] In another aspect, the invention includes a composition suitable for application to the skin. In aspects, such a composition is suitable for topical application. In aspects, the composition is a cream, aqueous gel, or other suitable formulation for topical application or cosmetic use. The composition may include other components frequently added to or present in cosmetics or makeup. The composition may include dyes or skin tints.[000175] Such compositions include compositions for therapeutic skincare. Therapeutic skincare and compositions thereof and therefor involve the use of high-quality ingredients and advanced treatments—such as serums, chemical peels, and laser resurfacing—to repair, protect, and heal the skin. Ingredients include antioxidants, peptides, vitamins, ceramides, and aloe vera, which target aging, acne, and inflammation.[000176] Compositions for therapeutic skincare help strengthen the skin barrier and improve overall texture. Active Ingredients include serums and creams comprising hyaluronic acid for hydration, retinols for renewal, and antioxidants for protection. Targeted Treatments include microneedling, chemical peels, and laser therapies are used to reduce scars, wrinkles, and pigmentation. Condition-Specific Care includes products tailored for specific needs, such as calming redness, treating acne, or intensely hydrating dry skin. Common Therapeutic Ingredients include: Ceramides & Shea Butter to restore and maintain a healthy skin barrier; Aloe Vera to soothe inflammation and promote healing; Pumpkin Enzymes to exfoliate and stimulate cell turnover; and / or Hyaluronic Acid to deliver deep hydration[000177] Compositions of the invention may comprise the engineered peptide combined with one or more ingredients associated with therapeutic skincare, cosmetics or treatments to reduce redness or swelling.[000178] Also provided are compositions containing nucleic acid molecules that, either alone or in combination with other nucleic acid molecules, are capable of expressing an effective amount of an engineered lysin-derived peptide(s) in vivo. Cell cultures containing these nucleic acid molecules, polynucleotides, and vectors carrying and expressing these molecules in vitro or in vivo, are also provided.[000179] Therapeutic or pharmaceutical compositions may comprise engineered lysin-derived peptide(s) combined with a variety of carriers to treat the illnesses caused by the susceptible bacteria. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine; amino acids such as glutamic acid, aspartic acid, histidine, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, trehalose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counter-ions such as sodium; non-ionic surfactants such as polysorbates, poloxamers, or polyethylene glycol (PEG); and / or neutral salts, e.g,, NaCl, KC1, MgCl2, CaCl2, and others. Glycerin or glycerol (1,2,3-propanetriol) is commercially available for pharmaceutical use. It may be diluted in sterile water for injection, or sodium chloride injection, or other pharmaceutically acceptable aqueous injection fluid, and used in concentrations of 0.1 to 100% (v / v), preferably 1.0 to 50% more preferably about 20%. DMSO is an aprotic solvent with a remarkable ability to enhance penetration of many locally applied drugs. DMSO may be diluted in sterile water for injection, or sodium chloride injection, or other pharmaceutically acceptable aqueous injection fluid, and used in concentrations of 0.1 to 100% (v / v). The carrier vehicle may also include Ringer's solution, a buffered solution, and dextrose solution, particularly when an intravenous solution is prepared.[000180] A engineered lysin-derived peptide(s) may be added to these substances in a liquid form or in a lyophilized state, whereupon it will be solubilized when it meets body fluids such as saliva. The engineered lysin-derived peptide(s) may also be in a micelle or liposome.[000181] The effective dosage rates or amounts of an engineered lysin-derived peptide(s) to treat the infection will depend in part on whether the engineered lysin-derived peptide(s) will be used therapeutically or prophylactically, the duration of exposure of the recipient to the infectious bacteria, the size and weight of the individual, etc. The duration for use of the composition containing the engineered lysin-derived peptide(s) also depends on whether the use is for prophylactic purposes, wherein the use may be hourly, daily or weekly, for a short time period, or whether the use will be for therapeutic purposes wherein a more intensive regimen of the use of the composition may be needed, such that usage may last for hours, days or weeks, and / or on a daily basis, or at timed intervals during the day. Any dosage form employed should provide for a minimum number of units for a minimum amount of time. The concentration of the active units of enzyme believed to provide for an effective amount or dosage of enzyme may be in the range of about 100 units / ml to about 500,000 units / ml of fluid in the wet or damp environment of the nasal and oral passages, and possibly in the range of about 100 units / ml to about 50,000 units / ml. Carriers that are classified as "long" or "slow" release carriers (such as, for example, certain nasal sprays or lozenges) could possess or provide a lower concentration of active units per ml, but over a longer period of time, whereas a "short" or "fast" release carrier (such as, for example, a gargle) could possess or provide a high concentration of active units per ml, but over a shorter period of time. The amount of active units per ml and the duration of time of exposure depend on the nature of infection, whether treatment is to be prophylactic or therapeutic, and other variables. There are situations where it may be necessary to have a much higher unit / ml dosage or a lower unit / ml dosage. In aspects, the dosage of the engineered peptide is 1 pg / ml to 100 pg / ml. In aspects, the dosage of the engineered peptide is 1 pg / ml to 200 pg / ml. In aspects, the dosage of the engineered peptide is 10 pg / ml to 100 pg / ml. In aspects, the dosage of the engineered peptide is 10 pg / ml to 200 pg / ml. In aspects, the dosage of the engineered peptide is 10 pg / ml to 300 pg / ml. In aspects, the dosage of the engineered peptide is 10 pg / ml to 400 pg / ml. In aspects, the dosage of the engineered peptide is 10 pg / ml to 500 pg / ml. In some aspects, the composition is administered, or is applied to the skin for example, at least once per day. In aspects, the dosage of the engineered peptide is 10 pg / ml to 500 pg / ml. In some aspects, the composition is administered, or is applied to the skin for example, 1-2 times per day.[000182] The engineered lysin-derived peptide(s) should be in an environment having a pH which allows for activity. Prior to, or at the time the engineered lysin-derived peptide(s) is put in the carrier system or oral delivery mode, it is preferred that the enzyme be in a stabilizing buffer environment for maintaining a pH range between about 4.0 and about 9.0, more preferably between about 5.5 and about 7.5. A stabilizeror pH modulator may be combined with the engineered peptide, in a composition for example, to serve to retain the pH.[000183] A stabilizing buffer may allow for the optimum activity of the engineered lysin-derived peptide(s). The buffer may contain a reducing reagent, such as dithiothreitol. The stabilizing buffer may also be or include a metal chelating reagent, such as ethylenediaminetetracetic acid disodium salt, or it may also contain a phosphate or citrate-phosphate buffer, or any other buffer.[000184] A mild surfactant can be included in a therapeutic or pharmaceutical composition in an amount effective to potentiate the therapeutic effect of the engineered lysin-derived peptide(s) may be used in a composition. Suitable mild surfactants include, inter alia, esters of polyoxyethylene sorbitan and fatty acids (Tween series), octylphenoxy polyethoxy ethanol (Triton-X series), n-Octyl-.beta.-D-glucopyranoside, n- Octyl-.beta.-D-thioglucopyranoside, n-Decyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D- glucopyranoside, and biologically occurring surfactants, e.g., fatty acids, glycerides, monoglycerides, deoxycholate and esters of deoxy cholate.[000185] Preservatives may also be used in this invention and preferably comprise about 0.05% to 0.5% by weight of the total composition. The use of preservatives assures that if the product is microbially contaminated, the formulation will prevent or diminish microorganism growth. Some preservatives useful in this invention include methylparaben, propylparaben, butylparaben, chloroxylenol, sodium benzoate, DMDM Hydantoin, 3-lodo-2-Propylbutyl carbamate, potassium sorbate, chlorhexidine digluconate, or a combination thereof.[000186] Pharmaceuticals for use in all embodiments of the invention include antimicrobial agents, anti¬ inflammatory agents, antiviral agents, local anesthetic agents, corticosteroids, destructive therapy agents, antifungals, and antiandrogens. In the treatment of acne, active pharmaceuticals that may be used include antimicrobial agents, especially those having anti-inflammatory properties such as dapsone, erythromycin, minocycline, tetracycline, clindamycin, and other antimicrobials. The preferred weight percentages for the antimicrobials are 0.5% to 10%.[000187] Local anesthetics include tetracaine, tetracaine hydrochloride, lidocaine, lidocaine hydrochloride, dyclonine, dyclonine hydrochloride, dimethisoquin hydrochloride, dibucaine, dibucaine hydrochloride, butambenpicrate, and pramoxine hydrochloride. A preferred concentration for local anesthetics is about 0.025% to 5% by weight of the total composition. Anesthetics such as benzocaine may also be used at a preferred concentration of about 2% to 25% by weight.1000188] Corticosteroids that may be used include betamethasone dipropionate, fluocinolone actinide, betamethasone valerate, triamcinolone actinide, clobetasol propionate, desoximetasone, diflorasone diacetate, amcinonide, flurandrenolide, hydrocortisone valerate, hydrocortisone butyrate, and desonide are recommended at concentrations of about 0.01% to 1.0% by weight. Preferred concentrations for corticosteroids such as hydrocortisone or methylprednisolone acetate are from about 0.2% to about 5.0% by weight.[000189] Means of application of the therapeutic composition comprising an engineered lysin-derived peptide(s) include, but are not limited to direct, indirect, carrier and special means or any combination of means. Direct application of the engineered lysin-derived peptide(s) may be by any suitable means to directly bring the polypeptide in contact with the site of infection or bacterial colonization, such as to the nasal area (for example nasal sprays), dermal or skin applications (for example topical ointments or formulations), suppositories, tampon applications, etc. Nasal applications include for instance nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, mouthwashes or gargles, or through the use of ointments applied to the nasal nares, or the face or any combination of these and similar methods of application. The forms in which the lytic enzyme may be administered include but are not limited to lozenges, troches, candies, injectants, chewing gums, tablets, powders, sprays, liquids, ointments, and aerosols.[000190] When the engineered lysin-derived peptide(s) is introduced directly by use of sprays, drops, ointments, washes, injections, packing and inhalers, the peptide(s) is preferably in a liquid or gel environment, with the liquid acting as the carrier. The engineered lysin-derived peptide(s) may be in the form of a cream, salve, or gel. A dry anhydrous version of the altered enzyme may be administered by the inhaler and bronchial spray, although a liquid form of delivery is preferred.[000191] Compositions for treating topical infections or contaminations comprise an effective amount of at least one engineered lysin-derived peptide(s) according to the invention and a carrier for delivering at least one peptide to the infected or contaminated skin, coat, or external surface of an animal or human. The mode of application for the engineered lysin-derived peptide(s) includes a number of different types and combinations of carriers which include, but are not limited to an aqueous liquid, an alcohol base liquid, a water soluble gel, a lotion, an ointment, a nonaqueous liquid base, a mineral oil base, a blend of mineral oil and petrolatum, lanolin, liposomes, protein carriers such as serum albumin or gelatin, powdered cellulose carmel, and combinations thereof. A mode of delivery of the carrier containing the therapeutic agent includes, but is not limited to a smear, spray, a time-release patch, a liquid absorbed wipe, and combinationsthereof. The engineered lysin-derived peptide(s) may be applied to a bandage either directly or in one of the other carriers. The bandages may be sold damp or dry', wherein the peptide is in a lyophilized form on the bandage. This method of application is most effective for the treatment of infected skin or affected / inflamed skin. The carriers of topical compositions may comprise semi-solid and gel-like vehicles that include a polymer thickener, water, preservatives, active surfactants or emulsifiers, antioxidants, sun screens, and a solvent or mixed solvent system. U. S. Pat. No. 5,863,560 (Osborne) discusses a number of different carrier combinations which can aid in the exposure of the skin to a medicament. Polymer thickeners that may be used include those known to one skilled in the art, such as hydrophilic and hydroalcoholic gelling agents frequently used in the cosmetic and pharmaceutical industries. CARBOPOLR™ is one of numerous cross-linked acrylic acid polymers that are given the general adopted name carbomer. These polymers dissolve in water and form a clear or slightly hazy gel upon neutralization with a caustic material such as sodium hydroxide, potassium hydroxide, triethanolamine, or other amine bases. KLUCELR™ is a cellulose polymer that is dispersed in water and forms a uniform gel upon complete hydration. Other preferred gelling polymers include hydroxyethylcellulose, cellulose gum, MVE / MA decadiene crosspolymer, PVM / MA copolymer, or a combination thereof.[000192] A composition comprising a engineered lysin-derived peptide(s) can be administered in the form of a candy, chewing gum, lozenge, troche, tablet, a powder, an aerosol, a liquid, a liquid spray, or toothpaste, such as for the prevention or treatment of bacterial infections associated with upper respiratory tract illnesses or with oral infections. The lozenge, tablet, or gum into which the engineered lysin-derived peptide(s) is added may contain sugar, com syrup, a variety of dyes, non-sugar sweeteners, flavorings, any binders, or combinations thereof. Similarly, any gum-based products may contain acacia, carnauba wax, citric acid, cornstarch, food colorings, flavorings, non-sugar sweeteners, gelatin, glucose, glycerin, gum base, shellac, sodium saccharin, sugar, water, white wax, cellulose, other binders, and combinations thereof. Lozenges may further contain sucrose, cornstarch, acacia, gum tragacanth, anethole, linseed, oleoresin, mineral oil, and cellulose, other binders, and combinations thereof. Sugar substitutes can also be used in place of dextrose, sucrose, or other sugars.[000193] Compositions comprising engineered lysin-derived peptide(s) can be directed to the mucosal lining, where, in residence, they kill colonizing disease bacteria. The mucosal lining, as disclosed and described herein, includes, for example, the upper and lower respiratory tract, eye, buccal cavity, nose, rectum, vagina, periodontal pocket, intestines and colon. Due to natural eliminating or cleansing mechanisms of mucosal tissues, conventional dosage forms are not retained at the application site for anysignificant length of time. It may be advantageous to have materials which exhibit adhesion to mucosal tissues, to be administered with one or more engineered lysin-derived peptide(s) and other complementary agents over a period of time. Materials having controlled release capability are particularly desirable, and the use of sustained release mucoadhesives has received a significant degree of attention. Micelles and multilamillar micelles may also be used to control the release of peptide(s). Other approaches involving mucoadhesives which are the combination of hydrophilic and hydrophobic materials, are known. Orahesive. RTM. from E. R. Squibb & Co is an adhesive which is a combination of pectin, gelatin, and sodium carboxymethyl cellulose in a tacky hydrocarbon polymer, for adhering to the oral mucosa. Therapeutic or pharmaceutical compositions can also contain polymeric mucoadhesives including a graft copolymer comprising a hydrophilic main chain and hydrophobic graft chains for controlled release of biologically active agents. The graft chains consist essentially of polystyrene, and the main polymer chain of hydrophilic monomeric moieties, some of which have acidic functionality. The weight percent of the polystyrene macromonomer in the graft copolymer is between about 1 and about 20% and the weight percent of the total hydrophilic monomer in the graft copolymer is between 80 and 99%, and wherein at least 10% of said total hydrophilic monomer is acidic, said graft copolymer when fully hydrated having an equilibrium water content of at least 90%. Compositions containing the copolymers gradually hydrate by sorption of tissue fluids at the application site to yield a very soft jelly like mass exhibiting adhesion to the mucosal surface. During the period of time the composition is adhering to the mucosal surface, it provides sustained release of the pharmacologically active agent, which is absorbed by the mucosal tissue.[000194] The compositions of this application may optionally contain other polymeric materials, such as poly(acrylic acid), poly-(vinyl pyrrolidone), and sodium carboxymethyl cellulose plasticizers, and other pharmaceutically acceptable excipients in amounts that do not cause deleterious effect upon mucoadhesivity of the composition.[000195] The dosage forms of the compositions of this invention can be prepared by conventional methods. In cases where intramuscular injection is the chosen mode of administration, an isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. A vasoconstriction agent can be added to the formulation. The pharmaceutical preparations according to this application are provided sterile and pyrogen free.[000196] A engineered lysin-derived peptide(s) of the invention may also be administered by any pharmaceutically applicable or acceptable means including topically, orally or parenterally. For example,the engineered lysin-derived peptide(s) can be administered intramuscularly, intrathecally, subdermally, subcutaneously, or intravenously to treat infections by bacteria. In cases where parenteral injection is the chosen mode of administration, an isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. A vasoconstriction agent can be added to the formulation. The pharmaceutical preparations according to this application are provided sterile and pyrogen free.[000197] The effective dosage rates or amounts of the engineered lysin-derived peptide(s) to be administered parenterally, and the duration of treatment will depend in part on the seriousness of the infection, the weight of the patient, particularly human, the duration of exposure of the recipient to the infectious bacteria, the number of square centimeters of skin or tissue which are infected, the depth of the infection, the seriousness of the infection, and a variety of a number of other variables. The composition may be applied anywhere from once to several times a day, and may be applied for a short or long term period. The usage may last for days or weeks. Any dosage form employed should provide for a minimum number of units for a minimum amount of time. The concentration of the active units of enzymes believed to provide for an effective amount or dosage of enzymes may be selected as appropriate. The amount of active units per ml and the duration of time of exposure depend on the nature of infection, and the amount of contact the carrier allows the engineered lysin-derived peptide(s) to have.[000198] The bacterial killing capability exhibited by the engineered lysin-derived peptide(s) of the invention provides for various methods based on the antibacterial effectiveness of the peptide(s) of the invention. Thus, the present invention contemplates antibacterial methods, including methods for killing of bacteria, for reducing a population of bacteria, for treating or alleviating a bacterial infection, for treating a human subject exposed to a pathogenic bacteria, and for treating a human subject at risk for such exposure. The susceptible bacteria are demonstrated herein to include Cutibacterium, Staphylococcus and Prevotella bacteria. Methods of treating various conditions are also provided, including methods of prophylactic treatment of Cutibacterium, Staphylococcus and / or Prevotella infections, reducing Cutibacterium, Staphylococcus and / or Prevotella population or carriage. In particular, methods of treating skin conditions associated with Cutibacterium and / or Staphylococcus are provided. In particular, methods of treating skin conditions associated with C. acnes and / or S. aureus are provided. In particular, methods of treating skin conditions associated with C. acnes and / or S. aureus and / or S. epidermidis are provided. Methods of treating acne are provided. Methods of treating or preventing acne vulgaris are provided.[000199] In aspects, the invention provides a method of killing bacteria or reducing a population of bacteria comprising the step of contacting the bacteria with an amount of one or more engineered peptide, or with a composition comprising one or more engineered peptide, effective to kill the bacteria.[000200] In an aspect of the method, the bacteria is gram negative or gram positive.[000201] In an aspect of the method, the bacteria is Cutibacterium, Staphylococcus, Acinetobacter, Escherichia, Pseudomonas, and / or Prevotella. In an aspect of the method, the bacteria is Cutibacterium. or Staphylococcus. In one such aspect, the bacteria is C. acnes, S. aureus or S. epidermidis. In one such aspect, the bacteria is C. acnes or S. aureus.[000202] In an aspect of the method, the bacteria is an antibiotic-resistant bacteria.[000203] In an aspect of the method, the bacteria is methicillin-resistant Staphylococcus aureus (MRSA), vancomycin intermediate-sensitivity Staphylococcus aureus (VISA), or vancomycin resistant Staphylococcus aureus (VRSA)[000204] In another aspect, a method is provided for treating acne vulgaris in a human comprising administering to a human having acne an effective amount of one or more engineered peptide, or a composition comprising one or more engineered peptide, whereby the acne vulgaris is reduced or controlled.[000205] In aspects, methods are provided wherein the number of C. acnes and / or S. aureus bacteria associated with the acne is reduced.[000206] In aspects, methods are provided wherein the composition further comprises one or more of retinoid, benzoyl peroxide, salicylic acid, or an antibiotic.[000207] A method for treating or controlling contaminations of or infections by one or more bacteria of Cutibacterium, Staphylococcus or Prevotella bacteria is provided, comprising administering an effective amount of one or more engineered peptide provided herein, or with the composition of one or more of the engineered peptide. A method for treating or controlling contaminations of or infections by one or more bacteria of Cutibacterium, Staphylococcus, Prevotella, Acinetobacter, Escherichia, and / or Pseudomonas bacteria is provided, comprising administering an effective amount of one or more engineered peptide provided herein, or with the composition of one or more of the engineered peptide.[000208] In an aspect of the method, the bacteria is C. acnes or S. aureus or S. epidermidis. In an aspect of the method, the bacteria is P. intermedia.[000209] In some aspects, the bacteria is Prevotella, particularly P. intermedia, and the disease or condition associated with the bacteria is an oral or dental disease or condition, such as periodontal infection,periodontal disease, dental caries, or is a lung disease or condition such as cystic fibrosis (CF) or chronic bronchitis. In an aspect of the method(s), the engineered peptide is effective in the presence of saliva (oral / mouth) and / or surfactant (lungs) which are present in the tissue, organ, location, area of the infection, disease or condition.[000210] Methods are provided for reducing the number of bacteria on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing the number of Cutibacterium, Staphylococcus or Prevotella bacteria on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing the number of C. acnes and / or S. aureus bacteria on skin.[000211] Methods are provided for reducing a bacterial infection on skin, particularly on exposed areas of the skin such as the face ( including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing the number of Cutibacterium, Staphylococcus or Prevotella bacteria in a bacterial infection on skin, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing the number of C. acnes and / or S. aureus bacteria in a bacterial infection on skin, particularly on exposed areas of the skin.[000212] In other aspects, methods are provided for reducing the size of a site of bacterial growth on skin, such as a red, warm, painful or swollen area, particularly on exposed areas of the skin such as the face (including nose, ears, cheeks, chin), neck, chest, back, arms, legs. In aspects, methods are provided for reducing the number of Cutibacterium, Staphylococcus or Prevotella bacteria associated with the site of bacterial growth on skin. In aspects, methods are provided for reducing the number of C. acnes and / or S. aureus bacteria associated with the site of bacterial growth.[000213] In some aspects, the bacterial growth on skin is associated with infections or conditions such as impetigo, cellulitis, or abscesses. In some aspects, the bacterial growth on skin is associated with infections or conditions such as folliculitis or rosacia. Folliculitis is a common skin condition characterized by inflammation or infection of hair follicles, resulting in small, red, or pus-filled bumps (pimples) that may itch or sting. Folliculitis is often caused by or associated with Staphylococcus aureus bacteria. It typically affects hair-bearing areas like the face, scalp, thighs, and groin. Treatment often involves warm compresses, topical antibiotics, or antiseptic washes. The engineered peptides of the invention have applicability to and use for folliculitis, including particularly wherein it is associated with Staphylococcus bacteria or infection. In methods and applications for treatment or alleviation of folliculitis, the engineeredpeptide(s) may be combined with one or more antibiotic. In methods and applications for treatment or alleviation of folliculitis, the engineered peptide(s) may be formulated in a cream, gel, salve, including in combination with an antiseptic, such as isopropyl alcohol, hydrogen peroxide, povidone-iodine, and chlorhexidine.[000214] Rosacea is a chronic inflammatory skin condition causing persistent facial redness, flushing, visible blood vessels, and acne-like bumps, primarily affecting the cheeks, nose, chin, and forehead.[000215] In accordance with the invention, methods are provided for balancing the microbiome or for correcting or mediating dysbiosis of the microbiome, particularly dysbiosis of the skin microbiome. The skin of the animal or human body is ordinarily colonized by a diverse milieu of microorganisms, some of which are harmless and some of which are beneficial to the animal or human host. Microorganisms associate with the skim microbiome include bacteria, as well as fungi and viruses. Three bacterial phyla, Actinobacteria, Firmicutes, and Proteobacteria, predominate on skin, particularly commensal species Corynebacterium tuberculostericum, Cutibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis. The skin microbiome contributes to the pathophysiology of various skin diseases and disorders, including atopic dermatitis, acne vulgaris, alopecia, Hidradenitis Suppurativa (HS), psoriasis, Seborrheic dermatitis, skin ulcers and wounds.[000216] In an aspect of the above methods of killing bacteria, the methods are performed in vitro or ex vivo. In one such aspect, the methods are performed so as to sterilize or decontaminate a solution, material or device, particularly intended for use by or in a human. In another aspect, the methods are performed to decontaminate or sterilize a surface, such as skin.[000217] In accordance with any of the methods of the invention, the composition thereof may further comprise a carrier, including a pharmaceutically acceptable carrier, additive or diluent. In accordance with any of the methods of the invention, the composition thereof may further comprise a suitable vehicle for delivery of the polypeptide to a site of infection. In accordance with any of the methods of the invention, the composition thereof may further comprise one or more antibiotic. In accordance with any of the methods of the invention, the composition thereof may further comprise one or more agent effective against bacteria or the condition associated with bacteria that are the target of killing by the engineered peptide(s).[000218] 'The term ‘agent’ means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules. In particular the term agent includes compounds such as test compounds, added additional compound(s), or lysin enzyme compounds.[000219] The term ‘agonist’ refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.[000220] The term ‘assay’ means any process used to measure a specific property of a compound. A ‘screening assay’ means a process used to characterize or select compounds based upon their activity from a collection of compounds.[000221] The term ‘preventing’ or ‘prevention’ refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.[000222] The term ‘prophylaxis’ is related to and encompassed in the term ‘prevention’, and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.[000223] ‘Therapeutically effective amount’ means that amount of a drug, compound, antimicrobial, antibody, polypeptide, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician, in particular, with regard to bacterial infections and growth of bacteria, the term “effective amount” is intended to include an effective amount of a compound or agent that will bring about a biologically meaningful decrease in the amount of or extent of infection of bacteria, including having a bacteriocidal and / or bacteriostatic effect. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the growth or amount of infectious bacteria, or other feature of pathology such as for example, elevated fever or white cell count as may attend its presence and activity.[000224] The term ‘treating’ or ‘treatment’ of any disease or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., arresting the disease or growth of the infectious agent or bacteria or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment ‘treating’ or ‘treatment’ refers to ameliorating at least one physical parameter, which may not be discernible by the subject. Tn yet another embodiment, ‘treating’ or ‘treatment’ refers to modulating the disease or infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In a further embodiment, ‘treating’ or ‘treatment’ relates to slowing the progression of a disease or reducing an infection.[000225] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.[000226] It is noted that in the context of treatment methods which are carried out in vivo or medical and clinical treatment methods in accordance with the present application and claims, the term subject, patient or individual is intended to refer to a human.[000227] The term “bacteriocidal” refers to capable of killing bacterial cells.[000228] The term “bacteriostatic” refers to capable of inhibiting bacterial growth, including inhibiting growing bacterial cells.[000229] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.[000230] The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the S phase activity of a target cellular mass, or other feature of pathology such as for example, elevated blood pressure, fever or white cell count as may attend its presence and activity.[000231] One method for treating skin, tissue or systemic bacterial infections caused by Cutibacterium or Staphylococcus or Prevotella bacteria comprises parenterally treating the infection with a therapeutic agent comprising an effective amount of one or more engineered peptide(s) of the invention, particularly an engineered lysin-derived peptide derived from SEQ ID NO:6, including variants thereof, including such polypeptides as provided herein in SEQ ID NO: 1. or any of SEQ ID NOS:2-5, and an appropriate carrier. A number of other different methods may be used to introduce the engineered peptide(s). These methods include introducing the engineered peptide(s) intravenously, intramuscularly, subcutaneously, intrathecal ly, and subdermally. One skilled in the art, including medical personnel, will be capable of evaluating and recognizing the most appropriate mode or means of administration, given the nature and extent of the bacterial condition and the strain or type of bacteria involved or suspected. For instance, topical use and administration of one or more engineered peptide(s) would be most beneficial for treatment of acne vulgaris.[000232] Infections may also be treated by injecting into the infected tissue of the human patient a therapeutic agent comprising the appropriate engineered peptide(s) and a carrier for the enzyme. The carrier may be comprised of distilled water, a saline solution, albumin, a serum, or any combinations thereof. More specifically, solutions for infusion or injection may be prepared in a conventional manner, e.g. with the addition of preservatives such as p-hydroxybenzoates or stabilizers such as alkali metal salts of ethylenediamine tetraacetic acid, which may then be transferred into fusion vessels, injection vials or ampules. Alternatively, the compound for injection may be lyophilized either with or without the other ingredients and be solubilized in a buffered solution or distilled water, as appropriate, at the time of use. Non-aqueous vehicles such as fixed oils, liposomes, and ethyl oleate are also useful herein. Other phage associated lytic enzymes, along with a holin protein, may be included in the composition.[000233] Various methods of treatment are provided for using an engineered peptide(s), such as SEQ ID NO:1, or any of SEQ ID NOS:2-5, as exemplified herein, as a prophylactic treatment for eliminating or reducing the carriage of susceptible bacteria, preventing those humans who have been exposed to others who have the symptoms of an infection from getting sick, or preventing those with a mild acne condition from progressing to acne vulgaris, or as a therapeutic treatment for those who have already become ill or have conditions, such as skin conditions, from the infection. In an aspect, the engineered peptide(s), can be used to treat, for example, respiratory tract illnesses, such as CF or bronchitis, particularly by the use of bronchial sprays or intravenous administration of the engineered peptide(s). In an aspect, an engineered peptide(s), can be used for the prophylactic and therapeutic treatment of eye infections or ocular conditions or diseases. The method of treatment comprises administering eye drops or an eye wash which comprise an effective amount of at least one engineered peptide(s) of the invention and a carrier capable of being safely applied to an eye, with the carrier containing the engineered peptide(s). The eye drops or eye wash are preferably in the form of an isotonic solution. The pH of the solution should be adjusted so that there is no irritation of the eye, which in turn would lead to possible infection by other organisms, and possible to damage to the eye. While the pH range should be in the same range as for other lytic enzymes, the most optimal pH will be in the range as demonstrated and provided herein. Similarly, buffers could also be used. Other antibiotics which are suitable for use in eye drops may be added to the composition containing the engineered peptide(s). Other antibiotics which are suitable for use in skin ointments or dermal gels or creams, such as for alleviation or treatment of skin conditions, inflammations or acne, may be added to the composition containing the engineered peptide(s). Bactericides and bacteriostatic compounds may also be added. The concentration of the engineered peptide(s) in the solution can be in the range of from about 100units / ml to about 500,000 units / ml, with a more preferred range of about 100 to about 5,000 units / ml, and about 100 to about 50,000 units / ml. Concentrations can be higher or lower than the ranges provided.[000234] The engineered lysin-derived peptide(s) of the invention may also be used in a contact lens solution, for the soaking and cleaning of contact lenses. This solution, which is normally an isotonic solution, may contain, in addition to the enzyme, sodium chloride, mannitol and other sugar alcohols, borates, preservatives, and the like.[000235] The carrier may comprise sterile aqueous or oily solutions or suspensions. The engineered peptide] s) may be added to the carrier, which may also contain suitable preservatives, and preferably a surface-active agent. Bactericidal and fungicidal agents preferably included in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol. Additionally, any number of other skin lotion or dermal cream carriers may be used. Additionally, the carrier may typically include vitamins, minerals, carbohydrates, sugars, amino acids, proteinaceous materials, fatty acids, phospholipids, antioxidants, phenolic compounds, isotonic solutions, oil based solutions, oil based suspensions, and combinations thereof.[000236] The diagnostic, prophylactic and therapeutic possibilities and applications that are raised by the recognition of and isolation of the engineered lysin-derived peptide(s) of the invention, derive from the fact that the polypeptides of the invention cause direct and specific effects (e.g. killing) in susceptible bacteria. Thus, the polypeptides of the invention may be used to eliminate, characterize, or identify the relevant and susceptible bacteria.[000237] Thus, a diagnostic method of the present invention may comprise examining a cellular sample or medium for the purpose of determining whether it contains susceptible bacteria, or whether the bacteria in the sample or medium are susceptible by means of an assay including an effective amount of one or more engineered lysin-derived peptide(s) and a means for characterizing one or more cell in the sample, or for determining whether or not cell lysis has occurred or is occurring. Patients capable of benefiting from this method include those suffering from an undetermined infection, a recognized bacterial infection, or suspected of being exposed to or carrying a particular bacteria. A fluid, food, medical device, composition or other such sample which will come in contact with a subject or patient may be examined for susceptible bacteria or may be eliminated of relevant bacteria. In one such aspect a fluid, food, medical device, composition or other such sample may be sterilized or otherwise treated to eliminate or remove any potential relevant bacteria by incubation with or exposure to one or more engineered lysin-derived peptide(s) of theinvention. The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. In one instance, the engineered lysin-derived peptide(s) of the invention complex(es) with, or otherwise binds or associates with, relevant or susceptible bacteria in a sample and one member of the complex is labeled with a detectable label. The fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.[000238] The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.EXAMPLE 1[000239] Acne vulgaris is a skin disorder that affects millions worldwide, with Cutibacterium acnes (C. acnes) playing a key role in its inflammation. Antibiotics have long been used to reduce C. acnes and inflammation, but growing antibiotic resistance has limited their recommended use to three months. Yet, clinical needs often extend treatments, increasing resistant strains. Additionally, other common acne treatments with bactericidal activity like benzoyl peroxide can cause irritation, dryness, and peeling. This growing problem underscores a need for alternative therapies with rapid bactericidal action to both control C. acnes quickly with reduced side effects and lower the risk of resistance. Our strategy focused on isolating a predicted cationic antimicrobial peptide from the C-terminal domain of a phage lysin derived from Prevotella intermedia lysogen and engineering synthetic variants by adding positively charged amino acids to boost its efficacy. One of these engineered peptides, Pl 56, containing 35 amino acid, proved highly effective in eradicating all tested strains of C. acnes, as well as Staphylococcus aureus, another skin bacterium associated with acne. Pl 56 acted rapidly, reducing C. acnes counts by >5-logs in 10 minutes, further reducing the potential of resistance development. Additionally, Pl 56 maintained its potency when used in conditions resembling those encountered on the skin surface and in hair follicles (e.g., temperature, pH and salt concentration), as well as in combination with retinoid treatment — all without being toxic to human cells. These features position peptide Pl 56 as a promising topical drug for clinical applications.[000240] Acne vulgaris is a very common skin problem, affecting most adolescents in the United States, and in some cases, may persists into adulthood. Globally, acne vulgaris ranks eighth in overall diseaseprevalence (1). Acne is a chronic inflammatory disease of the skin pilosebaceous unit that produces sebum to lubricate both the skin and hair, and acts as a natural barrier against external environmental factors (1, 2). While the pathogenesis of acne is multifactorial, dysbiosis of the skin microbiome is an important factor, and the bacterium Cutibacterium acnes (C. acnes, formerly Propionibacterium acnes) plays a key role (3). Mechanistically, C. acnes contribute to the inflammatory process that is typical of acne vulgaris, and therefore, the elimination of C. acnes is part of current therapeutic protocols (1, 2, 4-8). In some cases, other skin microbiome members, such as staphylococci, including Staphylococcus aureus (S. aureus), can contribute to skin inflammation (3). Moreover, ”. aureus is one of several microbes that can contribute to the skin microbiome imbalances described in acne vulgaris (9).[000241] C. acnes is a lipophilic Gram-positive bacterium (10). While C. acnes grow ideally in anaerobic lipid rich conditions, it is also an aerotolerant bacterium that can detoxify oxygen and, therefore, can be sustained on the surface of the skin (11). Although considered a commensal, C. acnes involvement in various infections - e.g., bone and prosthesis, spinal disk, eyes after cataract surgery, central nervous system catheters, and others - led to its emergence as an opportunistic pathogen (10).[000242] Current protocols for the treatment of acne vulgaris include the use of antibiotics to eradicate C. acnes, thereby mitigating the typical inflammatory process in acne lesions (3, 7). These antibiotics are often combined with topical benzoyl peroxide or retinoids to further mitigate inflammation (7). Due to concerns about the development of antibiotic resistance, current acne treatment guidelines recommend limiting antibiotic use to a duration of up to 3 months. However, in clinical practice, the length of antibiotic treatment is often significantly longer, and indeed, the prevalence of C. acnes strains that are resistant to various antibiotics is increasing (12-15). Taken together, the need for treatment alternatives is evident.[000243] Lysins are phage-encoded enzymes used by the phage to degrade the bacterial cell wall and promote hypotonic lysis, thereby releasing progeny virions from the phage-infected cells. When delivered externally as a purified recombinant protein, lysins may be used as efficient antimicrobials to rapidly lyse the target bacteria (16). Native lysins that kill gram-positive (G+) pathogens degrade the peptidoglycan while lysins directed to gram-negative (G-) bacteria must initially disrupt the outer membrane to subsequently access and degrade the peptidoglycan. Because of this dual action, most native G- lysins structurally comprise a single globular peptidoglycan-degrading catalytic domain with a C -terminal cationic region responsible for destabilizing the outer membrane (17). We found that when the C-terminal cationic segment is isolated and delivered externally on its own as a peptide, it kills the G- bacteria bydestabilizing both the outer and cytoplasmic membranes (18, 19). Further, we demonstrated that the latter effect enables strong activity against G+ bacteria as well (19).[000244] Currently, there is a lack of effective lysins for therapeutic use against C. acnes that exhibit >3 log colony forming units (CFU) reduction. Furthermore, no lysin-derived peptide has been shown to be active against C. acnes. In this study, we modified a lysin-derived peptide from a Prevotella intermedia phage by strategically introducing cationic amino acids to improve its bactericidal activity against C. acnes.RESULTS[000245] Bactericidal activity of the modified lysin-derived peptides[000246] To identify a potential lysin or lysin-derived peptide with activity against C. acnes, a bioinformatic search was restricted to anaerobic G- bacteria with <50% homology to our reported G- lysin PlyF307 from Acinetobacter baumannii (20). The potential candidate lysin had also to comprise a putative C-terminal cationic region with comparable physicochemical characteristics to that of P307 (18) including length, charge, hydropathicity and predicted structure. Ultimately, a 141 aa Prevotella intermedia lysin, PlyPiOl, from a P. intermedia lysogen (GenBank: MBQ0073608.1) with a predicted single globular muramidase domain was identified, expressed and purified. The sequence of this 141 amino acid peptide denoted PlyPiOl is as follows:[000247] However, this whole lysin (PlyPiOl) exhibited low antibacterial activity overall against C. acnes (Figure 7). We then synthesized the C-terminal positively charged region of PlyPiOl (aa 102-132) termed PiPOl (31aa peptide, KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6). The predicted helix-loop-helix hairpin structure of PiPOl was similar to other lysin-derived peptides, such as P307 and PaPl-2 (Figure 1), but showed less resemblance to the human cationic antimicrobial peptide LL37 (18, 19, 21). Despite this, PiPOl exhibited low bactericidal activity overall against C. acnes (Figure 7). In as much as the antibacterial effect of the parent lysin PlyPiOl and the lysin derived parent peptide PiPOl, it was necessary and warranted to modify the parent peptide PiPOl to achieve bactericidal activity (>+ 3 log CFU reduction) with the more difficult C. acnes strains such as HSS F.[000248] The amino acid sequences of the P307, PaPl-2 and LL-37 peptides are provided below.[000249] P307 corresponds to amino acids 108-138 of lysin PlyF307 (PlyF307 146 aa is Genbank accession KJ740396) and has the following sequence:NAKDYKGAAAEFPKWNKAGGRVLAGLVKRRK (SEQ ID NO: ).[000250] The amino acid sequence for PaPl-2 is NAGDYAGAAEQFERWNKAGGKVLPGLVRRRA (SEQ ID NO:9). This sequence was derived from amino acids 103-133 of the PlyPaOl lysin (accession WP_058157505).[000251] The sequence of the LL-37 peptide (which is an active C-terminal peptide of CAP18 native polypeptide) is LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 10).[000252] Modifying the positive net charge of antimicrobial peptides has been previously shown to greatly enhance its antimicrobial activity (23). Therefore, to improve the bactericidal activity of PiPOl, five modified versions of the peptide (PH, Pi ll, P16, P156 and P157) were synthesized with the random addition of positively charged amino acids (lysine and arginine) to the N- and / or C-terminal ends (see Table 3). The bactericidal potency of each peptide was evaluated against several clinical isolates of C. acnes using a dose-response killing assay. As shown in Figure 2, when assayed at >5 ug / mL, each peptide was capable of more than a log reduction in CPU counts of all C. acnes strains except for the HSS F strain. Compared to the other peptides, P156 and P157 exhibited high bactericidal potency. P156 was capable of lowering the viability of all C. acnes strains below the limit of detection (LOD) at >5 jig / mL.[000253] The Pl 56 amino acid sequence shown in Table 1 (SEQ ID NO: 1) is based on lysin-derived peptide (PiPOl ) sequence. For the engineered peptide, the bold amino acids corresponds to modified amino acid / s; pl, theoretical isoelectric point; MW, molecular weight; Net Charge, at pH 7.4; GRAVY, Grand Average of Hydropathicity.TABLE 1. Engineered peptide P156 from P. intermedia lysin PlyPiOl with the best killing activity among those tested in this study.Peptide Amino Acid Sequence pl MW Net GRAVY (kDa) Charge P156 RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR 11.90 4.15 +11.1 -1.149(SEQ ID NO: 1). _....[000254] The bacterial strains used in this study are shown below in Table 2:TABLE 2. Bacterial strains used in this study,Species Strain Source NotesReferences C. acnes ATCC 6919 ATCC Type IA; Source: facial acne (35) C. acnes HSS B HSS Source: 2015 clinical isolate This study C. acnes HSS C HSS Source: 2015 clinical isolate This study C. acnes HSS D HSS Source: 2015 clinical isolate This study C. acnes HSS E HSS Source: 2015 clinical isolate This study C. acnes HSS F HSS Source: 2015 clinical isolate This studyC. acnes KPA 171202 RUBC Type IB; Source: skin (36) S. aureus 8325 RUBC MSSA; Source: blood (37) S. aureus NRS384 NARSA USAS 00 pulsotype, MRSA; Source: wound (38) S. aureus MW2 ATCC USA400 pulsotype, MRSA; Source: blood (39)BAA- 1707ATCC, American Type Culture Collection; HSS, Hospital for Special Surgery, New York, NY; MRSA, methicillin-resistant S'. aureus', MSSA, methicillin-sensitive & aureus NARSA, Network on Antimicrobial Resistance in S. aureus RUBC, The Rockefeller University Bacterial Collection[000255] S. aureus constitutes a major component of the skin microbiome and can contribute to the inflammation of acne vulgaris. It colonizes the skin surface under aerobic conditions but can expand into the hair follicle, where anaerobic conditions may predominate (24, 25). To evaluate the bactericidal activity of the peptide derivatives against S. aureus, we used standard methicillin-resistant 5. aureus (MRSA) strains USA300 and USA400 (Table 2), as well as the methicillin-sensitive S. aureus (MSSA) strain 8325 in a dose-response killing assay. Under aerobic conditions (Figure 3A-C), all five peptide derivatives at >50 pg / ml demonstrated a strong antimicrobial activity against the MRSA and MSSA S. aureus strains, with >4 log CFU reduction, dropping counts to or below the LOD (10 CFU / ml). However, P156 and Pl 57 could also reduce CFU counts for all the S'. aureus strains below the LOD at. concentrations as low as 5 pg / ml.[000256] Taken together, the results from Figures 2 and 3 demonstrate that Pi 56 at 5 jxg / ml was bactericidal (>3 log kill) against all C. aeries and S. aureus strains tested. Additionally, at 25 pg / ml, it was capable of lowering the viability of all the bacterial strains below the LOD (>4 log CFU reduction). The sequence and properties of the selected best peptide P 156 are shown in Table 1.[000257] Since S. aureus growth may also occur within the hair follicle, we next aimed to study whether PI 56, at a concentration of 25 pg / ml, can also exhibit activity against S’, aureus grown under anaerobic conditions. As shown in Figure 3D, like the results observed under aerobic growth conditions, Pl 56 tested against the common MRSA USA300 strain under anaerobic conditions also achieved >4-log CFU reduction at 25 ug / ml, reducing counts to below the LOD (<10 CFU).[000258] In addition, Pl 56 was tested for activity against Staphylococcus epidermidis in a bacterial killing assay and was found to be very effective in killing 5. epidermidis (strains 5. epidermidis ATCC 1228 and ATCC 35984 were evaluated and killed to CFUs below detection (Figure 8).[000259] Bactericidal activity of P156 under conditions relevant to skin properties and C. acnes growth.[000260] An effective bactericidal agent for treating acne vulgaris should rapidly eliminate C. acnes upon skin application. Therefore, we evaluated the time kill kinetics of Pl 56 against C. acnes. As shown in Figure 4A, P156 demonstrated rapid action, achieving >3 log reduction in bacterial CFU within 1 minute and >5 log reduction in 10 minutes at 25 μg / mL[000261] Additionally, the bactericidal activity of Pl 56 was evaluated under conditions relevant to skin properties and optimal C. acnes growth. The human skin surface temperature typically ranges from 32°C to 37°C, close to the optimal growth temperature for C. acnes of 30°C to 37°C (26). To simulate these conditions, P156 was thawed from -80°C, stored for four weeks at 4°C, and then its bactericidal activity was assessed at various temperatures. Figure 4B shows that the peptide (at 25 μg / ml) maintained potent bactericidal activity across a broad range of temperatures (20°C to 40°C), reducing C. acnes CFU counts below the LOD (>5-logs). This finding also suggests that the peptide remains stable and active even after four- week storage at 4°C.[000262] The optimal pH for C. acnes growth ranges from 6.0 to 7.0, which is within the pH range found in hair follicles (6.3 to 6.6) (27, 28). However, the normal skin surface itself also tends to be slightly more acidic (28). P156, at 25 μg / mL, exhibited significant bactericidal activity across a broad pH range (5.5 to 8.0), achieving >4 log CFU reduction (Figure 4C).[000263] Next, NaCl concentration was considered in relation to conditions of sweat on the skin surface. While NaCl levels on skin in the absence of sweat are very low (<1 mM), concentrations within sweat glands can on average reach up to 50 mM (25). Although NaCl levels on sweat gland-adjacent skin or within hair follicles under sweat conditions are less known, considering the diffusion of sweat from sweat glands to these adjacent areas, they are estimated to range between 10 to 50 mM. Figure 4D illustrates that P156, at 25 μg / mL, achieved a reduction of >3 log CFU counts at ≤50 mM NaCl, and a reduction to below the detection limit (>4 log CFU) at concentration under 25 mM NaCl.[000264] Cytotoxicity[000265] To assess the cytotoxicity of Pl 56 on human cells, hemolysis of human red blood cells (hRBCs) was evaluated after incubation with a wide concentration range of P156 (0.5–256 μg / mL) in phosphate buffered saline (PBS). The human cationic AMP LL-37 was used as a positive control due to its known potential for inducing hRBCs toxicity' (hemolysis) (29). As seen in Figure 5, LL-37 exhibited a dosedependent increase in optical density at 405 nm (OD405), indicating progressive hemolysis. In contrast, P156 showed no RBC toxicity and maintained a consistent OD value across all peptide concentrations tested, comparable to the PBS negative control.[000266] Potential combination therapy with current common acne vulgaris treatments [000267] In addition to antibiotics to eradicate C. acnes, accepted treatment protocols for acne vulgaris include retinoids. The guidelines of the American Academy of Dermatology for the treatment of acne vulgaris indicate that retinoids are the core of topical therapy for acne due to their comedolytic and anti¬ inflammatory activity (30). Since retinoids are often used alongside antibiotics, the bactericidal activity of P156 was tested in combination with a retinoid to rule out any potential antagonism. Treating 5. aureus for 1-hour with retinoic acid alone, at concentrations typically used for acne vulgaris treatment (0.01-0.1%), exhibited no antibacterial effect (Figure 6). Conversely, in the presence of 0.01-0.1% retinoic acid, P156 (at 25 μg / mL) lowered S. aureus viability below the LOD (>4 log CFU reduction). This indicates that the bactericidal potency of P156 remains unaffected when used in combination with retinoic acid.MATERIALS AND METHODS[000268] Bacterial Strains and Culture Conditions[000269] Bacterial strains used in this study are outlined in Table 2 and were stored at -80°C. C. acnes strains were grown in a BACTRON900 anaerobic chamber (Sheldon Manufacturing Inc.) at 37°C to exponential phase in reduced Trypticase Soy Broth (rTSB, BD Biosciences) supplemented with 2% (vol / vol) glycerol. S. aureus strains were grown either aerobically or anaerobically at 37°C to exponential phase in TSB (aerobic growth) or rTSB (anaerobic growth).[000270] Peptide Synthesis and Properties[000271] The various engineered peptides were synthesized by Biomatik corporation. The table below (Table 3) shows the peptides that were synthesized for evaluation. Lysine (K) and / or arginine (R) amino acids added to the N or C terminal ends are shown in bold.TABLE 3" Pll. KAKAPRAE1YAQFNK WVYAGGKKLSG[JVKRRK (SEQ'7D NO: 2). ' Pili ' KAKAPI^P16 K AKAPRAEIYAQFNK W V Y AGGKKLSG1. VKRRRR (SFQ ID NO: 4)' O AQFNKWVYAGGKKLSGLV^R^(SEQ ID''N6: Tj.P157 RKAKAPR^..[000272] The properties of peptide P156 (shown in Table 1) were predicted with ProtParam-Expasy. Net charge at pH 7.4 was calculated using the Prot pi-Protein tool.[000273] One Hour Killing Assays[000274] Unless stated otherwise, the standard conditions for killing assays involved statically treating exponential phase bacteria at 5x105CFU / mL with a lysin-derived peptide at 25 μg / mL in a 96-well microtiter plate under aerobic (S. aureus) or anaerobic conditions (C. acnes and S. aureus) in 20 mM Tris (Fisher Scientific), pH 7.2, for 1 h at 37°C. For dose-response killing assays, peptide concentrations ranging from 1 up to 125 μg / mL were tested.[000275] To evaluate the effect salt has on the antibacterial activity of the peptide, C. acnes strain ATCC 6919 were treated with P156 in buffer supplemented with 0 to 500 mM NaCI (Fisher Scientific). The bactericidal potency of Pl 56 in broad pH range was assayed by treating C. acnes strain ATCC 6919 with the peptide in either 20 mM 2-(N-morpholino) ethanesulfonic acid (MES, pH 5.5 to 6.5; Fisher Scientific), sodium phosphate buffer (NaPO4, pH 6.5 to 7.0; both NaH2PO4and Na2HPO4components are from Fisher Scientific), or Tris (pH 7.0 to 8.0).[000276] Temperature-based killing assays were performed by initially thawing a frozen aliquot of P 156, which was then stored at 4°C for a month. Next, C. acnes strain ATCC 6919 was treated with the above- mentioned Pl 56 aliquot in buffer at 15°C to 40°C. For these experiments, separate aliquots of both the bacteria at 106CFU / mL and peptide at 50 μg / mL were initially equilibrated to the temperature being assayed for 5 min in 1.7 mL microcentrifuge tubes using an EchoTherm chilling / heating plate (Torrey Pines Scientific). The bacteria and peptide were then mixed together 1:1 in a microcentrifuge tube, yielding respective final concentrations of 5x105CFU / mL and 25 μg / mL, and subsequently incubated in the chilling / heating plate for 1 h.[000277] For effect of retinoid on peptide bactericidal activity, all-trans-retinoic acid (Sigma-Aldrich) at 0.01-0.1% (wt / vol) with or without P156 at 25 μg / mL.[000278] Each killing assay comprised an untreated control (bacteria absent peptide) for every condition tested. After 1 h, 100 pL directly from the sample, as well as 5 pL of each dilution from a 10-fold serial dilution, were plated on either TSB agar (for 5. aureus killing assays using aerobic conditions), rTSB agar (for S. aureus killing assays using anaerobic conditions), or rTSB agar with 2% glycerol (for C. acnes killing assays) in order to quantitate bacterial viability'. The limit of detection (LOD) for each experiment was 10 CFU / mL, with one exception. For the dose-response killing assay evaluating the collection of peptides against C. acnes, only the 10-fold serial dilutions from each sample were plated, resulting in a LOD of 200 CFU / mL. Experiments performed under anaerobic conditions used agar plates that were reduced overnight. Additionally, buffers for these particular assays were made fresh the day of theexperiment and reduced for 2 h prior to use. Error bars correspond to ± the standard error of the mean (SEM) of two or three biological replicates.[000279] Time-Kill Assay[000280] Using an EchoTherm chilling / heating plate, separate aliquots of both C. acnes strain ATCC 6919 at 106CFU / mL and P156 at 50 pg / mL in 20 mM Tris, pH 7.2, were equilibrated to 37°C for 10 min in 1.7 mL microcentrifuge tubes. The bacteria and peptide were then combined 1:1 in a microcentrifuge tube to obtain final concentrations of 5x105CFU / mL and 25 μg / mL, respectively. An untreated control (bacteria without peptide) was also included. The samples were incubated for a total of 30 min at 37°C. At 1, 5, 10, 15, 20, 25 and 30 min, an aliquot was removed. Both 100 pL directly from the sample, as well as 5 pL of each dilution from a 10-fold serial dilution, were plated on rTSB agar supplemented with 2% glycerol in order to assess bacterial viability. The LOD was 10 CFU / mL. Error bars correspond to ± SEM of three biological replicates.[000281][000282] Healthy donor blood was collected in an EDTA-containing conical tube at The Rockefeller University Hospital. This study was approved by our Institutional Review Board, and all adult subjects provided written informed consent. Human red blood cells (hRBCs) were harvested by low-speed centrifugation, washed three times, and resuspended in PBS, pH 7.4, to a 10% (vol / vol) concentration. Using a 96-well untreated microtiter plate, the hRBC solution was diluted 1:1 with the peptides at final concentrations from 0.5 to 256 μg / ml. hRBCs incubated in PBS with or without 0.1% (vol / vol) Triton X-100 represented positive and negative controls for hemolysis, respectively. Following a 4-h incubation at 37°C with 5% CO2, intact hRBCs were pelleted by low speed and the resulting supernatant was transferred to a new microtiter plate. Using a SpectraMax M5 microplate reader (Molecular Devices), the OD405nm of each supernatant was measured to quantitate the relative concentration of hemoglobin released.DISCUSSION[000283] Bactericidal activity of modified lysin-derived peptides as an antibiotic alternative [000284] Current therapeutic protocols for acne vulgaris aim to mitigate inflammation by eradicating the bacterial stimulus. However, prolonged antibiotic use has shown the development of resistance. In this paper we evaluated in vitro the future potential clinical use of a lysin-derived peptide as a possible antibiotic alternative to eradicate and control C. acnes on the skin surface, a major contributor to the pathogenesis of acne.[000285] Both the purified whole lysin from a lysogen found in P. intermedia and its native C-terminal cationic peptide did not have significant bactericidal effects against C. acnes. Thus, to improve the activity of the native cationic peptide, the cationic nature of the peptide was increased (23). By adding the positively charged amino acids (arginine) to the synthesized peptide, the resulting five modified peptides displayed improved bactericidal properties against C. acnes. For each modified peptide, bactericidal potency was dose- and, except for P156, strain-dependent (Figure 2). P156 demonstrated a strong bactericidal activity (>3 log kill) against all seven of the C. acnes strains as well as S. aureus at concentrations as low as 5 μg / mL and >5 log kill at 25μg / ml (Figures 2 and 3). As a result, this specific peptide was tested to evaluate its ability to kill C. acnes under conditions (pH, temperature and NaCl) found in the normal human skin.[000286] Lack of effective animal model and alternative approach to predict clinical efficacy [000287] To better predict its success in future clinical trials, the peptide, such as Pl 56, etc. should ideally be evaluated in an animal model. However, the absence of effective animal models for acne vulgaris presents a significant challenge in dermatological research (31, 32). This is primarily due to key biological differences (e.g., structure of sebaceous glands and hair follicle, and variation in the microbiome) between humans and animals, particularly mouse models, which fail to accurately mimic the condition of acne vulgaris. Due to these limitations and considering that treatment would occur on the skin surface rather than systemically, we instead evaluated the bactericidal activity of peptide (Pl 56) in vitro under conditions that closely simulate human skin and hair follicles. The experiments were carried out under optimal conditions for C. acnes growth, aiming to better approximate the real environment for therapeutic intervention.[000288] A major clinical challenge with current antibiotic treatments for acne vulgaris is the development of bacterial resistance. A rapid-acting bactericidal agent, such as Pl 56 and the other engineered peptides disclosed herein., can reduce the likelihood of resistance by quickly eliminating the bacteria before they are able to adapt to the drug. Rapid action also helps resolve inflammation quickly and prevent the formation or worsening of acne lesions. Pl 56 appears to kill C. acnes on contact, killing >3 logs within one minute and >5 logs in ten minutes, making it useful in a clinical setting (Figure 4A).[000289] Additionally, Pl 56, as exemplary of the engineered peptides disclosed herein, maintained bactericidal efficacy within the optimal temperature range for C. acnes growth (30°C to 37°C) and across a broad range of possible facial skin temperatures, reducing bacterial counts >4 logs at each temperature assayed (Figure 4B). Its stability, shown by its activity after thawing from -80°C to 4°C and 4-week storage at this temperature, suggests a long shelf life and high efficacy under practical conditions.[000290] The activity of Pl 56 was also quantitated against C. acnes at physiological pH and salt. The optimal pH range for C. acnes growth (pH 6-7) is similar to the pH found within hair follicles, while the normal skin surface tends to be more acidic. This suggests that C. acnes growth on the skin surface might be slower in more acidic, aerobic conditions. P156 exhibited a robust bactericidal effect, achieving a >4 log CFU reduction across a broad pH range, with complete bacterial eradication within the optimal pH range for C. acnes growth (pH 6-7). However, in patients with acne vulgaris, skin pH is often higher (up to 7.6 in (33)). Despite this, Pl 56 was able to eradicate C. acnes at these elevated pH levels. In addition to pH, the effect of salt on the antibacterial activity of Pl 56 was measured. The salt concentration in the skin and hair follicles is affected by sweat. Within NaCl levels typical for the skin — ranging from less than 1 mM in the absence of sweat to up to 50 mM with sweat — the peptide effectively reduced C. acnes populations by 3 logs in 50 mM NaCl. Moreover, the killing efficiency of Pl 56, improved significantly (>4 logs) at lower salt concentrations (Figure 4D).[000291] Cytotoxicity’[000292] The mechanism of action of cationic antimicrobial peptides (AMPs) has been previously described and includes destabilization of bacterial cell membranes (31). Since the cytoplasmic membrane of G+ bacteria, such as C. acnes and S. aureus, share similarities with human cell membranes (i.e., phospholipid bilayer), and other AMPs (e.g., LL-37 in Fig. 5 and (29), or colistin in (34)) have demonstrated cytotoxicity to human cells, it was crucial to rule out potential human cell toxicity associated with Pl 56. Results from a hemolytic assay revealed that, similar to the PBS negative control, Pl 56 does not disrupt the membrane of hRBCs (Figure 5). Like other lysin-derived peptides (18-19), the lack of cytotoxicity associated with Pl 56 indicates the peptide exhibits selective activity towards prokaryotic membranes.[000293] Combination with current standard treatment of acne vulgaris[000294] Drug combinations are often essential in the treatment of acne vulgaris due to the multifactorial nature of the condition. Acne results from a combination of factors like excess sebum production, bacterial colonization (particularly C. acnes), inflammation and abnormal keratinization of skin cells. Using multiple drugs targeting different aspects of acne can increase treatment efficacy and reduce the risk of resistance or side effects. The current therapeutic protocols include combination of antibiotic with at least one antiinflammatory component, most commonly retinoid. In this regard, P156 maintained its activity when combined with retinoids (Figure 6).[000295J In summary, a C-terminal cationic peptide derived from the P. intermedia lysin PlyPiOl, with a low antibacterial activity towards C. acnes, was engineered with several cationic modifications. Theresulting panel of modified peptides had varying antibacterial efficacy against C. acnes. One peptide, P156, uniquely demonstrated strong, rapid bactericidal activity against all tested C. acnes and S. aureus strains. Importantly, P156 remained effective under conditions relevant to acne-affected skin, including variations in temperature, salt, pH and in combination with retinoids. These properties support P156 as a potential drug for clinical use against C. acnes in context of acne vulgaris.REFERENCES1. Zaenglein AL. 2018. Acne Vulgaris. N Engl J Med 379:1343-1352.2. Williams HC, Dellavalie RP, Gamer S. 2012. Acne vulgaris. Lancet 379:361-72.3. Dessinioti C, Katsambas A. 2024. The Microbiome and Acne: Perspectives for Treatment. Dermatol Ther (Heidelb) 14:31-44.4. Moradi Tuchayi S, Makrantonaki E, Ganceviciene R, Dessinioti C, Feldman SR, Zouboulis CC. 2015. Acne vulgaris. Nat Rev Dis Primers 1:15029.5. Tanghetti EA. 2013. The role of inflammation in the pathology of acne. J Clin Aesthet Dermatol 6:27-35.6. Huang L, Yang S, Yu X, Fang F, Zhu L, Wang L, Zhang X, Yang C, Qian Q, Zhu T. 2024. Association of different cell types and inflammation in early acne vulgaris. Front Immunol 15:1275269.7. Hauk L. 2017. Acne Vulgaris: Treatment Guidelines from the AAD. Am Fam Physician 95:740-741.8. Reynolds RV, Yeung II, Cheng CE, Cook-Bolden F, Desai SR, Druby KM, Freeman EE, Keri JE, Stein Gold LF, Tan JKL, Tollefson MM, Weiss JS, Wu PA, Zaenglein AL, Han JM, Barbieri JS. 2024. Guidelines of care for the management of acne vulgaris. J Am Acad Dermatol 90: 1006 el-1006 e30. 9. Totte JE, van der Feltz WT, Bode LG, van Belkum A, van Zuuren EJ, Pasmans SG. 2016. A systematic review and meta-analysis on Staphylococcus aureus carriage in psoriasis, acne and rosacea. Eur J Clin Microbiol Infect Dis 35:1069-77.10. Mayslich C, Grange PA, Dupin N. 2021. Cutibacterium acnes as an Opportunistic Pathogen; An Update of Its Virulence- Associated Factors. Microorganisms 9.11. Gajdacs M, Spengler G, Urban E. 2017. Identification and Antimicrobial Susceptibility Testing of Anaerobic Bacteria: Rubik's Cube of Clinical Microbiology? Antibiotics (Basel) 6.12. Coates P, Vyakmam S, Eady EA, Jones CE, Cove JH, Cunliffe WJ. 2002. Prevalence of antibiotic-resistant propionibacteria on the skin of acne patients: 10-year surveillance data and snapshot distribution study. Br J Dermatol 146:840-8.13. Dessinioti C, Katsambas A. 2017. Propionibacterium acnes and antimicrobial resistance in acne. Clin Dermatol 35:163-167.14. Dessinioti C, Katsambas A. 2022. Antibiotics and Antimicrobial Resistance in Acne: Epidemiological Trends and Clinical Practice Considerations. 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Novel phage lysin capable of killing the multidrug-resistant gram-negative bacterium Acinetobacter baumannii in a mouse bacteremia model. Antimicrob Agents Chemother 59: 1983-91.21. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Zidek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D. 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596:583-589. 22. Ibrahim HR, Thomas U, Pellegrini A. 2001. A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action. J Biol Chem 276:43767-74.23. Islam MM, Asif F, Zaman SU, Arnab MK. H, Rahman MM, Hasan M. 2023. Effect of charge on the antimicrobial activity of alpha-helical amphibian antimicrobial peptide. Curr Res Microb Sci 4:100182.24. Piewngam P, Otto M. 2024. Staphylococcus aureus colonisation and strategies for decolonisation. Lancet Microbe 5:e606-e618.25. Nakamura K, Williams MR, Kwiecinski JM, Horswill AR, Gallo RL. 2021. Staphylococcus aureus Enters Hair Follicles Using Triacylglycerol Lipases Preserved through the Genus Staphylococcus. J Invest Dermatol 141:2094-2097.26. Schlecht S, Freudenberg MA, Galanos C. 1997. Culture and biological activity of Propionibacterium acnes. Infection 25:247-9.27. Korting HC, Lukacs A, Vogt N, Urban J, Ehret W, Ruckdeschel G. 1992. Influence of the pH-value on the growth of Staphylococcus epidermidis, Staphylococcus aureus and Propionibacterium acnes in continuous culture. Zentralbl Hyg Umweltmed 193:78-90.28. Kaden D, Dahne L, Knorr F, Richter H, Lademann J, Meinke MC, Patzelt A, Darvin ME, Jung S. 2020. Determination of the pH Gradient in Hair Follicles of Human Volunteers Using pH-Sensitive Melamine Formaldehyde-Pyranine Nile Blue Microparticles. Sensors (Basel) 20.29. Ciomei CD, Tapper H, Bjartell A, Stemby NH, Bodelsson M. 2006. Human antimicrobial peptide LL-37 is present in atherosclerotic plaques and induces death of vascular smooth muscle cells: a laboratory study. BMC Cardiovasc Disord 6:49.30. Zaenglein AL, Pathy AL, Schlosser BJ, Alikhan A, Baldwin HE, Berson DS, Bowe WP, Graber EM, Harper JC, Kang S, Keri JE, Leyden JJ, Reynolds RV, Silverberg NB, Stein Gold LF, Tollefson MM, Weiss JS, Dolan NC, Sagan AA, Stern M, Boyer KM, Bhushan R. 2016. Guidelines of care for the management of acne vulgaris. J Am Acad Dermatol 74:945-73 e33.31. McLaughlin J, Waterson S, Layton AM, Bjourson AJ, Barnard E, McDowell A. 2019. Propionibacterium acnes and Acne Vulgaris: New Insights from the Integration of Population Genetic, Multi-Omic, Biochemical and Host-Microbe Studies. Microorganisms 7.32. Mirshahpanah P, Maibach HI. 2007. Models in acnegenesis. Cutan Ocul Toxicol 26: 195-202.33. Prakash C, Bhargava P, Tiwari S, Majumdar B, Bhargava RK. 2017. Skin Surface pH in Acne Vulgaris: Insights from an Observational Study and Review of the Literature. J Clin Aesthet Dermatol 10:33-39.34. Lim LM, Ly N, Anderson D, Yang JC, Macander L, Jarkowski A, Forrest A, Bulitta JB, Tsuji BT. 2010. Resurgence of Colistin: A Review of Resistance, Toxicity, Pharmacodynamics, and Dosing. Pharmacotherapy 30:1279-1291.35. Douglas HC, Gunter SE. 1946. The taxonomic position of Corynebacterium acnes. J Bacteriol 52:15-23.36. Bruggemann H, Henne A, Hoster F, Liesegang H, Wiezer A, Strittmatter A, Hujer S, Durre P, Gottschalk G. 2004. The complete genome sequence of Propionibacterium acnes, a commensal of human skin. Science 305:671-3.37. Novick R. 1967. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology 33:155-66.38. McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC. 2003. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol 41:5113-20.39. Voyich JM, Otto M, Mathema B, Braughton KR, Whitney AR, Welty D, Long RD, Dorward DW, Gardner DJ, Lina G, Kreiswirth BN, DeLeo FR. 2006. Is Panton-Valentine leukocidin the major virulence determinant in community-associated methicillin-resistant Staphylococcus aureus disease? J Infect Dis 194:1761-70.Example 2[000296] As shown in Example 1, peptide Pl 57 (SEQ ID NO:5) exhibited high bactericidal activity. Additional studies were undertaken to further evaluate the Pl 57 peptide.[000297] Particulars of peptide Pl 57 are provided below in Table 4.[000298] Table 4. Engineered peptide Pl 57 from P. intermedia lysin PlyPiOl>Peptide Amino Acid Sequence _ pl MW (kPa) Net Charge GRAVY P157 RKAKAPRAEIYAQFNKVWYAGGKKLSGLVKRRR 11.52 3.85 +10 -0.945 Amino acid sequence, native lysin-derived peptide (PiPOJ) sequence (no underline) and modified amino acids (underlined); pI, theoretical isoelectric point; MW, molecular weight; Net Charge, at pH 7.4; GRAVY, Grand Average of Hydropathicity[000299] Bactericidal properties of Pl 57 were assessed under various relevant skin conditions. Killing kinetics was assessed in a time course evaluation (Fig. 9A). P157 (25 μg / mL) exhibited potent bactericidal activity against C. acnes, reducing bacterial viability by ~5 logs within 1 min. The rapid killing kinetics of the peptide, combined with its proposed antibacterial mechanism of action (membrane destabilization), are indicative of an antibacterial agent that kills on contact. These results suggest that, as a potential therapeutic for acne vulgaris, P157 could effectively eliminate accessible C. acnes upon application to the skin.[000300] Peptide Pl 57 was evaluated over different temperatures and it was demonstrated (Figure 9B) that Pl 57 (25 pg / mL) retained high bactericidal activity against C. acnes over a broad temperature range. At 15-40°C, the peptide reduced bacterial counts by ~5 logs. Pi 57 displays potent bactericidal activity across temperatures found on human skin (32-37°C) and those optimal for C. acnes growth (30-37°C).[000301] Peptide P157 activity was evaluated at different pH values (Figure 9C) and Pl 57 displayed strong bactericidal activity against C. acnes over a broad pH range. At pH 6.0-8.0, the peptide (25 pg / mL) reduced bacterial counts by >4 logs. Activity was modestly reduced at pH 5.5, with P157 decreasing viability by 2.3 logs. Overall, the peptide rapidly kills C. acnes in growth-optimal pH conditions (pH 6.0- 7.0) and remains highly active within the pH range of hair follicles (pH 6.3-6.6), as well as the slightly more acidic skin surface.[000302] Peptide Pl 57 activity was evaluated at different salt concentrations (Figure 9D) and Pl 57 (25 pg / mL) retained high bactericidal activity against C. acnes at NaCl concentrations up to 50 mM (~5-log CFU reduction), whereas killing decreased to 1-2 logs at 75-100 mM NaCl. The collective data suggest Pl 57 effectively eradicates C. acnes at salt concentrations found on the skin, both in the absence (<1 mM) and presence of sweat (10-50 mM).[000303] Given the peptide’s significant C. acnes killing, Pl 57 was evaluated against 5. epidermidis which often colonizes the skin, including in combination with C. acnes. As shown in Figure 10, P157 (25 μg / mL) exhibited potent bactericidal activity against both S. epidermidis strains tested, decreasing viability by >5 logs. The ability of the peptide to rapidly eradicate S. epidermidis is advantageous given its role in acne vulgaris. S. epidermidis can promote C. acnes biofilm formation and, during skin microbiome dysbiosis, can form biofilms that exacerbate inflammation and worsen acne lesions.[000304] Next, P 157 was assessed to evaluate any cytotoxicity toward eukaryotic cells. Human red blood cells (RBCs) were incubated with Pl 57 in comparison to the human antimicrobial peptide LL37 as a control, as well as PBS negative control, and detergent to lyse the cells as a positive control (Triton X- 100). Lysis was quantified via the relative concentration of hemoglobin released into the supernatant upon incubation. The results are depicted in Figure 11. Unlike LL37, P157 lacked cytotoxicity towards hRBCs at all concentrations tested (0.5-256 pg / mL). The absence of hemolytic activity indicates selective activity towards prokaryotic membranes and supports the potential suitability of P157 as a topical therapeutic for acne.[000305] Stability of the P157 peptide was assessed after freeze thaw and upon storage. P157 retained activity after a freeze thaw cycle from -80 °C to 4°C and 4-weeks storage at 4°C (data not shown).[000306] Methods[000307] Bacterial Strains and Culture Conditions[000308] C. acnes strain ATCC 6919 were grown in a BACTRON900 anaerobic chamber (Sheldon Manufacturing Inc.) at 37°C to exponential phase in reduced Trypticase Soy Broth (BD Biosciences) supplemented with 2% (vol / vol) glycerol (Fisher Scientific). S. epidermidis strains were grown aerobically at 37°C to exponential phase in Trypticase Soy Broth.[000309] Peptide Synthesis and Properties[000310] Pl 57 was synthesized by Biomatik Corporation. Physicochemical properties were predicted with ProtParam-Expasy (web.expasy.org / protparam). Net charge at pH 7.4 was calculated using the Prot pi-Protein tool, version 2.2.29.152.[000311] 30 min Time-Kill Assay[000312] Using an EchoTherm chilling / heating plate (Torrey Pines Scientific), separate aliquots of exponential phase Cutibacterium acnes strain ATCC 6919 at 106CFU / mL and Pl 57 at 50 pg / mL in 20 mM Tris (Fisher Scientific), pH 7.2, were equilibrated at 37°C for 10 min in 1.7 mL microcentrifuge tubes. The bacteria and peptide were then combined 1:1 in a microcentrifuge tube to obtain final concentrations of 5x105CFU / mL and 25 pg / rnL, respectively. An untreated control (bacteria without peptide) was also included. The samples were incubated for a total of 30 min at 37°C under anaerobic conditions. At various time points, an aliquot was removed, serial diluted and plated on reduced Trypticase Soy Agar (BD Biosciences) supplemented with 2% (vol / vol) glycerol to assess bacterial viability. The limit of detection was 10 CFU / mL. Error bars correspond to the ±standard error of the mean of three biological replicates.[000313] One-Hour Killing Assays[000314] The temperature-based killing assays were preformed using a EchoTherm chilling / heating plate. Separate aliquots of exponential phase Cutibacterium acnes strain ATCC 6919 at 106CFU / mL and Pl 57 at 50 pg / mL in 20 mM Tris (Fisher Scientific), pH 7.2, were equilibrated at temperatures ranging from 15-40°C for 10 min in 1.7 mL microcentrifuge tubes. The bacteria and peptide were then combined 1:1 in a microcentrifuge tube to obtain final concentrations of 5xl05CFU / mL and 25 pg / mL, respectively, and subsequently incubated for 1 h. An untreated control (bacteria without peptide) was also included for each temperature assayed.[000315] For the pH-based killing assays, exponential phase Cutibacterium acnes strain ATCC 691 at 5xl05CFU / mL were treated statically under anaerobic conditions in a 96-well microtiter plate with Pl 57 at 25 pg / mL in either 20 mM MES (pH 5.5-6.5), sodium phosphate (pH 6.5-7.0) or Tris (pH 7.0-8.0), for 1h at 37°C (all buffer chemicals were from Fisher Scientific). An untreated control (bacteria without peptide) was also included for each pH assayed.[000316] Using a 96-well microtiter plate, NaCl-based killing assays consisted of statically treating exponential phase Cutibacterium acnes strain ATCC 6919 at 5xl05CFU / mL under anaerobic conditions with Pl 57 at 25 pg / mL in 20 mM Tris, pH 7.2, supplemented with 0-300 mM NaCl (Fisher Scientific) for 1 h at 37°C. An untreated control (bacteria without peptide) was also included for each salt concentration assayed.[000317] For the S. epidermidis killing assays, exponential phase S. epidermidis strains ATCC 12228 and ATCC 35984 at 106CFU / mL were incubated in individual wells of a 96-well microtiter plate with Pl 57 at 25 pg / rnL in 20 mM Tris, pH 7.2, for 1 h at 37°C. An untreated control (bacteria without peptide) was included for each strain.[000318] At the culmination of each killing assay, an aliquot from each sample was removed, serial diluted and plated on either reduced Trypticase Soy Agar supplemented with 2% glycerol (C. acnes) or Trypticase Soy Agar (S. epidermidis) to enumerate bacterial viability. The limit of detection was 10 CFU / mL. Error bars correspond to the ±standard error of the mean of three biological replicates.[000319] Cytotoxicity Assays[000320] Healthy donor blood was collected in an EDTA-containing conical tube at The Rockefeller University Hospital. This study was approved by our Institutional Review Board, and all adult subjects provided written consent. Human red blood cells (hRBCs) were harvested by low-speed centrifugation, washed three times and resuspended in phosphate buffered saline (PBS; Fisher Scientific), pH 7.4, to a 10% (vol / vol) concentration. Using a 96-well untreated microtiter plate, the hRBC solution was diluted 1: 1 with either P157 or LL37 at final concentrations ranging from 0.5-256 pg / mL. hRBCs incubated in PBS with or without 0.1% (vol / vol) Triton X-100 (Fisher Scientific) represented positive and negative controls for hemolysis, respectively. Following a 4 h incubation at 37°C with 5% CO2, intact hRBCs were pelleted at low speed, and the resulting supernatant was transferred to a new' microtiter plate. Using a SpectraMax M5 microplate reader (Molecular Devices), the OD405nm of each supernatant was measured to quantify the relative concentration of hemoglobin released.[000321][000322] In summary, the peptide Pl 57. demonstrated strong, rapid bactericidal activity against C. acnes and S. aureus strains and remained effective under conditions relevant to acne-affected skin, including variations in temperature, salt, pH, storage. These properties support Pl 57 as a potential drug for clinicaluse against C. acnes in the context of acne vulgaris and other skin disorders and conditions associated with bacterial colonization.[000323] This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof The present disclosure is therefore to be considered as in all aspects illustrate and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.[000324] Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

Claims

WHAT IS CLAIMED IS:

1. An engineered lysin-derived peptide comprising the sequence KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRR (SEQ ID NO:6), or an amino acid sequence having at least 90% identity to SEQ ID NO:6 and having one or more cationic amino acids added at the N-terminus and / or C-terminus, wherein the peptide is effective to kill bacteria.

2. The engineered peptide of claim 1, wherein one or more lysine (K) or arginine (R) amino acid is added at the N-terminus and / or C-terminus.

3. The engineered peptide of claim 1, selected from KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRK (SEQ ID NO:2), KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRKK (SEQ ID NO:3), KAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:4), RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:1), and RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO: 5).

4. The engineered peptide of claim 3, wherein the peptide has the sequence of RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO:1), or a variant of SEQ ID NO: 1 having at least 90% identity to SEQ ID NO: 1, wherein the peptide is effective to kill C. acnes bacteria.

5. The engineered peptide of claim 3, wherein the peptide has the sequence of RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO:5), or a variant of SEQ ID NO:5 having at least 90% identity to SEQ ID NO:5, wherein the peptide is effective to kill C, acnes bacteria.

6. The engineered peptide of claim 3, wherein the peptide consists of RRKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRRR (SEQ ID NO: 1).

7. The engineered peptide of claim 3, wherein the peptide consists of RKAKAPRAEIYAQFNKWVYAGGKKLSGLVKRRR (SEQ ID NO: 5).

8. The engineered peptide of any one of claims 1-7, wherein the peptide is capable of killing gram¬ positive and gram-negative bacteria.

9. The engineered peptide of any one of claims 1-8, wherein the peptide is capable of killing pathogenic and antibiotic-resistant bacteria.

10. The engineered peptide of any one of claims 1-7, wherein the peptide is capable of killing Cutibacterium and Staphylococcus bacterial strains, particularly C. acnes and 5. aureus.

11. A composition comprising the engineered peptide of any one of claims 1-10.

12. The composition of claim 11. which is an antimicrobial composition further comprising a pharmaceutically acceptable carrier.

13. The composition of any one of claims 11 or 12, further comprising one or more antibiotic.

14. The composition of any one of claims 11 or 12, further comprising one or more of retinoid, benzoyl peroxide, or salicylic acid.

15. The composition of any one of claims 11-14 suitable for topical administration and effective to kill one or more type of bacteria.

16. A pharmaceutical composition for killing gram-positive or gram-negative bacteria comprising the engineered peptide of any of claims 1-7 in an amount effective to kill the gram-positive or gram-negative bacteria.

17. A nucleic acid capable of encoding the engineered peptide of any of claims 1-7.

18. The nucleic acid of claim 17, wherein said nucleic acid is operatively linked to an expression control sequence.

19. A unicellular host transformed with the nucleic acid of claim 17 or 18.

20. A method of killing bacteria or reducing a population of bacteria comprising the step of contacting the bacteria with a composition comprising an amount of an engineered peptide of any of claims 1-7, or with the composition of any of claims 11-14, effective to kill the bacteria.

21. The method of claim 20 wherein the bacteria is gram negative or gram positive.

22. The method of claim 20 or 21, wherein the bacteria is Cutibacterium or Staphylococcus.

23. The method of claim 22, wherein the bacteria is C. acnes, S. aureus or 5. epidermidis.

24. The method of claim 20 or 21 wherein the bacteria is an antibiotic-resistant bacteria.

25. The method of claim 24 wherein the bacteria is methicillin-resistant Staphylococcus aureus (MR. SA), vancomycin intermediate-sensitivity Staphylococcus aureus (VISA), or vancomycin resistant Staphylococcus aureus (VRSA).

26. A method for treating acne vulgaris in a human comprising administering to a human having acne an effective amount of a composition comprising an engineered peptide of any of claims 1-7, or with the composition of any of claims 11-16, whereby the acne vulgaris is reduced or controlled.

27. The method of claim 26, wherein the number of C. acnes and / or S. aureus bacteria associated with the acne is reduced.

28. The method of claim 26 wherein the composition further comprises one or more of retinoid, benzoyl peroxide, salicylic acid, or an antibiotic.

29. A method for treating or controlling contaminations of or infections by one or more bacteria of Cutibacterium, Staphylococcus or Prevotella bacteria, comprising administering an effective amount of a composition comprising an engineered peptide of any of claims 1 -7, or with the composition of any of claims 11-15.

30. The method of claim 29, wherein the bacteria is C. acnes, S. aureus or 5'. epidermidis.

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