Proteasomally-cleaved peptides as a novel class of antimicrobial peptides for therapeutic intervention

Abstract

Described herein are antimicrobial peptides comprising protein fragments having microbiocidal activity which are degradation products of the proteasome. The antimicrobial peptides are useful in treating bacterial infections, sepsis, and wounds or skin conditions.

Description

PROTEASOMALLY-CLEAVED PEPTIDES AS A NOVEL CLASS OFANTIMICROBIAL PEPTIDES FOR THERAPEUTIC INTERVENTIONSEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Januray 22, 2025, is named P-636278-PC_SL.xml and is 12,493,246 bytes in size.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to antimicrobial peptides comprising protein fragments having microbiocidal activity which are degradation products of the proteasome. The antimicrobial peptides are useful in treating bacterial infections, sepsis, and wounds or skin conditions.BACKGROUND

[0003] For decades, the prevailing paradigm has posited the primary function of proteasome- derived peptides as serving for antigen presentation via Major Histocompatibility Complex I (MHCI) molecules. One of the major defense mechanisms, that has been conserved through evolution, is the production and secretion of Antimicrobial peptides (AMPs). AMPs are naturally occurring molecules found in various organisms, including humans, which play a crucial role in the innate immune response against microbial pathogens. AMPs are synthesized by a wide range of cells and tissues throughout the body, including epithelial cells, leukocytes, and mucosal surfaces and they are present in various bodily fluids like saliva, sweat, and tears. Such AMPs have a broadspectrum antimicrobial activity against bacteria. They work by direct disruption of microbial cell membranes (e.g. cationic host defense peptides), or by modulating host transcription and translation. Many of the AMPs are initially produced as inactive precursor molecules called pro-AMPs and their shift to an active form requires enzymatic processing, often involving proteases.SUMMARY OF THE DISCLOSURE

[0004] In one aspect, disclosed herein is a modified antimicrobial peptide (AMP) comprising a protein fragment having microbiocidal activity wherein the protein fragment is the degradation product produced by the proteasome.

[0005] In a related aspect, the protein from which said AMP is derived is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB1B, RPLP0, EIF4A1, GAPDH and RPS29.

[0006] In a related aspect, the AMP comprises an amino acid sequence selected from the sequences set forth in any of SEQ ID NOs: 1 to 14,328.

[0007] In another aspect, disclosed herein is a pharmaceutical composition comprising one or more AMPs described herein and an acceptable carrier.

[0008] In another aspect, disclosed herein is a method of treating or preventing a microbial infection in a subject in need thereof, the method comprising administering to the subject the modified AMP or the pharmaceutical composition described herein.

[0009] In one related aspect, the bacterial infection is Gram-negative bacterial infection. In a related aspect, the Gram-negative bacteria is selected from the group consisting of Enterob acteriaceae, Pseudomonadaceae Bacteroidaceae, Streptobacillus, and Acinetobacter.

[0010] In another related aspect, the Gram-negative bacteria is selected from the group consisting of Escherichia, Salmonella, Shigella, Citrobacter, Edwardsiella, Enter obacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Serratia, Yersinia, Staphylococcus, Pseudomonas, Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas, Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella, Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella, Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae, in particular Treponema and Borrelia, Leptospiraceae, Campylobacter, Helicobacter, Spirillum, Bacteroides, Fusobacterium, Prevotella, Porphyromonas, A. baumanii. Mycobacterium avium, Mycobacterium tuberculosis, and Listeria monocytogenes

[0011] In another related aspect, the bacterial infection is Gram-positive bacterial infection. In a related aspect, the Gram-positive bacteria is selected from the group consisting of Staphylococcus haemolyticus, Micrococcus luteus, Mycobacterium, Pseudomonas aeruginosa, and Staphylococcus aureus.

[0012] In a related aspect, the bacterial infection is an infection of the skin, of soft tissues, the respiratory system., the lung, the digestive tract, the eye, the ear, the nasopharynx, the bones, and / or the vagina.

[0013] In another aspect, disclosed herein is a method of treating a wound or skin condition the method comprising administering to the subject the modified AMP or the pharmaceutical composition.

[0014] In a related aspect, the skin condition is selected from the group consisting of: wound, dermatological disorder, psoriasis, ichthyosis, sarcoidosis, impetigo, acne (including hidradenitis suppurativa), burns, diaper rash, Netherton's syndrome, actinic keratosis, dermatomycoses, dermatosis or ectodermal dysplasia, atopic dermatitis, contact dermatitis, seborrheic dermatitis, vulgaris, filaggrin deficiency, allergic dermatitis,dandruff, pemphigus vulgaris, lichen planus, scleroderma, dermatomyositis, alopecia, skin carcinoma, melanoma, eczema, squamous cell carcinoma, acne vulgaris, erythema toxicum neonatorum, folliculitis, autoimmune bullous skin disease, bullous pemphigoid, pemphigus foliaceus, dermatitis and other disorders associated with damage or breakdown of the skin.

[0015] In another aspect, disclosed herein is a method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis, the method comprising administering to the subject the modified AMP or the pharmaceutical composition described herein.

[0016] In a related aspect, the subject is a human or a non -human animal.

[0017] In another aspect, disclosed herein is a wound covering comprising the antimicrobial peptide (AMP) described herein. In a related aspect, disclosed herein is a method of treating a wound in a subject in need thereof, the method comprising applying the wound covering to the wound.

[0018] In another aspect, the AMP is administered as and expressed from a minigene.

[0019] In another aspect, the AMP is administered as and expressed from a vector comprising a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG) and a gene encoding an AMP-containing protein. In a related aspect, the expression of the nucleic acid sequence produces a protein-dTAG fusion protein. In a related aspect, the protein-dTAG fusion protein is ubiquitinated and then degraded by a proteasome. In a related aspect, the AMP-containing protein is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB1B, RPLP0, EIF4A1, GAPDH and RPS29.

[0020] In another aspect, disclosed herein is a Proteolysis-targeting chimera (PROTAC) comprising an E3 Ubiquitin Ligase binding moiety and a moiety that binds an AMP-containing target protein coupled together by a linker. In a related aspect, the AMP-containing target protein is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB IB, RPLP0, EIF4A1, GAPDH and RPS29.

[0021] In another aspect, disclosed herein is a method of treating or preventing a microbial infection in a subject in need thereof, the method comprising administering to the subject the PROTAC described herein.

[0022] In another aspect, disclosed herein is a method of treating a wound or skin condition, the method comprising administering to the subject the PROTAC described herein.

[0023] In another aspect, disclosed herein is a method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis, the method comprising administering to the subject the PROTAC described herein.

[0024] In another aspect, disclosed herein is a method of preserving a food product comprising applying a composition comprising the antimicrobial peptide (AMP) described herein to the food product.

[0025] In a related aspect, the food product is selected from fruits, vegetables, legumes, beverages, starch food products, wheat products, dairy products, processed food products, aquatic products, and meat products. In a related aspect, the food product is a raw meat product.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A, Normalized presentation of previously reported AMP sequences (dark gray) showing the AMP position relative to the full protein sequence (gray). In the outer ring, conserved AMPs are colored in light gray with black dot, not conserved in black, AMPs for which we do not have conservation data are marked as NA. Conserved AMPs are defined as the ones that consist of amino acids that have statistically significant (Mann-Whitney U-test, P-value < 0.05) lower Rate4site rates than other amino acids within the host protein. The P-values were adjusted for multiple testing by Benjamini-Hochberg.

[0027] Figure IB, Comparison of predicted local distance difference test (pLDDT, per amino acid) in putative AMPs compared to amino acids from other regions of the same proteins. Mann -Whitney U-test, **** P < 0.0001.

[0028] Figure 1C, Comparison of relative solvent accessible surface area (rASA, per amino acid) of the same regions as in B. Mann-Whitney U-test, **** P < 0.0001.

[0029] Figure ID, Distribution of peptides upon in silico proteasomal cleavage of the human proteome, shown are only the peptides above a score of 5, according to AMP biochemical characteristic and length (cationic and hysrophobic).

[0030] Figure IE, Intracellular colony-forming units (CFU) / ml count of .S'. Typhimurium from cells infection (MOI 15) normalized to cell count ( 2* 10A6) A549 cells in the presence or absence of Bortezomib (50 nM). Error bars indicate mean ± s.e.m. (n = 6 independent samples) ** P<0.01

[0031] Figure IF, Schematic representation of bacterial growth assay in conditioned medium from A549 cells.

[0032] Figure 1G, Growth of .S', enterica in condition medium of Epoxomicin-treated A549 cells (IpM) at different time points (1, 6, and 12 h). Data represent the mean of O.D. (optical density) values ± s.e.m. (n = 3 independent samples) **** P<0.0001. Normalized on the first time point.

[0033] Figure 1H, growth of .S'. enterica in condition medium of Bortezomib-treated HCT116 cells (50 nM). Data represent the mean of O.D. (optical density) values ± s.e.m. (n = 3 independent samples) ** P<0.0I. Normalized on the first time point.

[0034] Figure II, Growth of .S'. enterica in condition medium of Bortezomib-treated A549 cells (50 nM). Data represent the mean of O.D. values ± s.e.m. (n = 3 independent samples) * P<0.05. Normalized on the first time point.

[0035] Figure 1J, Growth of .S'. enterica in conditioned medium derived from A549 cells treated with or without Bortezomib (50 nM). Proteinase K was added to the conditioned medium before .S'. enterica growth. Data represent the mean of O.D. values ± s.e.m. (n = 3 independent samples), ns P > 0.05, * P<0.05. Normalized on the first time point.

[0036] Figure IK, .S', enterica growth in medium from HCT116 cells. The experiment was performed as described in Figure H. Data represent the mean of O.D. values ± s.e.m. (n = 3 independent samples), ns P> 0.05, ** P<0.01. Normalized on the first time point. In all the condition medium experiments cells were stimulated with bacterial TLR agonists: HKLM (TLR2), LPS-EK (TLR4), FLA-ST (TLR5), ATP and heat killed .S'. Typhimurium, S. enterica , P. aeruginosa, S. haemolyticus , M. luteus.

[0037] Figure 2C, the number of peptides that are overlapping between the MAPP and peptides identified in the A549 secretome.

[0038] Figure 2D, Distribution of MAPP peptides according to AMP score.

[0039] Figure 2E, Heatmaps of XTT assay of overnight growth of .S', enterica, S. haemolyticus, E. coli, Salmonella and M. luteus, in LB with 2 fold dilutions of numerous peptides shown in Table 2. Values normalized relative to Control (0 pg / ml).

[0040] Figure 2F, Pictures of LB agar plates withlO-fold serial dilutions of P. aeruginosa, S. enterica, E. coli, M. luteus and .S', haemolyticus, and with PPP1CB (at MBC concentration) treatment or DMSO.

[0041] Figure 2G, Representative transmission electron micrographs of .S', haemolyticus, M. luteus, E. coli, and .S', enterica incubated with PPPICb for 1 hour in LB (at MBC concentration) or without (Untreated). Arrows show morphological disruptions and cytoplasm release (n = 3 independent samples).

[0042] Figure 2H, Bacteria permeabilization assay showing the percentage of propidium iodide uptake of P. aeruginosa, S. enterica, E. coli, M. luteus, and .S', haemolyticus, over 40 minutes with increasing concentration of PPPICB. Data show mean ± s.e.m. (n = 3). one-way ANOVA **** P < 0.0001.

[0043] Figure 21, Schematic representation of permeabilization assay with Propidium iodide.

[0044] Figure 2J, permeabilization assay of P. aeruginosa, S. enterica, E. coli, M. luteus and .S'. haemolyticus. n=3; LK20 (positive control) and top scored peptides.

[0045] Figure 2K, Schematic representation of acute pneumonia infection model in mice with P. aeruginosa.

[0046] Figure 2L, CFU ofP. aeruginosa (Log 10) per lung in mice treated with PBS or PPPICb (5 mg / kg i.v.) or Tobramycin (50 mg / kg i.p.). The box plot shows mean ± s.d. (n = 8). **** P < 0.0001.

[0047] Figure 2M, Schematic representation of bacteremia (IP) infection model in mice with P. aeruginosa.

[0048] Figure 2N, CFU of P. aeruginosa (LoglO) per lung in mice treated with PBS or PPPICb (10 mg / kg i.v.) or Tobramycin (50 mg / kg i.p.). The box plot shows mean ± s.d. (n = 8). **** P < 0.0001. All statistical comparisons were made by the one-way ANOVA test.

[0049] Figure 3A, Scheme of the dTAG system for targeted proteasomal degradation.

[0050] Figure 3B, Western blot analysis of A549 cells expressing HA-GFP or HA-PPP1CB. Cells were treated with V-l drug or DMSO and / or proteasome inhibitor (Epoxomicin (IpM)) for 6 hours. Vinculin is used as a loading control.

[0051] Figure 3C, Intracellular CFU / ml counts of S. typhimurium (MOI 100) in A549 expressing HA-GFP or HA-PPP1CB. Bar graphs show the ratio of CFUs of A549 cells V-l-treated over CFUs of A549 cells DMSO-treated. Error bars show s.e.m. (n = 3) ** P<0.01.

[0052] Figure 3D, Intracellular CFU / ml counts of M. luteus (MOI 100) in A549 expressing HA- GFP or HA-PPP1CB. Normalized as in C. Error bars show s.e.m. (n = 3 independent samples) * P<0.05.

[0053] Figure 3E, Measurement (OD595) of .S', enterica growth over 270 minutes in conditioned media from A549 cells expressing HA-GFP (V-l treated or DMSO). Data represent the mean of O.D. (optical density) values ± s.e.m. (n = 4 independent samples) ns P > 0.05.

[0054] Figure 3F, .S'. enterica growth in conditioned media from A549 cells expressing HA- PPPlCb (V-l treated or DMSO) Data shows mean ± s.e.m. (n = 4 independent samples) ** P<0.01.

[0055] Figure 3G, Ratio of OD values at 270 min from V-l-treated over DMSO-treated A549 cells expressing HA-GFP or HA-PPP1CB. Data from Fig. 3 E-F. Error bars show s.e.m. (n = 4 independent samples) * P<0.05. All statistical comparisons were made by two-sided t-test for unpaired samples.

[0056] Figure 4A, Schematic representation of MAPP on A549 infected with .S', enterica.

[0057] Figure 4B, Bar plot of the frequency of shared peptides between conditions; unique peptides in control (black), infection (dark gray) or shared (light gray).

[0058] Figure 4C, Heat map showing the nature of C-terminus cleavages of peptides from A549 cells upon 1 or 4 hours of infection with .S', enterica (MOI 15), following MAPP.

[0059] Figure 4D, Bar graphs show the quantification of peptides with K, R, H cleavages in the infection (dark gray) and the controls (grey) for the same protein population for 1 hour or 4 hours. Error bars represent mean ± s.d. (n = 3 independent samples). **** P < 0.0001, Mann-Whitney U- test.

[0060] Figure 4E, Bar graphs show the W / F / Y cleavages in the infection (dark gray) and the controls (grey) for the same protein population for 1 hour or 4 hours. Error bars represent mean ± s.d. (n = 3 independent samples). **** P < 0.0001, Mann-Whitney U-test.

[0061] Figure 4F, Schematic representation of immunoprecipitation of proteasomes from A549 cells upon .S'. enterica infection. Immunoprecipitated proteasomes were used for Anorogenic assay, probing the cleavage activity of different model peptides.

[0062] Figure 4G, Trypsin-like proteasome activity of proteasomes from A549 cells upon 4 hours of .S', enterica infection (top line), 1 hour of .S', enterica infection (middle line) or in the absence of infections (light grey, botom line)l hour, dark grey (second from botom line) 4 hours) (MOI 15). Values show the Relative Fluorescence Unit (RFU) of the RLR peptide. Error bars represent mean ± s.e.m. (n = 6 independent samples), ONE-WAY ANOVA test.

[0063] Figure 4H, Bar graph showing the quantification of trypsin-like proteasome activity at a time point of 200 minutes. Error bars represent mean ± s.e.m. (n = 6). ns P > 0.05, *** P < 0.001, ONE-WAY ANOVA test.

[0064] Figure 41, Bar graph showing the quantification of chymotryptic-like proteasome activity at a time point 200 minutes. Error bars represent mean ± s.e.m. (n = 6). *** P < 0.001, one-way ANOVA test.

[0065] Figure 4J, Volcano plot of proteins co -immunoprecipitated with proteasomes from A549 cells infected for 4 hours with E. coli relative to uninfected cells. The graph shows the Log2 Fold change per -Log 10 P value.

[0066] Figure 4K, Bar graphs show relative PSME3 intensity in proteomics in A549 cells or upon proteasome pull down, infected for 1 hour with .S', enterica. Data show mean ± s.e.m (n = 3 independent samples), ns P>0.05, ** P<0.01, **** P < 0.0001.

[0067] Figure 4L, Bar graphs show relative PSME3 intensity in proteomics in A549 cells or upon proteasome pull down, infected for 4 hours with .S', enterica. Data show mean ± s.e.m (n = 3 independent samples), ns P>0.05, ** P<0.01, **** P < 0.0001.

[0068] Figure 4M, Pictures of colonies of .S'. typhimurium on agar plate after infection of A549 cells stable with empty vector (shCtrl) or with PSME3 Knock-Down (KD). 1: 10, 10-fold serial dilutions.

[0069] Figure 4N, Quantification of CFU of .S'. typhimurium (MOI 100) in A549 cells stable with empty vector (shCtrl) or with PSME3 Knock-Down (KD). Data show mean ± s.e.m (n = 6 independent samples). ** P<0.01.

[0070] Figure 40, Growth of .S', enterica, S. typhimurium, S. haemolyticus , and M. luteus in conditioned medium derived from A549 cells stable with empty vector (shCtrl) or with PSME3 Knock-Down (KD). Data represent the mean of O.D. values ± s.e.m. (n = 6 independent samples). *** P < 0.001, * P<0.05. All statistical comparisons that were not specified are made by t-test for unpaired samples.

[0071] Figures 5A-5C, mice lungs in Pneumonia model. Mice were divided into 3 groups: Uninfected and received PBS (Uninfected); infected with P. aeruginosa PAO1 and treated with PBS (P.A. PBS); or infected with P. aeruginosa PAO1 and treated with PPP1CB (P.A. PPP1CB).Hematoxylin and Eosin (H&E) staining of selected mice (Figure 5A), Arrows showing bacterial clusters; Bar plot showing mean ± s.e.m. of Neutrophilic infdtration severity (Figure 5B); Bar plot showing mean ± s.e.m. of tissue injury in the pneumonia model (Figure 5C). mean ± s.e.m., oneway ANOVA; ** P < 0.01, *** P < 0.001. Neutrophilic infdtration analysis in lung tissue graded based on severity: 1 = minimal (<1%), 2 = slight (1-25%), 3 = moderate (26-50%), 4 = moderate / severe (51-75%), 5 = severe / high (76-100%).

[0072] Figures 6A-6B, mice heart tissue in Bacteremia model. Mice were divided into 3 groups: Uninfected and received PBS (UNINFECTED); infected with P. aeruginosa PAO1 and treated with PBS (PBS); or infected with P. aeruginosa PAO1 and treated with PPP1CB (PPP1CB) (IV, 10 mg / kg). Hematoxylin and Eosin (H&E) and Gram staining of selected mice (Figure 6A); Scoring of Myonecrosis severity (Figure 6B). Degree of lesions was graded from one to five depending on severity: 1 = not present or minimal (< 1%); 2 = slight (1-25%); 3 = moderate (26-50%); 4 = moderate / severe (51-75%); 5 = severe / high (76-100%).

[0073] Figures 7A-7B, mice liver tissue in Bacteremia model. Mice were divided into 3 groups: Uninfected and received PBS (UNINFECTED); infected with P. aeruginosa PAO1 and treated with PBS (PBS); or infected with P. aeruginosa PAO1 and treated with PPP1CB (PPP1CB) (IV, 10 mg / kg). Hematoxylin and Eosin (H&E) and Gram staining of selected mice (Figure 7A); Scoring of Hepatocellular necrosis severity (Figure 7B). Degree of lesions was graded from one to five depending on severity: 1 = not present or minimal (< 1%); 2 = slight (1-25%); 3 = moderate (26- 50%); 4 = moderate / severe (51-75%); 5 = severe / high (76-100%).

[0074] Figures 8A-8B, mice spleen tissue in Bacteremia model. Mice were divided into 3 groups: Uninfected and received PBS (UNINFECTED); infected with P. aeruginosa PAO1 and treated with PBS (PBS); or infected with P. aeruginosa PAO1 and treated with PPP1CB (PPP1CB) (IV, 10 mg / kg). Hematoxylin and Eosin (H&E) and Gram staining of selected mice (Figure 8A); Scoring of lymphocytolysis severity (Figure 8B). Degree of lesions was graded from one to five depending on severity: 1 = not present or minimal (< 1%); 2 = slight (1-25%); 3 = moderate (26-50%); 4 = moderate / severe (51-75%); 5 = severe / high (76-100%).

[0075] Figures 9A-9B, mice kidney tissue in Bacteremia model. Mice were divided into 3 groups: Uninfected and received PBS (UNINFECTED); infected with P. aeruginosa PAO1 and treated with PBS (PBS); or infected with P. aeruginosa PAO1 and treated with PPP1CB (PPP1CB) (IV, 10 mg / kg). Hematoxylin and Eosin (H&E) and Gram staining of selected mice (Figure 9A); Scoring of tubular necrosis severity (Figure 9B). Degree of lesions was graded from one to five depending on severity: 1 = not present or minimal (< 1%); 2 = slight (1-25%); 3 = moderate (26-50%); 4 = moderate / severe (51-75%); 5 = severe / high (76-100%).

[0076] Figure 10, Survival plot of in-vivo sepsis mice model of P. aeruginosa PAO1 (6.7 x 10A7 CFU / mouse). Mice were divided into 3 groups: infected with P. aeruginosa PAO1 and treated withPBS (Vehicle); infected with P. aeruginosa PAO1 and treated with PPP1CB (PPP1CB) and infected with P. aeruginosa PAO1 and treated with Tobramycin. (n=15 in each group).DETAILED DESCRIPTION

[0077] The present subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this disclosure is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

[0078] The present disclosure shows that proteasome-derived peptides provide a defense mechanism against bacterial growth in vitro and in vivo. These peptides, collectively called PDDPs (proteasome-derived defense peptides), are constitutively made, their production is enhanced upon bacterial infection, by alteration of proteasomal composition and function governed by the recruitment of a regulatory subunit of the proteasome, PSME3. In turn, PSME3 -capped proteasomes promote tryptic-like peptide cleavage, enhancing the generation of peptides with cationic termini. Proteome-wide computational analysis of proteasomal cleavage uncovers the potential extent of this phenomenon, revealing that most proteins in the proteome contain putative PDDPs that would be made upon protein degradation across different tissues. These paptides lend themselves to be more stable (protease cleavage resistnat) and more tolerable by the immune system. The present inventors have uncovered a previously unrecognized role of proteasomal degradation in facilitating the generation of new defense peptides upon protein breakdown, as a novel mechanism in innate immunity.Antimicrobial peptides (AMPs)

[0079] In some embodiments, provided herein is an antimicrobial peptide (AMP). In some embodiments, the AMP exhibits antimicrobial activity. In some embodiments, the antimicrobial peptide (AMP) comprises a protein fragment having microbiocidal activity wherein the protein fragment is the degradation product produced by the proteasome.

[0080] In some embodiments, provided herein is a modified antimicrobial peptide (AMP) comprising a protein fragment having microbiocidal activity wherein the protein fragment is the degradation product produced by the proteasome.

[0081] In some embodiments, the term “antimicrobial peptide” (AMP) as used herein refers to any peptide that has microbiocidal activity. In some embodiments, the antimicrobial peptide comprises a peptide having anti-bacterial, anti-infectious, anti-infective and / or germicidal, bacteriocidal, properties. In some embodiments, the antimicrobial activity encompasses the ability to reduce or inhibit the survival of a microorganism by killing or irreversibly damaging it, or to reduce or inhibit the growth or proliferative ability of a target microorganism without necessarily killing it.

[0082] In some embodiments, the term "peptide" may encompass native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs. In some embodiments, the peptides described herein comprise modified peptides, which have modifications. In some embodiments, modifications render the peptides more stable upon administration or more capable of penetrating into target cells.

[0083] In some embodiments, the antimicrobial peptide (AMP) comprises a proteasome-derived defense peptide (PDDP). In some embodiments, the term “proteasome-derived defense peptide” (PDDP) may encompass peptides which are the degradation products produced by proteasomes which is an antimicrobial peptide.

[0084] In some embodiments, the AMP comprises a cationic peptide. In some embodiments, a cationic peptide has a positive charge (i.e., charge>0, e.g., +1, +2, +3, +4, +5, +6, +7, +8, +9, +10 or more). In some embodiments, the AMP comprises one or more polar cationic amino acid residues (i.e., residues having positively charged side chains), such as 2 or more, 3 or more, 4 or more, or 5 or more polar cationic amino acid residues. In some embodiments, amino acid residues having positively charged side groups include lysine, ornithine and arginine, and histidine.

[0085] In some embodiments, the AMP comprises a hydrophobic peptide. In some embodiments, the AMP comprises one or more hydrophobic amino acid residues (i.e., residues having hydrophobic side chains), such as 2 or more, 3 or more, 4 or more, or 5 or more hydrophobic amino acid residues. In some embodiments, amino acid residues having hydrophobic side groups include Alanine, Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Tyrosine, and Tryptophan.

[0086] In some embodiments, the AMP comprises a protein fragment derived from a protein. In some embodiments, the term “protein fragment” may encompass a peptide derived from a protein.

[0087] In some embodiments, the protein fragment comprises a modified derivative or modified fragment of a protein. As used herein, a “modified derivative” or “modified fragment” of a protein refers to polypeptides that are substantially homologous in primary structural sequence to a reference polypeptide sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications, include amino acid substitutions, or which incorporate or remove amino acids that are not found in the reference polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art.

[0088] In some embodiments, the AMP comprises a protein fragment derived from a protein, wherein said protein is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1,GRHPR, RPL21, MOB1B, RPLP0, EIF4A1, GAPDH and RPS29 (Table 2). In some embodiments, the protein from which the protein fragment is derived is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB1B, RPLP0, EIF4A1, GAPDH and RPS29. In someembodiments, the protein from which the AMP is derived is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB IB, RPLPO, EIF4A1, GAPDH and RPS29.

[0089] In some embodiments, the AMP comprises a protein fragment derived from GRHPR. In some embodiments, the AMP comprises a protein fragment derived from RPL21. In some embodiments, the AMP comprises a protein fragment derived from MOB 1 B . In some embodiments, the AMP comprises a protein fragment derived from RPLPO. In some embodiments, the AMP comprises a protein fragment derived from EIF4A1. In some embodiments, the AMP comprises a protein fragment derived from GAPDH. In some embodiments, the AMP comprises a protein fragment derived from DFNA5. In some embodiments, the AMP comprises a protein fragment derived from DCTN4. In some embodiments, the AMP comprises a protein fragment derived from RPL41. In some embodiments, the AMP comprises a protein fragment derived from PSMG2. In some embodiments, the AMP comprises a protein fragment derived from GLB1. In some embodiments, the AMP comprises a protein fragment derived from RPS4X. In some embodiments, the AMP comprises a protein fragment derived from PPP1CB. In some embodiments, the AMP comprises a protein fragment derived from CHMP2A. In some embodiments, the AMP comprises a protein fragment derived from ETS1. In some embodiments, the AMP comprises a protein fragment derived from RPS29. In some embodiments, the AMP comprises Protein phosphatase 1 catalytic subunit beta (PPP1CB).

[0090] In some embodiments, the AMP comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1 to 14,328. In some embodiments, AMP comprises an amino acid sequence selected from the sequences set forth in Table 1. In some embodiments, AMP comprises an amino acid sequence selected from the sequences set forth in Table 2.

[0091] The exemplary antimicrobial polypeptide sequences described herein (e.g., Table 1) are merely examples and are not the only antimicrobial polypeptides provided herein. Indeed, fragments and variants of the sequences of the disclosed peptides are within the scope of the present disclosure.

[0092] In some embodiments, the term “fragment” may encompass at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 contiguous amino acid residues of an AMP disclosed herein and retains at least one antimicrobial property of the AMP.

[0093] In some embodiments the AMP comprises at least 10 amino acids. In some embodiments the AMP comprises at least 15 amino acids. In some embodiments the AMP comprises at least 20 amino acids. In some embodiments the AMP comprises at least 25 amino acids. In some embodiments the AMP comprises at least 30 amino acids. In some embodiments the AMP comprises at least 35 amino acids. In some embodiments the AMP comprises at least 40 amino acids. In some embodiments the AMP comprises at least 50 amino acids.

[0094] In some embodiments the AMP sequence is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homologous to an amino acid sequence selected from: A. the amino acid sequence of a fragment of the amino acid sequence of a protein selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB1B, RPLP0, EIF4A1, GAPDH and RPS29; B. an amino acid sequence comprising a modified fragment of the amino acid sequence of a protein selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB1B, RPLP0, EIF4A1, GAPDH and RPS29; C. an amino acid sequence comprising the amino acid sequence of a sequence selected from SEQ ID NOs: 1-14,328; and D. an amino acid sequence comprising a modified amino acid sequence of a sequence selected from SEQ ID NOs: 1-14,328.

[0095] In some embodiments the AMP sequence is selected from: A. an amino acid sequence comprising a fragment of the amino acid sequence of a protein selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB1B, RPLP0, EIF4A1, GAPDH and RPS29; B. an amino acid sequence comprising a modified fragment of the amino acid sequence of a protein selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB IB, RPLP0, EIF4A1, GAPDH and RPS29; C. an amino acid sequence comprising the amino acid sequence of a sequence selected from SEQ ID NOs: 1-14,328; and D. an amino acid sequence comprising a modified amino acid sequence of a sequence selected from SEQ ID NOs: 1-14,328.

[0096] In some embodiments the first polypeptide sequence is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homologous to an amino acid sequence selected from the amino acid sequence of a sequence selected from SEQ ID NOs: 1-14,328.

[0097] In some embodiments, fragments include peptides that are missing one, two, three, four, or more amino acids from the N-terminus and / or the C-terminus relative to an AMP disclosed herein.

[0098] In some embodiments the AMP comprises an isolated AMP. As used herein, the term "isolated" means that the peptide is removed from its native enviromnent, e.g., a cell.

[0099] In some embodiments the AMP comprises a recombinant AMP.

[0100] In some embodiments the AMP comprises a synthetic AMP. As used herein, the term “synthetic” used in expressions such as “synthetic peptide” or “synthetic polypeptide” is intended to refer to non-naturally occurring molecules that can be produced in vitro (e.g., synthesized chemically and / or produced using recombinant DNA technology). In some embodiments, the AMP comprises a synthetic peptide. In some embodiments, the AMP comprises synthetic amino acids. In some embodiments, the aromatic amino acids, Trp, Tyr and Phe, may be substituted with synthetic non-natural amino acids such as TIC, naphthylelanine (Nol), ring -methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

[0101] In some embodiments the AMP comprises a modified AMP. In some embodiments the synthetic AMP comprises a modified AMP. In some embodiments the modified AMP comprises an N terminus modification. In some embodiments the modified AMP comprises a C terminus modification.

[0102] A person of skill in the art is familiar with the methods of modifying the peptides described herein by using one or more modified amino acids (e.g., non -naturally-occurring amino acids), or by chemically modifying the synthetic AMP, to suit particular needs of stability or other needs.

[0103] In some embodiments, the modified AMP comprises N-terminal acetylation. In some embodiments, the modified AMP comprises C-terminal amidation.

[0104] In some embodiments the AMP comprises one or more deletion, insertion, or modification of any amino acid residue, including the N- or C-terminal amino acids. In some embodiments the AMP comprises an acetylated, acylated, acryloylated, alkylated, glycosylated (e.g., glucosylated), PEGylated, myristylated, and the like N-terminal amino acid modification having an esterified, amidated, homoserine / homoserine lactone, or caprolactam C-terminal amino acid modification; or having a polyalkylene glycol (e.g., polyethylene glycol) conjugated to any free amino group. In some embodiments the AMP comprises the C-terminal amino acid amidation.

[0105] In some embodiments, the modified AMP comprises adding one or more Alanines at the C- terminal of the AMP. In some embodiments, the modified AMP comprises adding one or more Alanines at the N-terminal of the AMP.

[0106] In some embodiments, the modified AMP comprises adding one or more Arginines at the C-terminal of the AMP. In some embodiments, the modified AMP comprises adding one or more Arginines at the N-terminal of the AMP.

[0107] In some embodiments, the modified AMP comprises a N-mono substitution. In some embodiments, the N-mono substitution comprises N-benzyl substitution. In some embodiments, the N-mono substitution comprises N-phenethyl substitution. In some embodiments, the modified AMP comprises N, N-disubstitutions. In some embodiments, the N, N-disubstitution comprises N- methyl-N-benzyl disubstitution. In some embodiments, the N, N-disubstitution comprises N-ethyl- N-benzyl disubstitution. In some embodiments, the N, N-disubstitution comprises N, N-dibenzyl disubstitution.

[0108] In some embodiments, the modified AMP comprises 1 -amino-cyclohexane-carboxylic acid at the N-terminus. In some embodiments, the modified AMP comprises a Dap (Ac) group (N-acetyl- 2,3-diamino propionic acid at the C- terminus.

[0109] N-methylation and N-alkylation, sulfonation, and the addition of phosphate groups and other PTMs.

[0110] In some embodiments, the modified AMP comprises non-canonical amino acids with bioorthogonal groups. In some embodiments, bioorthogonal groups comprise azides. In someembodiments, bioorthogonal groups comprise ketones. In some embodiments, bioorthogonal groups comprise alkynes.

[0111] In some embodiments, the modified AMP comprises bioisosteres in N- or C-capping regions.

[0112] In some embodiments, the modified AMP comprises divalent metal ions. In some embodiments, divalent metal ions comprise Zn2+. In some embodiments, divalent metal ions comprise Cu2+.

[0113] In some embodiments, the modified AMP comprises a peptide bond modification. In some embodiments the peptide bond modification comprises CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. An artisan in the field would be familiar with the methods for preparing peptidomimetic compounds. In some embodiments, the peptide bonds (-CO-NH-) within the peptide may be substituted. In some embodiments, the peptide bonds are substituted by N- methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (- C0-CH2-), a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2- NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)- CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. In some embodiments, the peptide bond modifications can occur at any of the bonds along the peptide chain. In some embodiments, the peptide comprises at least one peptide bond modification. In some embodiments, the peptide comprises at least two peptide bond modifications. In some embodiments, the peptide comprises at least three peptide bond modifications. In some embodiments, the peptide comprises at least four peptide bond modifications.

[0114] In some embodiments, the AMP comprises one or more modified amino acids or one or more non-amino acid monomers. In some embodiments, the AMP comprises one or more fatty acids. In some embodiments, the AMP comprises one or more complex carbohydrates.

[0115] In some embodiments, the AMP comprises a linear peptide. In some embodiments, the AMP comprises a cyclic peptide. In some embodiments, the AMP comprises at least two cysteine residues flanking the core peptide sequence (i.e. cyclization can be generated via formation of S-S bonds between the two Cys residues). In some embodiments, side chain to side chain cyclization can also be generated via formation of an interaction bond of the formula -(-CH2-)n-S-CH-2-C-, wherein n = 1 or 2. In some embodiments, the AMP comprises Cys or homoCys. In some embodiments, the AMP comprises GIu, Asp, Lys, Om, di-amino butyric (Dab) acid, diaminopropionic (Dap) acid at various positions in the chain (-CO-NH or -NH-CO bonds). Backbone to backbone cyclization can also be obtained through incorporation of modified amino acids of the formulas H-N((CH2)n-COOH)-C(R)H-COOH or H-N((CH2)n-COOH)-C(R)H-NH2, wherein n = 1-4, and further wherein R is any natural or non -natural side chain of an amino acid.

[0116] In some embodiments, the AMP comprises analogs of AMPs having an amino acid sequence selected from SEQ ID NOs: 1-14,328. Analogs may differ from naturally occurring peptides by conservative amino acid sequence substitutions or by modifications which do not affect sequence, or by both. For example, conservative amino acid substitutions may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups: (a) glycine, alanine; (b) valine, isoleucine, leucine; (c) aspartic acid, glutamic acid; (d) asparagine, glutamine; (e) serine, threonine; (f) lysine, arginine; (g) phenylalanine, tyrosine.

[0117] In some embodiments, modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also included are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0118] Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.Nucleotides, Vectors, and Host Cells

[0119] Disclosed herein, in one aspect, is a nucleotide or nucleic acid sequence encoding the AMPs disclosed herein. The term “nucleotide”, “nucleotide sequence” or “nucleic acid molecule” as used herein is intended to include DNA molecules and RNA molecules or modified RNA molecules. A nucleic acid molecule may be single -stranded or double-stranded. In some embodiments, a nucleotide comprises a modified nucleotide. In some embodiments, a nucleotide comprises an mRNA. In some embodiments, a nucleotide comprises a modified mRNA. In some embodiments, a nucleotide comprises a modified mRNA, wherein the modified mRNA comprises a 5 '-capped mRNA. In some embodiments, a modified mRNA comprises a molecule in which some of the nucleosides have been replaced by either naturally occurring modified or synthetic nucleosides. In some embodiments, a modified nucleotide comprises a modified mRNA comprising a 5 '-capped mRNA and wherein some of the nucleosides have been replaced by either naturally occurring modified or synthetic nucleosides.

[0120] The term “isolated nucleotide” or “isolated nucleic acid molecule” as used herein refers to nucleic acids encoding the AMPs disclosed herein.

[0121] Disclosed herein, in one aspect, is a vector comprising the nucleic acid construct encoding the AMPs disclosed herein. In some embodiments, the vector is an expression vector.

[0122] As used herein, the term “vector” refers to discrete elements that are used to introduce heterologous nucleic acids into cells for either expression or replication thereof. An expression vector includes vectors capable of expressing nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of affecting expression of such nucleic acids. Thus, an expression vector may refer to a DNA or RNA construct, such as a plasmid, a phage, recombinant virus, or other vector that, upon introduction into an appropriate host cell, results in expression of the nucleic acids. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in prokaryotic cells and / or eukaryotic cells, and those that remain episomal orthose which integrate into the host cell genome.

[0123] The term “recombinant host cell” (or simply “host cell”) as used herein refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell.

[0124] Disclosed herein, in one aspect, is a host cell comprising an expression vector carrying the nucleic acid construct encoding the AMPs disclosed herein. In one embodiment, the cell or host cell is a prokaryotic cell or a eukaryotic cell.Compositions

[0125] In some embodiments, provided herein is a composition comprising one or more AMPs described herein in detail.

[0126] In some embodiments, provided herein is a composition comprising one or more AMPs, where the AMP comprises an amino acid sequence selected from the sequences set forth in any of SEQ ID NOs: 1 to 14,328. In some embodiments, provided herein is a composition comprising one or more AMPs. In some embodiments, the AMPs comprise an amino acid sequence selected from the sequences set forth in any of SEQ ID NOs: 1 to 14,328.

[0127] In some embodiments, provided herein is a pharmaceutical composition comprising one or more AMPs described herein in detail and an acceptable carrier. In some embodiments, the composition further comprises a pharmaceutical excipient. In some embodiments, the present invention further relates to a pharmaceutical composition comprising AMP for use as a medicament. In some embodiments, the AMP comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1 to 14,328.

[0128] In some embodiments, the pharmaceutical composition comprises at least one AMP. In some embodiments, the pharmaceutical composition comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 25, or at least 50 different AMPs. In some embodiments, the composition comprises at least two different AMPs. In some embodiments, the composition comprises at least threedifferent AMPs. In some embodiments, the composition comprises at least four EOs. In some embodiments, the composition comprises at least five different AMPs. In some embodiments, the composition comprises at least six different AMPs. In some embodiments, the composition comprises at least seven different AMPs. In some embodiments, the composition comprises at least eight different AMPs. In some embodiments, the composition comprises at least nine different AMPs. In some embodiments, the composition comprises at least ten different AMPs. In some embodiments, the composition comprises at least 50 different AMPs. In some embodiments, the composition comprises at least 100 different AMPs. In some embodiments, the composition comprises at least 1000 different AMPs.

[0129] In some embodiments, the composition comprises one or more AMPs appearing in Table 1. In some embodiments, the composition comprises one or more different AMPs selected from the AMP amino acid sequences set forth in any of SEQ ID NOs: 1 to 14,328.

[0130] In some embodiments the AMP composition further comprises antibiotics. In some embodiments, the antibiotics include, without limitation, one or more antibiotics selected from the group consisting of erythromycin, ampicillin, vancomycin, linezolid, methicillin, oxacillin, cefotaxime, rifampicin, amikacin, gentamicin, amikacin, kanamycin, tobramycin, neomycin, ertapenem, doripenem, imipenem / cilastatin, meropenem, ceftazidime, cefepime, ceftaroline, ceftobiprole, aztreonam, piperacillin, polymyxin b, colistin, ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, tigecycline, a conjugate thereof, and derivatives thereof.

[0131] In some embodiments, the composition is a veterinary composition. In some embodiments, the composition further comprises a veterinary excipient.

[0132] In some embodiments, the composition is an animal feed composition. In some embodiments, the composition is a fish feeding composition used to feed live fish.Compound / composition for use

[0133] In some embodiments, disclosed herein is a method of treating a disorder, disease or condition in a subject in need of treatment and or prevention thereof, wherein said method comprises the step of administering to the subject an effective amount of an antimicrobial peptide (AMP). In some embodiments, disclosed herein is a method of treating a disorder, disease or condition in a subject in need of treatment and or prevention thereof, wherein said method comprises the step of administering to the subject an effective amount of pharmaceutical composition comprising the antimicrobial peptide (AMP) described herein. In some embodiments, disclosed herein is a method of treating a disorder, disease or condition in a subject in need of treatment and or prevention thereof, wherein said method comprises the step of administering to the one or more of the antimicrobial peptides (AMPs) described herein.

[0134] In some embodiments, the AMP comprises a protein fragment derived from GRHPR. In some embodiments, the AMP comprises a protein fragment derived from RPL21. In someembodiments, the AMP comprises a protein fragment derived from MOB 1 B . In some embodiments, the AMP comprises a protein fragment derived from RPLPO. In some embodiments, the AMP comprises a protein fragment derived from EIF4A1. In some embodiments, the AMP comprises a protein fragment derived from GAPDH. In some embodiments, the AMP comprises a protein fragment derived from DFNA5. In some embodiments, the AMP comprises a protein fragment derived from DCTN4. In some embodiments, the AMP comprises a protein fragment derived from RPL41. In some embodiments, the AMP comprises a protein fragment derived from PSMG2. In some embodiments, the AMP comprises a protein fragment derived from GLB1. In some embodiments, the AMP comprises a protein fragment derived from RPS4X. In some embodiments, the AMP comprises a protein fragment derived from PPP1CB. In some embodiments, the AMP comprises a protein fragment derived from CHMP2A. In some embodiments, the AMP comprises a protein fragment derived from ETS1. In some embodiments, the AMP comprises a protein fragment derived from RPS29. In some embodiments, the AMP comprises Protein phosphatase 1 catalytic subunit beta (PPP1CB).

[0135] In some embodiments, the AMP comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1 to 14,328. In some embodiments, AMP comprises an amino acid sequence selected from the sequences set forth in Table 1.

[0136] In some embodiments, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Thus, in some embodiments, “treating”, has the same meaning as “ameliorating”, and “alleviating” and refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, or a combination thereof.

[0137] As used herein, the terms “administering”, “administer”, or “administration” refer to the delivery of the AMP or compositions described herein to a subject. In some embodiments, the compositions described herein can be administered either parenterally, enterally, or topically. Illustrative examples of parenteral administration include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Illustrative examples of enteral administration include, but are not limited to, sublingual, and oral administration.

[0001] In some embodiments, the AMPs or compositions described herein are administered in a therapeutically effective amount. The terms “effective”, “efficacy”, or “effectiveness” are used herein to refer to the ability of a therapy to obtain beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, inthe context of administering an AMP or a composition that treats infection, an effective amount of an AMP or composition is, for example, an amount sufficient to achieve treatment, as defined herein, as compared to the response obtained without administration of the AMP or composition. In some embodiments, a therapeutically effective amount is an amount of an AMP or composition to be delivered that is sufficient, when administered to a subject suffering from infection, to treat, improve the condition or inhibit the decline of a subject afflicted with infection.Treatment of bacterial infections

[0138] In some embodiments, antimicrobial peptides are used for treating or preventing a microbial infection in a subject. In some embodiments provided herein are methods of eliminating, reducing the number of, or significantly reducing the replication of at least one microbial organism in a subject. The AMPs have broad-spectrum antibacterial activity. In some embodiments, the AMPs and methods provide a reduced risk of development of pathogen resistance.

[0139] In some embodiments provided herein is a method of treating or preventing a microbial infection in a subject in need thereof, the method comprising administering to the subject the antimicrobial peptide (AMP) described herein. In some embodiments provided herein are methods of treating or preventing a microbial infection in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising one or more antimicrobial peptides (AMPs) described herein.

[0140] In some embodiments provided herein are methods of treating an infection comprising administering to the subject an antimicrobial peptide (AMP). In some embodiments provided herein are methods of treating an infection comprising administering to the subject a pharmaceutical composition comprising the antimicrobial peptide (AMP) described herein.

[0141] In some embodiments, the present invention further relates to a pharmaceutical composition comprising one or more AMPs for use in the treatment of an infection.

[0142] In some embodiments, the infection is a bacterial infection. In some embodiments, bacterial infection is a chronic bacterial infection. In some embodiments, the bacterial infection is associated with Pseudomonas (e.g. Pseudomonas aeruginosa), Salmonella (e.g. .S', enterica ), Staphylococcus spp. (e.g. Staphylococcus aureus), Acinetohacter (e.g. Acinetobacter baumanii), Mycobacterium spp. (e.g. Mycobacterium avium, Mycobacterium tuberculosis), Listeria monocytogenes and / or Salmonella bacteria.

[0143] In some embodiments, the bacterial infection is a Gram -negative bacterial infection. In some embodiments, the bacterial infection is a Gram-positive bacterial infection.

[0144] In some embodiments, the bacterial infection is selected from the group consisting of Enterobacteriaceae, Pseudomonadaceae Bacteroidaceae, Streptobacillus, and Acinetobacter.

[0145] In some embodiments, the Gram-negative bacteria is selected from the group consisting of Escherichia, Salmonella, Shigella, Citrobacter, Edwardsiella, Enterobacter, Hafnia, Klebsiella,Morganella, Proteus, Providencia, Serratia, Yersinia, Staphylococcus, Pseudomonas, Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas, Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella, Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella, Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae, in particular Treponema and Borrelia, Leptospiraceae, Campylobacter, Helicobacter, Spirillum, Bacteroides, Fusobacterium, Prevotella, Porphyromonas, A. baumanii. Mycobacterium avium, Mycobacterium tuberculosis, and Listeria monocyte.

[0146] In some embodiments, the Gram -positive bacteria is selected from the group consisting of Staphylococcus haemolyticus , Micrococcus luteus, Mycobacterium, Pseudomonas aeruginosa, and Staphylococcus aureus. In some embodiments, the Gram-positive bacteria is Methicillin-resistant Staphylococcus aureus.

[0147] In some embodiments, the bacterial infection is an infection of the skin, of soft tissues, the respiratory system, the lung, the digestive tract, the eye, the ear, the nasopharynx, the bones, and / or the vagina.

[0148] In some embodiments, infections include, but are not limited to, toxic shock syndrome, diphtheria, cholera, typhus, meningitis, whooping cough, botulism, tetanus, pyogenic infections, sinusitis, pneumonia, gingivitis, mucitis, folliculitis, cellulitis, acne and acne vulgaris, impetigo, osteomyelitis, endocarditis, ulcers, bums, dysentery, urinary tract infections, gastroenteritis, anthrax, Lyme disease, syphilis, rubella, septicemia, Buruli ulcer, mycetoma, chromoblastomycosis, vaginal candidiasis, tuberculosis, otitis media, eczema (atopic dermatitis), diabetic ulcers, venous ulcers, infected bums, infected wounds, infected ballistic wounds and plague; as well as primary, secondary, and opportunistic infections associated with, for example, trauma, surgery, endotracheal intubation, tracheostomy, and cystic fibrosis.

[0149] In some embodiments, the subject suffering from a microbial infection is immunosuppressed. As used herein, “immunosuppressed” or “immunocompromised” may encompass a condition where the immune system function of a subject is reduced or absent. Thus, in some embodiments, the treated subject is immunosuppressed. In some embodiments, the subject has undergone immunosuppressive treatments post organ transplantation or bone marrow transplantation, or is immunosuppressed due to side effects of drugs or therapy including T-cell therapy, radiotherapy, inherited immunosuppressive genetic traits or diseases, acquired immunosuppressive diseases such as AIDs, cancers such as leukemia or lymphoma.Treatment of sepsis

[0150] In some embodiments, disclosed herein is a method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need, comprising the step of administering an effective amount of an antimicrobial peptide (AMP) to said subject, wherein said administering treats, prevents, inhibits, reduces the incidence of, ameliorates, oralleviates sepsis in said subject. In some embodiments, disclosed herein is a method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need, comprising the step of administering a composition comprising an effective amount of an antimicrobial peptide (AMP) to said subject, wherein said administering treats, prevents, inhibits, reduces the incidence of, ameliorates, or alleviates sepsis in said subject.

[0151] In some embodiments, the present invention further relates to a pharmaceutical composition comprising one or more AMPs for use in treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need. In some embodiments, the present invention further relates to one or more AMPs for use in treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need.

[0152] In some embodiments, sepsis comprises severe sepsis. In some embodiments, sepsis comprises mild sepsis.

[0153] In some embodiments, treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need comprises prevention, inhibiting, reducing the incidence of organ failure. In some embodiments, treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need comprises prevention, inhibiting, reducing the incidence of organ dysfunction. In some embodiments, treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need comprises prevention, inhibiting, reducing the incidence of organ failure. In some embodiments, treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need comprises prevention, inhibiting, reducing the incidence of organ damage. In some embodiments, treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need comprises prevention, inhibiting, reducing the incidence of acute multiple organ failure. In some embodiments, treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need comprises a reduction in mortality of a subject suffering from sepsis. In some embodiments, treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need comprises improving the survival time in the subject in need.

[0154] In some embodiments, the method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need, increases the survival time in said subject by more than 60% compared with a subject not administered the AMP. In some embodiments, the method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need, increases the survival time in said subject by more than 70% compared with a subject not administered the AMP. In some embodiments, the method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need, increases the survival time in said subject by more than 80% compared with a subject not administered the AMP. In some embodiments, the method of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need, increases the survival time in said subject by more than 90% compared with a subject not administered the AMP. In some embodiments, the method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need, increases the survival time in said subject by more than 100% compared with a subject not administered the AMP.

[0155] As used herein, a “subject” or “patient” refers in one embodiment, to a human or any other animal. In some embodiments, the subject is a human. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is an animal. In some embodiments, the subject is a dog. In some embodiments, the subject is a cat.

[0156] The present disclosure further provides, in another aspect, a method treatment of a bacterial infection in a population of animals, comprising administering to the animals the composition described herein. In some embodiments, the method of treating a bacterial infection is for treating a population of animals, comprising administering to the animals the composition described herein.

[0157] The present disclosure further provides, in another aspect, a method treatment of a bacterial infection in an animal, comprising administering to the animal the composition described herein.

[0158] The present disclosure further provides, in another aspect, a method treatment of sepsis in an animal, comprising administering to the animal the composition described herein.

[0159] In some embodiments, the method comprises adding the composition to the water and / or food of the animal. In some embodiments, the method comprises adding the composition to the water of the animal. In some embodiments, the method comprises adding the composition to the food of the animal.

[0160] In some embodiments, the animal is a domesticated animal, a farm animal and a zoo animal. In some embodiments, the animal is selected from the group consisting of chickens, turkeys, cattle, sheep, goats, pigs, horses, cats, dogs, and rabbits.

[0161] The present disclosure further provides, in another aspect, a method treatment of a bacterial infection in fish, comprising administering to the fish the composition described herein.

[0162] In some embodiments, the term "fish" encompasses both vertebrate and invertebrate species of marine animals, such as fish, mollusks, for example, octopus, squid and cuttlefish, or crustaceans, for example, krill, shrimp, crabs, lobsters, and mantis shrimp.

[0163] A person of skill in the field would understand that administering the composition described herein to fish can be done in a variety of methods and steps, as long known in the field. In some embodiments, the composition described herein may be directly administered to the outer, skin surface of the fish, or alternatively, the composition may be indirectly administered to the fish by adding the the composition to the water medium of the fish. In some embodiments, the composition described herein may be administered to the fish e.g. by feeding the fish with the composition.Wound coverings

[0164] In some embodiments, provided herein are wound coverings comprising one or more antimicrobial peptides (AMPs).

[0165] In some embodiments provided herein are wound coverings comprising the antimicrobial peptide (AMP) described herein. In some embodiments, the AMP comprises a modified AMP. In some embodiments, the modified AMP comprises an amino acid sequence selected from the sequences set forth in any of SEQ ID NOs: 1 to 14,328.

[0166] In some embodiments, the present invention further relates to a wound covering comprising one or more AMPs for use in the treatment of a skin condition.

[0167] An artisan would appreciate that the term “wound covering”, having the same meaning as “wound bandages” and “wound dressings,” may encompass bandages, sutures, sterile pads, compresses, gauze, and the like. Wound coverings can be provided for temporary (e.g., 1 minute- several days) or more permanent (e.g., 1-3 months) treatment. In some embodiments, a wound covering includes a material that can be applied on or to a wound to treat the wound, and the wound covering may or may not be in direct contact with a wound.

[0168] In some embodiments, the wound coverings further comprise a bioactive agent. In some embodiments, the wound coverings further comprise antibiotics, anti-inflammatories, or combinations thereof.

[0169] In some embodiments, provided herein is a method of treating a skin condition comprising administering to the subject an antimicrobial peptide (AMP). In some embodiments, provided herein is a method of treating a skin condition comprising administering to the subject the composition comprising an effective amount of an antimicrobial peptide (AMP).

[0170] In some embodiments, provided herein is a method of treating a skin condition comprising applying a wound covering to a wound site on a subject.

[0171] In some embodiments provided herein is a method of treating a wound comprising administering to the subject an antimicrobial peptide (AMP). In some embodiments, provided herein is a method of treating a wound comprising administering to the subject the composition comprising an effective amount of an antimicrobial peptide (AMP).

[0172] In some embodiments provided herein is a method of treating a wound or skin condition in a subject in need thereof, the method comprising administering to the subject the modified AMP or the composition described herein.

[0173] In some embodiments, provided herein is a method of treating a wound comprising applying a wound covering to a wound site on a subject.

[0174] In some embodiments the wound site comprises a surgical site or a site of tissue injury. In some embodiments, provided herein is a wound covering comprising one or more AMPs for use intreating a wound. In some embodiments, provided herein is a wound covering comprising one or more AMPs for use in treating a skin condition.

[0175] In some embodiments, a skin condition comprises wound. In some embodiments, a skin condition comprises a dermatological disorder. In some embodiments, a skin condition comprises disorders associated with disruption of the skin, including, without limitation psoriasis, ichthyosis, sarcoidosis, impetigo, acne (including hidradenitis suppurativa), bums, diaper rash, Netherton's syndrome, actinic keratosis, dermatomycoses, dermatosis or ectodermal dysplasia, atopic dermatitis, contact dermatitis, seborrheic dermatitis, vulgaris, filaggrin deficiency, allergic dermatitis, dandruff, pemphigus vulgaris, lichen planus, scleroderma, dermatomyositis, alopecia, skin carcinoma, melanoma, eczema, squamous cell carcinoma, acne vulgaris, erythema toxicum neonatorum, folliculitis, autoimmune bullous skin disease, bullous pemphigoid, pemphigus foliaceus, dermatitis and other disorders associated with damage or breakdown of the skin.Vaccine adjuvant

[0176] In some embodiments, provided herein is a vaccine adjuvant comprising one or more antimicrobial peptides (AMPs).

[0177] In some embodiments provided herein is a vaccine adjuvant comprising the antimicrobial peptide (AMP) described herein.

[0178] In some embodiments, the AMPs described herein in detail are added as adjuvants to vaccines. In some embodiments, the present invention provides vaccines comprising one or more AMPs described herein in detail and one or more antigens. In some embodiments, provided herein is a composition comprising one or more AMPs for use as a vaccine adjuvant.

[0179] In some embodiments provided herein are methods of vaccinating a subject against a specific antigen or a group of specific antigens, said method comprising administering to the subject a vaccine comprising an antimicrobial peptide (AMP). In some embodiments, the method comprises administering an effective amount of an adjuvant comprising the AMP described herein. In some embodiments, the method comprises administering an effective amount of an adjuvant comprising AMP, after which a vaccine is administered. In some embodiments, the method comprises administering an effective amount of an adjuvant comprising one or more AMPs.

[0180] Delivery systems by cationic peptides

[0181] In some embodiments, the AMPs described herein in detail deliver drugs into cells. In some embodiments, the AMPs described herein in detail are added as vehicles to pharmaceutical compositions. In some embodiments, provided herein is a composition comprising one or more AMPs for use in drug delivery.

[0182] In some embodiments provided herein are methods of delivering drugs to a subject comprising administering to the subject an antimicrobial peptide (AMP) and a drug. In someembodiments, provided herein are methods of delivering drugs to a subject comprising administering to the subject a composition comprising the antimicrobial peptide (AMP) described herein in detail and a drug.

[0183] In some embodiments, drugs can be effectively “loaded” onto the AMP described herein, allowing successful delivery of the drug to a desired site, for example, without limitation, a site of infection. In some embodiments, the AMPs described herein are used as “carrier-peptides” for drugs, allowing specific targeting of drugs to a desired organ or site.

[0184] In some embodiments, provided herein is a composition comprising one or more antimicrobial peptides (AMPs) and a drug. In some embodiments, provided herein is a composition comprising one or more antimicrobial peptides (AMPs) and one or more drugs.

[0185] In some embodiments provided herein is a composition comprising the antimicrobial peptide (AMP) described herein and a drug. In some embodiments provided herein is a composition comprising the antimicrobial peptide (AMP) described herein and one or more drugs.Minigene expression of AMPs

[0186] As used herein the term "minigene" refers to a minimal gene fragment that excludes one or more components of a native gene locus but includes the necessary elements for expression of the gene product or some portion of the gene product or a synthetic construct.

[0187] In some embodiments, the pharmaceutical composition comprising the AMP is administered as and expressed from a minigene. In some embodiments, the AMP is administered as and expressed from a minigene. In some embodiments, the AMP is administered as a minigene. In some embodiments, the AMP is expressed from a minigene. In some embodiments, the AMP is expressed from a minigene upon administration to the subject.

[0188] In some embodiments, a minigene encoding the AMP is provided in a pharmaceutical composition and / or administered to a subject. In some embodiments, the minigene comprises a nucleic acid sequence encoding the AMP. In some embodiments, the minigene comprises a nucleic acid sequence encoding one or more AMPs. In some embodiments, the minigene comprises a nucleic acid sequence encoding one or more AMPs.

[0189] In some embodiments, the term "minigene" may encompass a minimal gene fragment that excludes one or more components of a native gene locus but includes the necessary elements for expression of the gene product or some portion of the gene product or a synthetic construct. In some embodiments, the minigene excludes at least one intron, or portion thereof. In some embodiments, the minigene comprises at least one intron, or portion thereof. In some embodiments, the minigene comprises at least some regulatory sequence that controls or enhances the expression of the minigene transcript. In some embodiments, the minigene regulatory sequence comprises a promoter. An artisan would appreciate that many promoters are available and suitable for use in a minigene, and the selection of such a minigene promoter will depend on, for example, the desired expressionlevel of the minigene transcript, the desired control of minigene expression, the desired size of the overall mini gene, the intended use of the mini gene, including the subject to which the minigene may be delivered. In some embodiments, the minigene comprises a native promoter, a non-native, a heterologous promoter, a minimal promoter, a minipromter, a constitutive promoter, a tissue specific promoter, an inducible promoter, a synthetic promoter, and the like. dTAG expression of AMP-containing proteins

[0190] In some embodiments, the pharmaceutical composition comprising the AMP is administered as and expressed from a vector comprising a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG) and a gene encoding an AMP-containing protein. In some embodiments, a vector comprising a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG) is in -frame in a 5' or 3' orientation with the AMP-containing protein.

[0191] In some embodiments, the AMP is expressed from a vector comprising a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG) and a gene encoding an AMP-containing protein. In some embodiments, the AMP-containing protein is expressed from the vector upon administration to the subject and is targeted to the proteosome.

[0192] In some embodiments, the expression of the nucleic acid sequence produces a protein - dTAG fusion protein. In some embodiments, the protein-dTAG fusion protein is ubiquitinated and then degraded by the proteasome. In some embodiments, the expression of the nucleic acid sequence is inducible. In some embodiments, the degradation of the protein-dTAG fusion protein by the proteosome, generates AMPs.

[0193] In some embodiments, the heterobifunctional compound targeting protein comprises any amino acid sequence to which a heterobifunctional compound can be bound, leading to the ubiquitination and degradation of the expressed protein-dTAG fusion protein when in contact with the heterobifunctional compound.

[0194] In some embodiments, the dTAG is derived from cytosolic signaling protein FKBP12. In some embodiments, the dTAG is a modified or mutant cytosolic signaling protein FKBP12.

[0195] Any vector known in the art is suitable for the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector can be, but is not limited to, a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector (AAV), a lentiviral vector, or any combination thereof.

[0196] A “fusion” or “hybrid” protein is a protein in which two or more polypeptides are linked, preferably covalently.

[0197] In some embodiments, the AMP -containing protein is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB IB, RPLPO, EIF4A1, GAPDH and RPS29 (Table 2).

[0198] In some embodiments, a heterobifunctional compound can bind the protein-dTAG fusion protein and recruit an E3 ligase e.g. the cereblon-containing CRL4A E3 ubiquitin ligase complex. This recruitment induces ubiquitination of the protein-dTAG fusion protein (on either the dTAG or on the AMP -containing protein) and subsequent degradation via the ubiquitin proteasome pathway. Through this approach the AMP -containing protein can be targeted for rapid ubiquitin mediated degradation to produce AMPs. In some embodiments, the heterobifunctional compound capable of binding the dTAG and degrading the protein-dTAG fusion protein is further administered to the subject. In some embodiments, the pharmaceutical composition further comprises a heterobifunctional compound. In some embodiments, the heterobifunctional compound comprises an endogenous heterobifunctional compound. In some embodiments, the heterobifunctional compound comprises any heterobifunctional compound capable of binding to a dTAG to induce degradation. In some embodiments, the heterobifunctional compound comprises any PROTAC capable of targeting a molecule to degradation thereby generating AMPs. Heterobifunctional compounds are generally known in the art.

[0199] In some embodiments, the term “binding” may encompass the non-covalent interaction between macromolecules.PROTACs targeting AMP-containing proteins

[0200] In some embodiments, provided herein is a PROteolysis TArgeting Chimera (PROTAC) comprising an E3 Ubiquitin Ligase binding moiety and a moiety that binds a target protein coupled together by a linker. In some embodiments, the target protein comprises an AMP-containing protein.

[0201] In some embodiments, provided herein is a PROteolysis TArgeting Chimera (PROTAC) comprising an E3 Ubiquitin Ligase binding moiety and a moiety that binds an AMP-containing target protein coupled together by a linker. In some embodiments, the present disclosure provides PROTACs useful for generating AMPs by inducing the degradation of AMP-containing target proteins.

[0202] In some embodiments, the linker is of a length appropriate to bring together the AMP- containing target protein and ubiquitinating machinery and thereby elicit the ubiquitination of the AMP-containing target protein and its subsequent degradation in the proteasome.

[0203] In some embodiments, the E3 Ubiquitin Ligase binding moiety comprises any E3 Ubiquitin Ligase binding moiety, including those that bind cereblon. In some embodiments, the E3 Ubiquitin Ligase binding moiety comprises a VHL binding compound, a CRBN binding compound, an IAP binding compound or a MDM2 binding compound.

[0204] In some embodiments, the PROTAC further comprises recruitment of the regulatory subunit of the proteasome. In some embodiments, the PROTAC further comprises recruitment of PSME3.

[0205] In some embodiments, the AMP -containing target protein is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB1B, RPLPO, EIF4A1, GAPDH and RPS29 (Table 2).

[0206] In some embodiments, provided herein is a method of treating or preventing a microbial infection in a subject in need thereof, the method comprising administering to the subject the PROTAC described herein. In some embodiments, provided herein is a PROTAC comprising an E3 Ubiquitin Ligase binding moiety and a moiety that binds an AMP -containing target protein coupled together by a linker, for use in the treatment of a bacterial infection.

[0207] In some embodiments, the bacterial infection is selected from the group consisting of Enterobactericicecie, Pseudomonadcicecie Bacteroidcicecie, Streptobacillus, and Acinetobacter .

[0208] In some embodiments, the Gram-negative bacteria is selected from the group consisting of Escherichia, Salmonella, Shigella, Citrobacter, Edwardsiella, Enterobacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Serratia, Yersinia, Staphylococcus, Pseudomonas, Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas, Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella, Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella, Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae, in particular Treponema and Borrelia, Leptospiraceae, Campylobacter, Helicobacter, Spirillum, Bacteroides, Fusobacterium, Prevotella, Porphyromonas, A. baumanii. Mycobacterium avium, Mycobacterium tuberculosis, and Listeria monocyte.

[0209] In some embodiments, the bacterial infection is a Gram-positive bacterial infection.

[0210] In some embodiments, the Gram -positive bacteria is selected from the group consisting of Staphylococcus haemolyticus, Micrococcus luteus, Mycobacterium, Pseudomonas aeruginosa, and Staphylococcus aureus.

[0211] In some embodiments, the bacterial infection is an infection of the skin, of soft tissues, the respiratory system., the lung, the digestive tract, the eye, the ear, the nasopharynx, the bones, and / or the vagina.

[0212] In some embodiments, provided herein is a method of treating a wound or skin condition in a subject in need thereof, the method comprising administering to the subject the PROTAC described herein. In some embodiments, provided herein is a PROTAC comprising an E3 Ubiquitin Ligase binding moiety and a moiety that binds an AMP -containing target protein coupled together by a linker, for use in treating a wound or skin condition.

[0213] In some embodiments, the skin condition is selected from the group consisting of: wound, dermatological disorder, psoriasis, ichthyosis, sarcoidosis, impetigo, acne (including hidradenitis suppurativa), bums, diaper rash, Netherton's syndrome, actinic keratosis, dermatomycoses,dermatosis or ectodermal dysplasia, atopic dermatitis, contact dermatitis, seborrheic dermatitis, vulgaris, filaggrin deficiency, allergic dermatitis, dandruff, pemphigus vulgaris, lichen planus, scleroderma, dermatomyositis, alopecia, skin carcinoma, melanoma, eczema, squamous cell carcinoma, acne vulgaris, erythema toxicum neonatorum, folliculitis, autoimmune bullous skin disease, bullous pemphigoid, pemphigus foliaceus, dermatitis and other disorders associated with damage or breakdown of the skin.

[0214] In some embodiments, provided herein is a method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need thereof, the method comprising administering to the subject the PROTAC described herein. In some embodiments, provided herein is a PROTAC comprising an E3 Ubiquitin Ligase binding moiety and a moiety that binds an AMP -containing target protein coupled together by a linker, for use in treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis.Preserving food products

[0215] In some embodiments, the present invention provides a method of preserving a food product, comprising applying a composition comprising the antimicrobial peptide (AMP) described herein to the food product. In some embodiments, provided herein is a composition comprising one or more AMPs for use in preserving food products. In some embodiments, provided herein is an antimicrobial peptide (AMP) as described herein in detail, for use in preserving food products.

[0216] In some embodiments, the term “food product” encompasses all foodstuffs including beverages. In some embodiments, the food product is coated with the AMP composition. In some embodiments, the food product is mixed with the AMP composition.

[0217] In some embodiments, the food product is selected from fruits, vegetables, legumes, beverages, starch food products, wheat products, dairy products, processed food products, aquatic products, and meat products. In some embodiments, the food product is a fresh food.

[0218] In some embodiments, the food product is fruit. In some embodiments, the food product is a vegetable. In some embodiments, the food product is a legume. In some embodiments, the food product is a beverage. In some embodiments, the food product is a starch food product. In some embodiments, the food product is a wheat product. In some embodiments, the food product is a dairy product. In some embodiments, the food product is a processed food product. In some embodiments, the food product is an aquatic product. In some embodiments, the food product is a meat product.

[0219] In some embodiments, the food product is a raw meat product. In some embodiments, the raw meat products including meat of cattle, swine, goats, sheep, deer, buffaloes; yaks; or the like, and also including poultries: chicken, fowls, ducks, turkeys, guinea fowls; or the like. In some embodiments, applying a composition comprising AMPs promotes sterilization of the raw meat and that can preserve the raw meat.

[0220] In some embodiments, the composition comprising AMPs can be applied by a variety of means. In some embodiments, the composition can be applied by spraying food. In some embodiments, the food could be dipped, coated, and the like. In some embodiments, devices or articles touching the food during preparation or processing can be treated using the present compositions.

[0221] In some embodiments, provided herein is a method for the treatment or prevention of bacterial contamination comprising applying a composition comprising the antimicrobial peptide (AMP) described herein to food processing equipment, food packaging material, or surfaces coming into contact with food.

[0222] The pharmaceutical compositions provided herein may also include other pharmaceutically-acceptable ingredients known to those skilled in the art, including, but not limited to, pharmaceutically-acceptable carriers, adjuvants, additives, excipients, diluents, fdlers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, and colouring agents. The formulations may further comprise other active agents including, for example, other therapeutic or prophylactic agents.

[0223] As provided herein, “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0224] “Pharmaceutically acceptable excipient, carrier or adjuvant” refers to an excipient, carrier or adjuvant that can be administered to a subject, together with an active ingredient, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compositions provided herein.

[0225] In one embodiment, the pharmaceutical composition of the present disclosure is administered in a therapeutically effective amount. In one embodiment, a “therapeutically effective amount” is intended to include an amount of the pharmaceutical composition effective to treat, improve the condition or inhibit the decline of a disease. In one embodiment, a “therapeutically effective amount” of the pharmaceutical composition of the disclosure is that amount of the composition which is sufficient to provide a beneficial effect to the subject being administered.

[0226] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0227] In the present disclosure the singular forms “a”, “an”, and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment incudes from the one particular and / or to the otherparticular value. Similarly, when values are expressed as approximations, by use of the antecedent “about”, it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In some embodiments, the term “about”, refers to a deviance of between 0.0001-5% from the indicated number or range of numbers . In some embodiments, the term “about”, refers to a deviance of between 1-10% from the indicated number or range of numbers. In some embodiments, the term “about”, refers to a deviance of up to 25% from the indicated number or range of numbers. The term “comprises” means encompasses all the elements listed, but may also include additional, unnamed elements, and it may be used interchangeably with the terms “encompasses”, “includes”, or “contains” having all the same qualities and meanings. The term “consisting of’ means being composed of the recited elements or steps, and it may be used interchangeably with the terms “composed of’ having all the same qualities and meanings.

[0228] It should be understood that the disclosure presented herein is not limited to the particular methodologies, protocols and reagents, and examples described herein. The terminology and examples used herein is for the purpose of describing particular embodiments only, for the intent and purpose of providing guidance to the skilled artisan, and is not intended to limit the scope of the disclosure presented herein.EXAMPLESEXAMPLE 1: Materials and Methods

[0229] Bioinformatic analysis of AMPs

[0230] All the computational analyses presented were done using Python 3.11 or R 4.3. 1. Python Packages used: beautifulsoup4 ver.4.12.2, bio ver.1.6.2, gseapy, ver.1.1.0, matplotlib ver.3.7.1, numpy ver. 1.24.3, pandas ver.2.0.2, scipy ver. 1.10.1, seaborn ver.0.12.2, skleam ver.0.0.post5, urllib3 ver.2.0.3. R Libraries used: BiocManager 1.30.22, circlize 0.4.15, ComplexHeatmap 2.16.0, drawProteins 1.20.0, dplyr 1.1.2, ggplot2 3.4.4, ggnewscale 0.4.10, ggrepel 0.9.4, PerformanceAnalytics 2.0.4, RColorBrewer 1.1-3, stringr 1.5.1, tidyr 1.3.0, tidyverse 2.0.0, ggplot2 3.4.4

[0231] Data retrieval and collection of experimentally-validated AMPs

[0232] Antimicrobial peptide (AMP) data were retrieved from four publicly available databases: CAMP(R4), DRAMP, dbaasp, and dbAMP. Data retrieval was conducted in July 2023, utilizing the respective web interfaces of each database. The databases were accessed through the following URLs: CAMP(R4) (https: / / camp.bicnirrh.res.in), DRAMP (http: / / dramp.cpu-bioinfor.org), dbaasp (https: / / dbaasp.org / home), and dbAMP (http: / / csb.cse.yzu.edu.tw / dbAMP / index.php).

[0233] For each database, AMP sequences were downloaded along with associated metadata, including but not limited to Protein ID, Sequence, organism source, and activity. The downloadeddatasets were saved in commonly used formats compatible with bioinformatics analysis, such as FASTA or comma-delimited text fdes. Following data retrieval, filtering was implemented to isolate natural AMP sequences from the downloaded datasets. Natural AMPs were defined as peptides derived from naturally occurring sources, excluding synthetic or engineered peptides.

[0234] BLAST Analysis

[0235] The combined dataset of AMPs underwent sequence similarity analysis using the Basic Local Alignment Search Tool (BLAST) version 2.2.26. This analysis was conducted against the human proteome database obtained from SWISS -PROT as of September 2023.

[0236] Peptides exhibiting a complete sequence identity match with human proteins were identified and subsequently retained for further analysis. To ensure stringent criteria, peptides shorter than five amino acids were excluded from the dataset to maintain consistency and reliability in subsequent analyses.

[0237] The analysis unveiled AMPs that had not been previously associated with human sources but have now been authenticated as constituents of the human proteome. The BLAST analysis was conducted against the entire SWISS -PROT database as well as the bacteria proteome specifically.

[0238] AMP accessibility analysis

[0239] To analyze the position of known AMPs within the structures of mature proteins, we used AlphaFold-predicted monomeric structures for human proteome (uploaded 14.01.24). For each protein that includes a known AMP sequence within its polypeptide chain, we calculated a relative solvent-accessible surface area (rASA) using the FreeSASA library. The predicted local distance difference test (pLDDT) of amino acids from AlphaFold was used to measure residue-wise disordemess (the more disordered region, the smaller the pLDDT score). To demonstrate the general trends for amino acids within AMP, we compared the rASA and pLDDT of amino acids within and outside of AMP regions for the whole set of AMP -containing proteins by the Mann-Whitney U-test.

[0240] AMP conservation analysis

[0241] For AMP conservation analysis, we used orthogroups of the Euarchontoglires subclade which include orthologs for H. sapiens. For every orthogroup, we aligned the protein sequences with MUSCLE (v5) using default parameters and quantified the amino-acid conservation rate by Rate4Site. To identify AMPs, which consist of more conservative amino acids than other parts of the protein, we compared per amino acid Rate4Site for amino acids within AMP with other amino acids from the same protein by Mann-Whitney U-test with further adjustment for multiple comparisons by Benjamini- Hochberg.

[0242] Analysis of AMP Gene expression in different tissues

[0243] RNA-seq data for human tissues (RNA HPA tissue gene data) were obtained from the Human Proteome Atlas website, with the dataset corresponding to January 2024. We identified genes encoding known Antimicrobial Peptides (AMPs) present in the human genome. These genes were thencross-referenced with the transcriptomic data obtained from the Human Proteome Atlas dataset to assess their expression profiles across different tissues. Transcript abundance for each AMP gene was determined based on protein-transcripts per million (pTPM) values. The data was normalized per gene by aZ score.

[0244] Enrichment Analysis using KEGG Pathways

[0245] Enrichment analysis employed the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database version 2021 and GO Molecular Function version 2023. To execute this analysis, the GSEAPY Python package vO. 10.5 was utilized. Appropriate statistical tests, consistent with enricher methodology, were applied, with a predetermined significance threshold set at a = 0.05. The proportion of genes associated with the identified pathways was compared to the entire gene repertoire cataloged in the SWISS-PROT database as of September 2023.

[0246] Bioinformatic analysis of potential proteasome-derived AMPs within human degradome

[0247] Scoring proteasome-derived peptides for their pore-forming probability

[0248] To get the list of potential AMPs produced by proteasome, data was accumulated from multiple mass spectrometry analysis of proteolytic peptides (MAPP) (Wolf-Levy, H. et al. Revealing the cellular degradome by mass spectrometry analysis of proteasome -cleaved peptides. Nat. Biotechnol., 2018) experiments conducted in the laboratory. Subsequently, this dataset underwent filtering utilizing scoring criteria derived from MaxQuant analysis, retaining only peptides that met specific thresholds. Following the filtration process, all peptides within the dataset were subjected to a scoring algorithm proposed by Pane et al. (Pane, K. et al. Antimicrobial potency of cationic antimicrobial peptides can be predicted from their amino acid composition: application to the detection of “cryptic” antimicrobial peptides. J. Theor. Biol. 419, 254-265, 2017), as implemented by Torres and colleagues (Torres, Marcelo DT, et al. "Mining for encrypted peptide antibiotics in the human proteome." Nature Biomedical Engineering 6.1 (2022): 67-75).

[0249] In silico proteasome cleavage of the human proteome

[0250] To get the whole list of AMPs potentially produced by proteasome, the entire human proteome downloaded from the SWISS-PROT was used (Sep 23), ensuring a comprehensive representation of protein sequences for analysis. This dataset served as the primary source for identifying potential AMPs. To identify potential proteasomal cleavage sites within protein sequences, the Pepsickle algorithm was employed, a specialized tool tailored for this purpose (Weeder, B. R., Wood, M. A., Li, E., Nellore, A. & Thompson, R. F. pepsickle rapidly and accurately predicts proteasomal cleavage sites for improved neoantigen identification. Bioinformatics 37, 3723-3733, 2021). Pepsickle utilizes advanced computational techniques to predict sites susceptible to proteasomal cleavage with high accuracy. The results obtained from pepsickle were utilized to generate peptide combinations ranging from 7 to 50 amino acids in length. These combinations represent putative proteasome-generated peptides and were further analyzed.

[0251] The identified peptides were cross-referenced with previously reported AMPs toascertain their potential efficacy. Mean scores were computed for peptides present in the known datasets to choose the minimal AMP score as a threshold for PDDPs (excluding score of zero) Peptides scoring above the threshold of 5, representing the mean score of reported AMPs, were considered as PDDPs.

[0252] Peptides exhibiting cationic cleavage properties were identified. Overlapping peptides were analyzed to identify the most promising candidates, considering both individual peptide scores and their collective contribution to antimicrobial properties.

[0253] Human cell lines

[0254] HCT116 and A549 and MDA MB231 cell lines were obtained from ATCC (CC1-185). A549 and HCT116 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; 41965039; Gibco) or McCoy's 5 A Medium (M8403; Sigma) respectfully, supplemented with 10% Fetal Bovine Serum (F7524; Sigma), Sodium Pyruvate (03-042; Sartorius) and Glutamine (03-020; Sartorius). Cells were cultured and passed in accordance with standard procedures at +37°C with 5% CO2. The absence of mycoplasma contamination was verified monthly.

[0255] Bacterial strains

[0256] Pseudomonas aeruginosa strain CHA-OST, Pseudomonas aeruginosa PAO1, Escherichia coli kl2, Micrococcus luteus NCTC 2665, Staphylococcus haemolyticus JCSC1435, Salmonella enterica suhsp. enterica and Salmonella typhimurium SL1344 were used. All the bacteria were cultured in Lysogeny broth (LB; Sigma) according to standard procedures.

[0257] Assessment of Minimal Inhibitory Concentration (MIC)

[0258] Bacteria overnight cultures were diluted in LB to 10A5 bacteria and treated with serial dilution (2 fold) of the peptides. The treated bacteria were incubated over 15h in 37c with breathing tape. OD was assured using the plate reader following XTT (Biological industries) 50 ul ofthe mix was added to per well.

[0259] Peptide synthesis

[0260] To access the antibacterial activity of selected MAPP-derived peptides, which were predicted to have antibacterial activity, we used chemically synthesized peptides with the same amino acid sequences ordered in GenScript. The sequences of peptides used in this study are presented in Table 2.

[0261] Bacterial killing assay

[0262] The bacterial culture was prepared as described previously and washed 3 times with phosphate-buffered saline (PBS; Sartorius). AMPs were diluted to a concentration of 32 ug / ml. Bacteria were then introduced to the AMPs with a bacterial load of 5x107CFU / ml. The plate was incubated for 1 hour at 37°C. Each treatment was serially diluted 10 -fold in PBS and spot-plated in LB -agar plates.

[0263] Membrane permeabilization assay

[0264] To measure the effect of putative AMPs on bacterial and human cell lines (A549 andHCT116) viability and permeabilization we used propidium iodide (PI; Sigma) assay. For this, bacteria were grown to midlogarithmic phase, washed with assay buffer (10 mM Mes pH 5.5, 25 mM NaCl; Sigma), diluted into assay buffer to 5xl08cells / ml, and mixed with PI to 5.5 pg / ml (8.3 pM) final concentration. 100 pl of bacteria with PI were transferred to 96-well plates and mixed with putative AMPs in various concentrations and with negative (DMSO) and positive (0.05% SDS; Bio-Lab) controls. Then the plates were incubated for 2 hours at 37°C in a fluorescent reader and the fluorescence output (excitation, 535 run; emission, 617 run) was measured every 5 min. Bacterial permeabilization activity is measured against the maximum fluorescence output from the positive control. For human cell lines, cells were collected and washed 2 times in PBS then suspended in PBS containing PI 5.5 pg / ml (8.3 pM). Measurements were done the same as for the bacteria.

[0265] Transmission electron microscopy

[0266] To directly assess the putative AMPs pore-forming ability we used transmission electron microscopy (TEM). For this, the bacteria were grown to midlogarithmic phase, incubated with the putative AMPs for 30 minutes and fixed with 4% paraformaldehyde, 2% glutaraldehyde (EMS) in 0.1 M cacodylate buffer (Sigma) containing 5 mM CaCL (pH 7.4), postfixed in 1% osmium tetroxide (EMS) supplemented with 0.5% potassium hexacyanoferrate tryhidrate and potassium dichromate (BDH chemicals) in 0.1 M cacodylate for 1H, stained with 2% uranyl acetate (EMS) in double distilled water for 1H, dehydrated in graded ethanol solutions and embedded in epoxy resin (Agar scientific Ltd.). Ultrathin sections (70 run) were obtained with a Leica EMUC7 ultramicrotome and transferred to 200 mesh copper transmission electron microscopy grids (SPI). Grids were stained with lead citrate and examined with a Tecnai T12 transmission electron microscope (Thermo Fisher Scientific). Digital electron micrographs were acquired with a bottom -mounted TVIPS TemCam-XF416 4k x 4k CMOS camera.

[0267] Bacterial growth in conditioned medium

[0268] .S', enterica, S. typhimurium, M. luteus and Staphylococcus haemolyticus were used for an extracellular infection in which the effect of conditioned medium low molecular weight fraction on bacterial growth was estimated. For this, cells were cultured until 90% confluency in standard media without antibiotics and then the conditioned media was filtered through ultrafiltration filters with a lOkDa cutoff (PES; 964014; Analytical Sales and Services or Vivaspin; VS0202). Then, 100 ul of the filtered conditioned media were mixed with the bacteria prediluted in DMEM to obtain the final bacterial dilution ~5xl07bacteria / ml, (for Micrococcus luteus 5xl06CFU / ml). The bacteria were incubated in 96 well plates at the plate reader for 2h or 4 h, and the OD 595 was measured every 10 minutes with shaking.

[0269] Intracellular cell infection assay

[0270] .S', typhimurium and M. luteus were used for intracellular infection in which the ability of bacteria to infect the human cell lines was estimated. An overnight culture of bacteria was grown to the mid-logarithmic stage in fresh LB media with 0.3M NaCl.Bacteria were washed 3 times withphosphate-buffered saline (PBS; Sartorius) and added to human cells with 100 or 15 Multiplicity of Infection (MOI). Then, the cells were incubated with bacteria for 1 hour, washed twice with PBS, and incubated for 1 hour in DMEM with Gentamycin (100 ug / ml final concentration). After incubation, cells were washed twice with PBS and lysed with 0.1% Triton X-100 (Sigma) solution for 15 minutes. Then, the cell lysate-containing bacteria was transferred to LB Agar plates with a serial 10-fold dilution.

[0271] Targeted protein degradation assay

[0272] PPP1CB and eGFP were cloned from pEGFP(N3)-PPlbeta (plasmid #44223, addgene) intoN terminal dTAG plasmid pLEX_305-N-dTAG (plasmid #91797, addgene) cut by restriction enzymes clal and agel followed by ligation with T4 ligation enzyme (NEB). Stable cell lines were generated with 3rd generation lentiviral infection. Cells were treated with 500nM of the dTAG V-l compound (Tocris #6914) for 6 hours for conditioned media experiments or 1-hour pre-infection with intracellular bacteria and DMSO was used as a control. The efficiency of d-TAG degradation was assessed by western blot against HA-tag using the anti-HA-tag antibody (SAB2702196; Sigma) and anti-vinculin antibody as a loading control (abl29002; Abeam). Bacterial infection was assessed in the medium and cells as described above.

[0273] PSME3 knockout

[0274] To access the role of PSME3 (PA28y) in generating proteasome -derived antibacterial peptides, we performed the experiments with its knockdown by MISSION short hairpin RNAs targeting human PSME3, or RFP, as a control obtained from Sigma (TRCN0000290025, TRCN000002209). The plasmids were stably expressed in A549 and then the cells were used for bacteria-killing assay as described above.

[0275] Immunoblotting

[0276] Protein concentration was assessed using a BC Assay Protein Quantitation Kit (Interchim). First, 20 pg of total protein was separated by SDS-PAGE on a 4-20% gradient 4-20% Criterion™ TGX™ protein gels (Bio-Rad) and transferred onto NC membranes using an iBlot 2 Gel Transfer Device (Thermo Fisher Scientific). The membranes were blocked in 5% milk prepared in TBS-0.1% Tween 20 (Sigma) and incubated in primary antibodies overnight at 4 °C, followed by washing and incubation with secondary antibody. Blots were developed using the ChemiDoc XRS+ Imaging System (Bio-Rad).

[0277] Mass-spectrometry analysis of proteasomal-cleaved peptides

[0278] Cell infection for MAPP analysis

[0279] A549 cells were seeded and grown to 90% confluency and washed 2 times with phosphate- buffered saline (PBS; Sartorius). Then cells were incubated in DMED without serum with or without .S', enterica bacteria (MOI 15) for 1 or 4 hours then washed and collected for processing.

[0280] Proteasome immunoprecipitation

[0281] MAPP was performed as previously described (Javitt, A. et al. The proteasome regulator PSME4 modulates proteasome activity and antigen diversity to abrogate antitumor immunity inNSCLC. Nat Cancer, 2023; Wolf-Levy, H. et al. Revealing the cellular degradome by mass spectrometry analysis of proteasome-cleaved peptides. Nat. Biotechnol., 2018). Cells were lysed with 25 mMHEPES, pH 7.4, 10% glycerol, 5 mM MgC12, l mMATP, and 1:400 protease-inhibitor mixture (Calbiochem), then homogenized through freeze-thaw cycles and passed through a needle. The lysates were cleared by 30-min centrifugation at 21,130g at 4 °C. Pellets were lysed again with 0.5 mM ammonium persulfate to enrich the nuclear fraction, followed by centrifugation. Mixed lysates were cross-linked as previously described. For immunoprecipitation, the lysates were then incubated with Protein G-Sepharose beads (Santa Cruz) with antibodies to PSMA1 and eluted with 100 mM Tris-HCl, pH 8, 8M urea, and 50 mM DTT for 30 min at 37 °C. Elution fractions were analyzed by SDS-PAGE to evaluate yield and purity.

[0282] Purification and concentration of proteasome peptides

[0283] A critical step in the procedure is the separation of peptides from the proteins eluted in the proteasome pulldown. MAPP analyzes endogenously cleaved peptides, whereas the proteasome complex and associated proteins are physically excluded. Immunoprecipitated proteasomes and their encompassed peptides were loaded on C18 cartridges (Waters) that were prewashed with 80% acetonitrile (ACN) in 0.1% TFA and then washed with 0.1% TFA only. After loading, the cartridges were washed with 0.1% TFA. Peptides were eluted with 30% ACN in 0.1% TFA.

[0284] Assessing proteasome composition

[0285] Following proteasome immunoprecipitation, proteasomes were denatured by 8M Urea for 30 min at room temperature, reduced with 5 mM dithiothreitol (Sigma) for Ihr at room temperature, and alkylated with 10 mM iodoacetamide (Sigma) in the dark for 45 min at room temperature. Samples were diluted to 2M urea with 50 mM ammonium bicarbonate. Proteins were then subjected to digestion with trypsin (Promega; Madison, WI, USA) overnight at 37°C at 50: 1 protein: trypsin ratio, followed by a second trypsin digestion for 4 hr. The digestions were stopped by the addition of trifluoroacetic acid (1% final concentration). Following digestion, peptides were desalted using Oasis HLB, pElution format (Waters, Milford, MA, USA). The samples were vacuum -dried and stored at -80°C until further analysis.

[0286] Total protein isolation

[0287] Lysates in 5% SDS in 50 mM Tris-HCl were incubated at 96 °C for 5 min, followed by six cycles of 30 s of sonication (Bioruptor Pico, Diagenode, USA). Proteins were reduced with 5 mM dithiothreitol and alkylated with 10 mM iodoacetamide in the dark. Each sample was loaded onto S- Trap microcolumns (Protifi, USA) according to the manufacturer’s instructions. In brief, after loading, samples were washed with 90: 10% methanol / 50 mM ammonium bicarbonate. Samples were then digested with trypsin for 1.5 h at 47 °C. The digested peptides were eluted using 50 mM ammonium bicarbonate; trypsin was added to this fraction and incubated overnight at 37 °C. Two more elutions were made using 0.2% formic acid and 0.2% formic acid in 50% acetonitrile. The three elutions were pooled together and vacuum -centrifuged to dry. Samples were kept at -80 °C until analysis.

[0288] Liquid chromatography-mass spectrometry

[0289] ULC / MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nano-Ultra Performance Liquid Chromatography (10 kpsi nanoAcquity; Waters, Milford, MA, USA). The mobile phase was: A) H2O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed -phase Symmetry Cl 8 trapping column (180 pm internal diameter, 20 mm length, 5 pm particle size; Waters). The peptides were then separated using a T3 HSS nano-column (75 pm internal diameter, 250 mm length, 1.8 pm particle size; Waters) at 0.35 pL / min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 35%B in 120 min, 35% to 90%B in 5 min, maintained at 90% for 5 min, and then back to initial conditions.

[0290] The nanoLC (Ultimate3000, Thermo Scientific) was coupled online through a nESI emitter (10 pm tip; FossillonTech) to a quadrupole Orbitrap mass spectrometer (Exploris480, Thermo Scientific).

[0291] Data was acquired in data-dependent acquisition (DDA) mode, using a Top 10 method. MS 1 resolution was set to 70,000 (at 400m / z), a mass range of 375-1650m / z, AGC of 3e6, and a maximum injection time was set to 100msec. MS2 resolution was set to 17,500, quadrupole isolation 1.7m / z, AGC of le5, dynamic exclusion of 40 sec, and a maximum injection time of 150 msec.

[0292] Mass spectrometry data analysis and label-free quantification

[0293] Raw data were analyzed in MaxQuant software (version 1.6.0.16) with the default parameters for the analysis of the proteasomal peptides, except for the following: unspecific enzyme, LFQ minimum ratio count of 1, minimum peptide length for unspecific search of 6, maximum peptide length for unspecific search of 40, and match between runs enabled. A stringent false discovery rate (FDR) of 1% was applied for peptide identification. For the analysis of tryptic digests, the default parameters were set, apart from a minimum peptide length of 6. Masses were searched against the human proteome database from UniProtKB (last update April 2020).

[0294] Peptides resulting from MaxQuant were initially filtered to remove reverse sequences and known MS contaminants. For the MAPP peptide fraction, we removed antibody and proteasome peptides as contaminants. To decrease ambiguity, we allowed peptides that had at least two valid label- free quantification intensities out of the sample replicates, and we included razor peptides, which belong to a unique MaxQuant ‘protein group’. MAPP protein intensities were inferred with MaxQuant. For graphical representation, intensities were log -transformed, and zero intensity was imputed to a random value chosen from a normal distribution of 0.3 s.d. and downshifted 1.8 s.d.

[0295] Peptide Cleavage Analysis

[0296] An analysis was conducted on proteasomal -derived peptides by MAPP described above. For each peptide, its absence or presence in each sample was annotated (scored 0 or 1), and the C- terminal amino acid was determined (N -terminal amino acid is the amino acid before the peptide start / cleavage site). Cysteines were not quantified for their frequency as they may be affected by thecrosslinker. Per sample, the relative frequency of each amino acid was calculated and standardized on the amino acid level for the heatmap representation (z-scores). Heatmaps were generated with the ComplexHeatmap (v2.18.0) package with row clustering using euclidean distances.

[0297] Proteasome activity assay on proteasome pulldowns

[0298] MAPP was performed as previously described (Javitt, A. et al. The proteasome regulator PSME4 modulates proteasome activity and antigen diversity to abrogate antitumor immunity in NSCLC. Nat Cancer, 2023; Wolf-Levy, H. et al. Revealing the cellular degradome by mass spectrometry analysis of proteasome-cleaved peptides. Nat. Biotechnol., 2018). Cells were lysed with 25 mMHEPES, pH 7.4, 10% glycerol, 5 mM MgC12, l mMATP, and 1:400 protease-inhibitor mixture (Calbiochem), then homogenized through freeze-thaw cycles and passed through a needle. The lysates were cleared by 30-min centrifugation at 21,130g at 4 °C. Protein concentration was assessed using a BC Assay Protein Quantitation Kit (Interchim). For immunoprecipitation, the lysates were incubated with Protein G-MagBeads (GeneScript) with antibodies to PSMA1 Proteasome subunit, in a Black 96 well plate and incubated overnight on an orbital shaker. The next day the beads were washed three times in PBS and incubated in a reaction buffer (50 mM HEPES, pH 7.5, 1 mM dithiothreitol, 5 mM MgC12, and 2 mM ATP). Proteasome activity was determined by cleavage of the Anorogenic precursor substrates Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC) and Ac-Arg-Leu-Arg-AMC (Ac-RLR- AMC)(Bachem). Fluorescence increase resulting from degradation of peptide-AMC at 37 °C was monitored over time by means of a duorometer (Synergy Hl Hybrid Multi-Mode Microplate Reader, BioTek) at 340 nm excitation and 460 nm emission. The resulting product curves were followed for up to 3.5 hours. Each duorescence intensity value represents a mean value obtained from three or more independent experiments.

[0299] Mouse models of Pseudomonas acute pneumonia and bacteremia

[0300] For the acute pneumonia model, CD-I mice (n = 8 / group) were intranasally-inoculated with the indicated concentration of the P. aeruginosa strain PAO1. Infected mice were treated twice daily with intravenous injection of 5 mg / kg of PPP1CB. Lungs were harvested and processed at 48 hours post-infection (hpi). For the bacteremia model, CD-I mice (n = 8 / group) were intraperitoneally infected with P. aeruginosa strain PAO1(2.5 x 10A7 CFU / mouse). Infected mice were treated twice daily with intravenous injection of 5 mg / kg of PPP1CB, and spleens were analyzed at 24-hpi. The control group, in both mouse models, was treated with the same volume of sterile PBS or the antibiotic tobramycin (50 mg / kg, intraperitoneal, once daily). Mouse tissues were homogenized in 1 mb sterile PBS using an Omni Sod Tissue Tip™ Homogenizer (Genizer LLC, Irvine, CA, USA). Bacterial burden was determined through serial dilution plating of the homogenate onto LB agar plates . For the mouse model of bacteremia-induced sepsis mortality, CD-I mice (7-week-old, males and females, 15 per cohort) were anesthetized by isodurane and intraperitoneally inoculated with a higher dose of P. aeruginosa strain PAO1 (6.7 x 10A7 CFU / mouse, in 100 pL), which resulted in bacteremia-derived sepsis mortality. Infected mice were treated twice daily retro -orbitally beginning at 2-hpi, for 3 days withPPP1CB IV (10 mg / kg, in 50 pl). The antibiotic tobramycin (50 mg / kg once daily, in 50 pl) and the same volume of vehicle (sterile PBS once daily, in 50 pl) were used as both positive and negative controls. Mouse mortality was monitored for 6 days (120 hours). Survival analysis was performed with a Kaplan-Meier Log Rank Survival Test using GraphPad Prism (Version 9.0.2). All collected mouse tissues were submitted for H&E and Gram staining and the pathological scoring was done manually (Extended File 1). The degree of lesions was graded from one to five based on severity: 1 = not present or minimal (< 1%); 2 = slight (1-25%); 3 = moderate (26-50%); 4 = moderate / severe (51-75%); 5 = severe / high (76-100%).EXAMPLE 2: Identification of proteasome degraded AMPs

[0301] Although many AMPs are canonically encoded and translated as intact functional entities, numerous AMPs are embedded within protein sequences and require further post-translational processing for their maturation). To examine the prevalence of AMPs that are contained within human proteins, we compared a list of experimentally validated AMPs to the human proteome. 308 putative unique AMPs (within 273 proteins) identified were identical in sequence (Fig. 1A). When the conservation of the AMP sequence across orthologs was examined and compared to other regions in the host protein, we found that 28.5% of the putative AMPs (28 out of 98 for which data was available) were more conserved (Fig. 1A). AMP -containing proteins included diverse protein classes such as histones and transcription factors. Notably, these proteins exhibited diverse tissue specificities, with some predominantly expressed in one or two tissue types. Importantly, the positions of putative human AMPs were predominantly located within a structured region and their accessibility was slightly reduced compared to all other areas of the protein (Fig. IB and Fig. 1C), indicating that such AMPs may not be readily clipped, without subsequent protein unfolding or degradation. Together, these data suggest that proteasomal degradation may be a novel mechanism for the maturation of AMPs.

[0302] To explore the capacity of protein degradation to yield protective peptides, we artificially cleaved the human proteome using predictive proteasomal cleavage sites. This approach generated a comprehensive database containing approximately 34 million peptides. Next, we scored these peptides based on their pore-forming cationic properties and compared them with the score of experimentally validated AMPs. Indeed, when the human proteome was digested in silico, we found that hundreds of thousands of the resultant peptides have cationic AMP characteristics, compromising around 1.2% of the peptide pool (Fig. ID). Considering that the proteasomal machinery constitutes around 1-2% of the total cellular protein content (by volume; 28-30), this suggests that the proteasome could be a major source of AMPs, potentially endowing cells with an enormous number of protective peptides at a basal state.

[0303] To test the hypothesis that putative AMPs are generated upon degradation of cellular proteins, bacterial infection with intracellular gram -negative bacteria (Salmonella Typhimurium) wastested, with or without prior inhibition of the proteasome. First, a significant increase in intracellular colony forming units (CFU) count was found when proteasome activity was inhibited (Fig. IE). Next, to assess the impact of proteasomal inhibition on the generation of AMPs, the antimicrobial killing activity of the peptide pool (<10 KDa fraction) that is secreted by cells to the extracellular milieu (Fig. IF) was tested. The growth of Escherichia coli, a mainly extracellular gram-negative pathogen, was examined. Inhibiting proteasomal activity enhanced bacterial growth (Figs. 1G-1I). To confirm that the bacterial growth inhibition was mediated by peptide activity, and not by other molecules, such as secreted metabolites, conditioned media was treated with proteinase-K, which is known to efficiently cleave peptides. Indeed, proteinase-K treatment abolished the effect confirming that the attenuated bacterial growth was due to the peptidic component in the conditioned medium (Fig. 1 J and Fig. IK).EXAMPLE 3: Newly identified proteasomal-derived defense peptides (PDDPs) attenuate bacterial growth

[0304] Mass-spectrometry analysis of proteasomal-cleaved peptides (MAPP) (Fig. 2A) is uniquely designed to capture a snapshot of the active degradation landscape. Specifically, it allows for the identification of the degradation products within or near the proteasome barrel, through immunoprecipitation of proteasomes, followed by the elution of the naturally-cleaved protein fragments. To explore the potential downstream functions of MAPP -identified peptides, degradation products were examined from different cell types and across different conditions.

[0305] Most of the AMPs characterized to date are cationic hydrophobic peptides that typically contain 10-50 amino acids in length, and these may be scored for their potential effect on bacterial growth by way of perturbing bacterial membranes.

[0306] To validate the secretion of proteasome-cleaved peptides, peptidomics analysis of the secretome collected from A549 cells was performed. When the MAPP and secretome peptides were compared, 110 peptides (11%) were identified as identical and 383 (40%) were contained within MAPP peptides, which may indicate further processing (Fig. 2C).

[0307] To uncover putative proteasomal -derived defense peptides (PDDPs), identified by MAPP, the cationic scoring system that assesses the biochemical properties of the peptides was used. From 50251 peptides identified in MAPP, peptides that had a score of higher than five (see materials and methods for threshold determination) and obtained with pore-forming properties were selected. The peptides were prioritized based on their scores (Fig. 2B and Fig. 2D, Table 1), and synthesized (Table 2). The top-scored peptides were contained within proteins with different functions such as PPP1CB, a catalytic subunit beta of a serine / threonine -specific phosphatase 1, PSMG2, a chaperone that promotes assembly of the 20S proteasome, DCTN4, dynactin subunit that is associated with the centrosome and DFNA5 also known as Gasdermin E, which is known to promote pyroptosis of mammalian cells.

[0308] Table 1: Representative PDDPs with cationic scoring

[0309] To examine the ability of the MAPP -identified peptides to attenuate bacterial growth, a subset of bacterial species was chosen. Specifically, the peptides were synthesized and their effect on bacterial growth was examined (Table 2). The growth of different strains was tested to assess the effect on diverse types of bacteria, including the gram -positive extracellular opportunistic Staphylococcus haemolyticus, the gram-positive intracellular commensal Micrococcus luteus, gram-negativeextracellular pathogenic Escherichia coli, Salmonella Typhimurium and Salmonella enterica. All bacteria were delayed in a dose-dependent and selective manner (Fig. 2E).

[0310] Table 2: Peptides used in antibacterial tests

[0311] The PPP1CB peptide (SEQ ID NO: 1033) exhibited the most prominent attenuation in the growth of diverse bacterial species (Fig. 2F). Mechanistically, it was suggested that cationic peptides may exert their function by biophysical disruption of bacterial membranes, which contain anionic phospholipids on their surface. To confirm the mechanism of action of the PPP1CB peptide, transmission electron microscope (TEM) images of the different bacteria demonstrate significant membrane aberrations upon treatment with the synthetic peptide. The examined bacterial species displayed membrane rupture and subsequent cytoplasm release (Fig. 2G). Furthermore, we used a propidium iodide assay, reflecting the penetrance of the dye as a function of perturbed membranes, across different bacterial species. Permeabilization of the bacterial membranes and the killing of three bacterial species was demonstrated, in a dose -dependent manner (Fig. 2H-J). Importantly, no detectable effects were observed on the permeabilization of mammalian cells treated with similar concentrations of the peptide, reflecting the selectivity in their activity towards microbial membranes (data not shown).EXAMPLE 4: Targeted protein degradation of PPP1CB attenuates intracellular and extracellular bacterial growth

[0312] Goal: over-expressing the host protein (PPP1CB) and targeting it to proteasomal degradation, to generate a “pool” of its degradation products.

[0313] To show a direct effect of the requirement of proteasomal degradation for generating functional defense peptides, we harnessed the dTAG system which targets a protein of interest, ‘on demand’, to degradation (Fig. 3A). The dTAG is based on a heterobifunctional dTAG molecule that induces binding of FKBPF36V tag and an E3 ubiquitin ligase complex (CRBN). Then, upon treatment with the drug (dTAGV-1), the dTAG system is activated, leading to the complete degradation of PPP1CB (Fig. 3B). Notably, PPP1CB degradation significantly inhibited intracellular bacterial infection, whereas the overexpression of PPP1CB alone, did not (Fig. 3C and Fig. 3D).

[0314] These results exemplify the ability of PPP1CB degradation products to provide intracellular protection against pathogens. The up-regulation and degradation of PPP1CB alone led to 20% attenuation in bacterial growth, when only conditioned medium was collected after activation of the dTAG system (Figs. 3E-3G), supporting that the proteasomal-cleaved peptides can be secreted, as demonstrated above. In all cases, the degradation of GFP, serving as a control, did not affect bacterial infection or growth. These results mechanistically confirm the ability of PDDPs to exert bacterial killing by pore formation in bacterial membranes. Notably, one of the PDDPs derived from PPP1CB shows higher potency in vivo compared to Tobramycin, an antibiotic that is clinically used. The ability of the dTAG system to attenuate bacterial growth supports their use as natural intracellular antibiotics. Other technologies for expressing multiple PDDPs can serve for targeting bacterial infections, across different tissues and human pathologies.EXAMPLE 5: Bacterial infection enhances PDDPs formation by augmenting tryptic-like cleavages of the proteasome

[0315] Goal: test whether PDDPs may be induced upon bacterial infection.

[0316] The degradation landscape was analysed via MAPP, in response to Salmonella enterica infection (Fig. 4A). A significant increase in the frequency of unique proteasome-derived peptides upon infection was found (Fig. 4B), leading to the study of the sequences of the identified peptides. Since no external protease (like trypsin) was used, the cleavage patterns observed reflect the catalytic activity of the proteasome. A significant change in peptide cleavage properties was seen during bacterial infection; the tryptic-like activity of the proteasome, which cleaves cationic residues (K, R, H), was notably enhanced, whereas the chymotryptic-like activity, which produces hydrophobic peptides preferred for MHC I presentation (F, Y, W), was reduced (Fig. 4C).

[0317] Specifically, within an hour of infection, the number of peptides with cationic termini almost doubled compared to the non-infected control (1981 versus 1141 peptides, respectively), while hydrophobic / aromatic termini peptide levels were decreased (1285 in infection versus 1580 in the control). Analysing only proteins identified in both experimental conditions showed a significant increase in the percentage of peptides with cationic C -termini per protein after infection, particularly when analyzing the unique peptide population (Fig. 4D and Fig. 4E). Conversely, for N-termini cleavages, which are not associated with proteasomal cleavage, we did not observe such a phenomenon. This observation suggests that the shift in peptide termini is directly associated with changes in proteasome cleavage patterns and not merely due to the selection of proteins targeted for degradation. We further confirmed the sensitivity of the assay to detect changes in the cleavage activity of different catalytic subunits by pharmacological agents, blocking either the constitutive or the immunoproteasome (data not shown). Inhibiting the constitutive proteasome led to reduced levels of caspase-like cleavages while inhibition of the immunoproteasome reduced both the tryptic-like and chymotryptic-like activities.

[0318] The change in proteasomal activity, upon bacterial infection, was further validated by a Anorogenic assay probing the cleavage activity of different model peptides (Fig. 4F). Proteasomes isolated from cells that were infected with bacteria exhibited increased tryptic-like activity and reduced chymotryptic-like activity compared to those from control cells (Figs. 4H-4J). Overall, these results suggest that during bacterial infection, proteasome activity shifts to favour the generation of cationic peptides through increased trypsin-like catalytic activity.

[0319] Further, an acute pneumonia infection model in mice was utilized (Fig. 2K). The infection of the lung was significantly reduced after 48 hours of treatment with the synthetic PPP1CB peptide (SEQ ID NO: 1033) administrated intravenous (IV), compared to the PBS control. Remarkably, a single peptide, at a concentration of 5 mg / kg (IV) had a comparable effect, albeit less efficacious, to a broad spectrum of activity antibiotics (Tobramycin; administrated at 50 mg / kg (I P)) (Fig. 2L).

[0320] Finally, the PPP1CB peptide was tested in a bacteremia model (intraperitoneal (IP) (Fig. 2M). PPP1CB (SEQ ID NO: 1033) was administered by IV at a concentration of 10 mg / kg, which resulted in significantly reduced bacterial infection after 24 hours, compared to PBS control. Strikingly, in this model, PPP 1 CB had greater efficacy than the antibiotics (T obramycin; administrated at 50 mg / kg (IP)) (Fig. 2N).EXAMPLE 6: PSME3 is upregulated upon bacterial infection and promotes tryptic-like cleavages

[0321] The function of cellular proteasomes is governed not only by the catalytic subunits of the constitutive- and the immune-proteasomes, but also by the binding of the catalytic core to different regulatory subunit caps (i.e., 19S subunits, PSME1-4, and PI31), adding to the heterogeneity ofproteasome populations and their activity under different conditions. To examine the alterations in the proteasome responsible for cationic cleavage patterns, proteasomes from infected cells and uninfected controls were immunoprecipitated.

[0322] All the subunits of the proteasome, including the 20S core particle and the 19S regulatory particle were identified. Further, PSME3, one of the proteasome regulatory caps, exhibited significantly increased binding to the proteasome upon infection (Figs. 4J-4L). Increased counts of colony formation units (CFU) were found upon intracellular salmonella infection of PSME3 -deficient A549 cells (Fig. 4M and Fig. 4N), compared to controls. Next, we examined whether PSME3 was also required for the generation of secreted PDDPs, to impede the extracellular bacterial growth. Indeed, a significant increase in bacterial growth in conditioned media collected from PSME3 -deficient cells was found (Fig. 40).

[0323] Taken together, these results suggest that an altered proteasome composition, marked by an increase in PSME3-capped proteasomes upon bacterial infection, may serve a protective role by promoting tryptic-like cleavages and the generation of PDDPs.EXAMPLE 7: Proteasomal degradation as a cell-autonomous mechanism of innate immunity

[0324] To explore the capacity of protein degradation to yield protective peptides, the human proteome was artificially cleaved using predictive proteasomal cleavage sites as was previously discussed (Fig. ID). Subsequently, these peptides were scored using the predefined scoring system mentioned above and additionally filtered to exclude those lacking cationic termini (either amino or carboxy terminus). The analysis revealed that 92% of annotated human genes harbored at least one peptide with a cationic terminus, resulting in the identification of 270,872 putative PDDPs. Surprisingly, 66.7% (182 out of 273) of known AMP -containing proteins could be attributed to putative PDDPs identified through the in silico cleavage approach.EXAMPLE 8: PPPICB-proteasomal cleaved peptide attenuates bacterial growth in an Acute pneumonia lung model

[0325] Mice were infected with P.aeruginosa PAO1 to generate the Acute pneumonia lung model. Control (uninfected) mice received PBS. The P. aeruginosa PAO1 groups were then treated with either PBS or PPP1CB peptide (SEQ ID NO: 1033) (10 mg / kg) for 3 days. The degree of lesions on mice lungs was graded following staining with hematoxylin and eosin (H&E).

[0326] Lung histopathology revealed no significant findings in uninfected mice. Mice infected withP. aeruginosa PAO1 and treated with PBS displayed alveoli multifocally infiltrated by numerous degenerate and viable neutrophils, beaded fibrillary material (fibrins), proteinaceous exudates, and occasional intralesional small clusters of short rods, as highlighted by Gram stain. The third group ofmice infected with P. aeruginosa PAO1 and treated with PPP1CB, to a lesser extent, showed alveoli multifocally infiltrated by low numbers of neutrophil with no apparent evidence of intralesional bacterial colonies (Fig. 5A- 5C, and Table 3).

[0327] Table 3: Individual histopathological findings and scoring of acute pneumonia model by H&E staining.degree of lesions was graded from one to five depending on severity: = not present or minimal (< 1%); 2 = slight (1-25%); 3 = moderate (26-50%); 4 = moderate / severe (51-75%); 5 = severe / high (76- 100%).EXAMPLE 9: PPPICB-proteasomal cleaved peptide attenuates bacterial growth in a bacteremia murine model

[0328] Mice were infected with P. aeruginosa PAO1 to generate the bacteremia murine model. Control (uninfected) mice received PBS. The bacteria infected groups were then treated with either PBS or PPP1CB peptide (SEQ ID NO: 1033) (10 mg / kg) for 3 days. The degree of lesions in mice heart, liver, spleen, and kidney was graded following staining with hematoxylin and eosin (H&E).

[0329] Heart: Multifocally colonizing the coronary arteries and medium to large-sized vessels of the myocardium are occasional numbers of intravascular bacterial colonies. The subjacent myofibers are segmentally disrupted and hyalinized with increased sarcoplasmic eosinophilia and absent cross striations (myonecrosis and myolysis) (Fig. 6A, Fig. 6B, and Table 4).

[0330] Liver: Extensively filling the hepatic sinusoids and vessels are myriad numbers of 2-5 pm long short rods and increased erythrocytes. Occasionally the surrounding hepatocytes are variably shrunken (atrophic) and angular with hypereosinophilic cytoplasm and pyknotic, karyorrhectic to completely faded nuclei (single cell death) (Fig. 7A, Fig. 7B, and Table 4).

[0331] Spleen: Multifocally scattered throughout the red pulps and within central arteries are small clusters of 2-5 pm long short rods. Diffusely, there is moderate to marked necrosis and collapse of the white pulp and germinal center lymphocytolysis within splenic corpuscles (Fig. 8A, Fig. 8B, and Table 4).

[0332] Kidney: The peri -tubular capillaries and inter-lobular and arcuate arteries and veins are diffusely distended and distributed with small clumps of short rods. Multifocally the renal tubules within the cortex and medulla exhibit variably microvacuolated to hypereosinophilic cytoplasm with pyknotic nuclei. The third group of mice treated with PPP1CB following infection, showed no apparent evidence of intralesional bacterial colonies is revealed in lungs, heart, liver, spleen, and kidney (Fig. 9A, Fig. 9B, and Table 4).

[0333] Table 4: Individual histopathological findings and scoring of bacteremia model by H&E staining.Degree of lesions was graded from one to five depending on severity: 1 = not present or minimal (< 1%); 2 = slight (1-25%); 3 = moderate (26-50%); 4 = moderate / severe (51-75%); 5 = severe / high (76- 100%).EXAMPLE 10: PPPICB-proteasomal cleaved peptide prolong survival of mice in sepsis model

[0334] Mice were infected with P .aeruginosa PAO1 for sepsis model. Mice were treated retro- orbitally twice daily, beginning at 2 hours post-infection (10 mg / kg), for 3 days with PPP1CB peptide (SEQ ID NO: 1033) or Tobramycin. Control mice were treated with PBS (Vehicle).

[0335] Mice infected with P .aeruginosa PAO1 and treated with PPP1CB peptide showed significant prolonged survival with 4 deaths within the 6 days experiment while all mice treated with PBS died within 6 days post infection. Remarkably, a single peptide, at a concentration of 10 mg / kg had a comparable effect, albeit less efficacious, to a broad spectrum of activity antibiotics (Tobramycin) (Fig. 10)

Claims

CLAIMS1. A modified antimicrobial peptide (AMP) comprising a protein fragment having microbiocidal activity wherein the protein fragment is the degradation product produced by the proteasome.

2. The modified AMP of claim 1, wherein the protein from which said AMP is derived is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, M0B1B, RPLPO, EIF4A1, GAPDH and RPS29.

3. The modified AMP of claim 1, wherein said modified AMP comprising an amino acid sequence selected from the sequences set forth in any of SEQ ID NOs: 1 to 14,328.

4. A pharmaceutical composition comprising one or more modified AMPs of any one of claims 1- 3 and an acceptable carrier.

5. A method of treating or preventing a microbial infection in a subject in need thereof, the method comprising administering to the subject the modified AMP of claims 1 to 3 or the composition of claim 4.

6. The method according to claim 5, wherein the bacterial infection is Gram -negative bacterial infection.

7. The method according to claim 6, wherein the Gram -negative bacteria is selected from the group consisting of Enterobacteriaceae, Pseudomonadaceae Bacteroidaceae, Streptobacillus, and Acinetobacter.

8. The method according to claim 6, wherein the Gram -negative bacteria is selected from the group consisting of Escherichia, Salmonella, Shigella, Citrohacter, Edwardsiella, Enterohacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Serratia, Yersinia, Staphylococcus, Pseudomonas, Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas, Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella, Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella, Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae, in particular Treponema and Borrelia, Leptospiraceae, Campylobacter, Helicobacter, Spirillum, Bacteroides, Fusobacterium, Prevotella, Porphyromonas, A. baumanii. Mycobacterium avium, Mycobacterium tuberculosis, and Listeria monocytogenes .

9. The method according to claim 5, wherein the bacterial infection is Gram-positive bacterial infection.

10. The method according to claim 9, wherein the Gram -positive bacteria is selected from the group consisting of Staphylococcus haemolyticus, Micrococcus luteus, Mycobacterium, Pseudomonas aeruginosa, and Staphylococcus aureus.

11. The method according to claim 6 or 9, wherein the bacterial infection is an infection of the skin, of soft tissues, the respiratory system., the lung, the digestive tract, the eye, the ear, the nasopharynx, the bones, and / or the vagina.

12. A method of treating a wound or skin condition in a subject in need thereof, the method comprising administering to the subject the modified AMP of claims 1 to 3 or the composition of claim 4.

13. The method according to claim 12 wherein the skin condition is selected from the group consisting of: wound, dermatological disorder, psoriasis, ichthyosis, sarcoidosis, impetigo, acne (including hidradenitis suppurativa), bums, diaper rash, Netherton's syndrome, actinic keratosis, dermatomycoses, dermatosis or ectodermal dysplasia, atopic dermatitis, contact dermatitis, seborrheic dermatitis, vulgaris, filaggrin deficiency, allergic dermatitis, dandruff, pemphigus vulgaris, lichen planus, scleroderma, dermatomyositis, alopecia, skin carcinoma, melanoma, eczema, squamous cell carcinoma, acne vulgaris, erythema toxicum neonatorum, folliculitis, autoimmune bullous skin disease, bullous pemphigoid, pemphigus foliaceus, dermatitis and other disorders associated with damage or breakdown of the skin.

14. A method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need thereof, the method comprising administering to the subject the modified AMP of claims 1 to 3 or the composition of claim 4.

15. The method according to any one of claims 5-14, wherein the subj ect is a human or a non -human animal.

16. A wound covering comprising the modified antimicrobial peptide (AMP) of any one of claims 1-3.

17. A method of treating a wound in a subject in need thereof, the method comprising applying the wound covering of claim 16 to the wound.

18. The method according to claims 5-14, wherein the modified AMP is administered as and expressed from a minigene.

19. The method according to claims 5-14, wherein the modified AMP is administered as and expressed from a vector comprising a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG) and a gene encoding an AMP -containing protein.

20. The method according to claim 19, wherein expression of the nucleic acid sequence produces a protein-dTAG fusion protein.

21. The method according to claim 20, wherein the protein-dTAG fusion protein is ubiquitinated and then degraded by the proteasome.

22. The method according to claim 19, wherein said AMP-containing protein is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, MOB IB, RPLPO, EIF4A1, GAPDH and RPS29.

23. A Proteolysis-targeting chimera (PROTAC) comprising an E3 Ubiquitin Ligase binding moiety and a moiety that binds an AMP-containing target protein coupled together by a linker.

24. The PROTAC of claim 23, wherein the AMP -containing target protein is selected from the group consisting of PPP1CB, DFNA5, PSMG2, CHMP2A, RPL41, DCTN4, GLB1, RPS4X, ETS1, GRHPR, RPL21, M0B1B, RPLPO, EIF4A1, GAPDH and RPS29.

25. A method of treating or preventing a microbial infection in a subject in need thereof, the method comprising administering to the subject the PROTAC of claim 23 or 24.

26. A method of treating a wound or skin condition in a subject in need thereof, the method comprising administering to the subject the PROTAC of claim 23 or 24.

27. A method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need thereof, the method comprising administering to the subject the PROTAC of claim 23 or 24.

28. A method of preserving a food product comprising applying a composition comprising the antimicrobial peptide (AMP) of any one of claims 1 -3 to the food product.

29. The method according to claim 28, wherein the food product is selected from fruits, vegetables, legumes, beverages, starch food products, wheat products, dairy products, processed food products, aquatic products, and meat products.

30. The method according to claim 29, wherein the food product is a raw meat product.

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