Defensin fragments for use in therapy or prophylaxis

Abstract

The present invention relates to new peptides derived from HD-5 or HNP-4 having antimicrobial activity, said peptides for use in modulating the microbiome of the gut, lung, skin, mouth, eye, ear, vagina or other body surface, and / or as antimicrobial agents in humans or other mammals; and to medicaments containing these peptides.

Description

[0001] The present invention relates to specific peptide sequences derived from alpha defensins and their use in medical therapy and / or prophylaxis.

[0002] Anaerobic and aerobic microorganisms, especially bacteria and yeasts (i.e. unicellular microorganisms that survive in the presence of oxygen, survive in the absence of oxygen or are able to tolerate oxygen) can cause various clinical signs (e.g. wound infections and abscesses, sepsis, infections), especially in the abdominal cavity, in the urogenital tract, on the skin or in the oral cavity, the eye, the ear and the mandibular region. Thus, these pathogenic species are often already found in the skin and oral cavity region, in particular in inflamed skin / eczema, periodontal tissue, the eye and the ear and in the stomach region in the gastric mucosal folds and in the duodenum, where they can then cause local inflammation, but in some cases also systemic acute and chronic inflammation. Even in the small intestine, which is rather sparsely colonized, many facultative anaerobes can cause pathological changes in the highly sensitive small intestinal mucosa; in the rectum, the main site of the bacterial flora, it is accepted that aerobic bacteria predominate, but here too, anaerobes can cause severe inflammatory reactions in the colonic mucosa. Some species of Candida are also found in the feces of many individuals and are potentially pathogenic.

[0003] At present, in particular such diseases are treated with antibiotics, which mainly attack and destroy the cell wall of the bacteria. A major problem in the treatment of these inflammatory diseases with antibiotics is the development of resistance to the antibiotics used, which has further advanced in recent times. This enables pathogenic bacteria / microorganisms to attenuate or completely neutralize the effect of the antibiotic substance. If it subsequently turns out that the microorganisms are resistant to common antibiotics, the disease can become life-threatening. The reason for the considerable increase in the number of multi-resistant bacterial strains in the past is that, due to their rapid growth and their short cultivation period, bacteria are constantly able to develop new strategies to neutralize antibiotics. Therefore, in addition to antibiotics, for example, natural, in particular plant, and synthetic oils and emulsions are also used at present.

[0004] In recent years, antimicrobial peptides, which are part of the innate immune system and are essential for the defense of epithelia against microbial infections, have gained interest for research and therapeutic applications.

[0005] In healthy humans, the skin and mucosa form a physical barrier against infections caused by microorganisms. The physical barrier consists of the stratum corneum in healthy skin and the mucus layer in mucosa, in which desquamation and mucus secretion cause a constant renewal of the surface, while microorganisms adhering to the surface are constantly removed. This physical barrier can prevent microorganisms from penetrating into the living epidermis when interacting with the lipids also present in the skin.

[0006] However, leaving this physical barrier aside, for healthy skin and mucosa to resist infection, additional factors are required; these include endogenous antimicrobial peptides (AMPs). Lysozyme is an antimicrobial peptide that is present in nasal secretions and can kill, among others, gram-positive bacteria. Also known as antimicrobial peptides in the intestinal mucosa are the defensins, whose presence appears to be essential, especially considering the intestinal epithelium's exposure to extremely large numbers of bacteria. In addition to having a mucus layer that is difficult for microorganisms to penetrate, the intestinal mucosa also contains Paneth cells that secrete human defensin 5, an alpha defensin that, among other functions, protects stem cells that are important for the continuous renewal of the intestinal mucosa. In humans, only alpha- and beta-defensins are expressed. While alpha defensins are mainly expressed in neutrophil granulocytes as well as in NK cells and certain T-lymphocyte subgroups, human defensin 5 and defensin 6 are expressed only in the Paneth cells of the small intestine, where they help to regulate and maintain the microbial balance in the intestinal lumen. On the other hand, beta-defensins are the most widely distributed, being secreted by many types of white blood cells and epithelial cells. Further known AMPs are the peptides called psoriasins, and RNas-7, which represent an effective endogenous broad-spectrum antibiotic in humans.

[0007] In addition to the known endogenous antimicrobial peptides, a number of antibiotics are known in the prior art; these include substances of biological origin and synthetically manufactured substances, thus they are either (in the original sense) naturally occurring low-molecular-weight metabolites of fungi or bacteria, or chemically synthesized therapeutic agents.

[0008] In particular in view of the development of resistances to natural and synthetic antibiotics is making microbial infectious diseases increasingly difficult to treat, there is therefore often a need for new antimicrobial active agents that are notable for fewer side effects and simple manufacture and handling.

[0009] The gastrointestinal microenvironment consists of a single-cell layer epithelium, a mucus layer, a local immune system and a microbiome, and these four components together play a crucial role in maintaining homeostasis in a healthy period. The human colon has a high-density microbial community of 10 11 -10 12 cells per gram of intestinal content, and human health is closely related to the diverse microbial community collectively known as the gut microbiota in the intestine. While on the one hand their abundance and prevalence are associated with disease - as is the case, for example, with Prevotella copri in inflammatory bowel disease (IBD) and infectious colitis - on the other hand, mucosal species such as Bacteroides fragilis and Lactobacillus reuteri have been shown to be resistant to colitis.

[0010] Thus, the microbiome composition in the human colon is heavily influenced and the microbial balance is disturbed when antibiotics are used in the context of an infection. Defensins are small cationic molecules, characterized by three conserved disulfide bonds, and represent a major group of AMPs. So far, six a-defensins have been identified in humans, namely four human neutrophil peptides (HNPs) 1, 2, 3, and 4, and two human defensins (HDs) 5 and 6. While HNPs form part of the armamentarium of neutrophils, here they are involved in systemic innate immunity, HDs are expressed in intestinal Paneth cells. As mentioned above, in the small intestine, Paneth cells play a key role in balancing the microbiota composition and protecting the host from invading pathogens by secreting a variety of AMPs, but most abundant are two a-defensins 5 (HD 5) and -6 (HD 6).

[0011] While HNP-1, HNP-2, and HNP-3 differ only in a single amino acid, HNP-4 differs in its sequence, has one additional positive charge and exhibits improved bactericidal activity compared to HNP-1-3 1,2 The activity of full-length antimicrobial peptides is influenced by environmental conditions including salt concentration, pH, or redox potential 3-6 Based on its strong antimicrobial activity, we used HNP-4 as a precursor for a new therapeutic agent with antimicrobial capacity. While large-scale expression of accurately folded defensins is a major problem, we focused on small fragments of HNP-4. We used naturally occurring proteases to digest the full-length peptide and subsequently identified the generated fragments. We tested the antibacterial and antifungal potential of these fragments and analyzed their cytotoxicity and hemolytic capacity.

[0012] The antimicrobial activity of a-defensins has been intensively studied in the past, and it has been recognized that alterations of their specific sequence can lead to major changes in their activity and can even lead to a complete loss of antimicrobial activity. SUMMARY

[0013] Thus, the problem to be solved by the present invention is to provide a new or alternative prophylactic and / or therapeutic approach with which infectious diseases as well as other diseases associated with an ecological disorder condition, such as metabolic diseases, diseases of the lungs, urogenital system, oral cavity, eyes and ears, and skin diseases, can be prevented and / or effectively treated.

[0014] According to the present invention, this and other problems are solved by providing peptides having antimicrobial activity and having an amino acid sequence derived from a- defensin fragments, said peptides consisting of between 6 and 27, in particular shorter, peptide fragments, which can be synthesized as linear peptides, e.g. linear peptides having 7, 9, 11 or 13 consecutive amino acids.

[0015] The peptides have in common that they are fragments of the naturally occurring a defensins HD-5 and HNP-4 and can be produced by reducing the naturally occurring peptides and subjecting them to cleavage using protease activity. Surprisingly, the related peptide HD-6, which contains a number of predicted cleavage sites, could not be cleaved under the same conditions.

[0016] The advantage of these short fragments of naturally occurring a-defensins is that they can be chemically synthesized as small linear peptides, thus significantly reducing the costs compared to the manufacture of full-length peptides. Furthermore, several of the peptides retain the antibiotic effect of the naturally occurring full-length defensins, while being non-toxic at effective concentrations.

[0017] The peptides can be used to modulate the microbiome of the gut and / or as antimicrobial agents without causing major changes to the healthy microbiome / disturbing the balance of the healthy microbiome.

[0018] Within the present invention, peptide sequences of a-defensins have been identified which, on the one hand, exhibit an increased antimicrobial effect against certain (in particular: pathogenic) bacteria in antimicrobial tests compared to full-length peptides, while, on the other hand, the newly identified peptides have no effect on microbial diversity.

[0019] These results allow the peptides according to the present invention to be used not only for the treatment of microbial infections and diseases caused by bacteria, even those caused by antibiotic-resistant bacteria, but also for the prevention of bacterial infections and for the modulation of the gut microbiome and potentially other epithelial surfaces (e.g. lungs, skin, urogenital tract, oral cavity, eyes, ears, etc.).

[0020] Thus, within the present invention, and as generally understood in the art, "modulation of the microbiome" means a beneficial influence of the peptides on the microorganisms present in the gut and on epithelial surfaces. As mentioned above, gut microorganisms are key to many aspects of human health, including immune, metabolic and neurobehavioral properties. With the peptides used according to the present invention, the bacterial diversity of the gut microbiome and potentially other epithelial surfaces can be supported and promoted.

[0021] "Gut microbiome" means the gut of a mammal, in particular a human being. Thus, preferred embodiments of the present invention relate to peptides for the modulation of the human gut microbiome.

[0022] According to an embodiment of the peptide according to the present application, the alpha-defensin fragment is a fragment of HD-5 or HNP4.

[0023] As mentioned in the outset, HD-5 is expressed in the Paneth cells of the small intestine. HD-5 comprises 94 amino acids including a signal peptide and a prodomain, wherein the mature peptide comprises amino acids number 63 to 94.

[0024] HNP4, also as mentioned in the outset, is expressed in the granules of neutrophilic granulocytes. HNP4 comprises 97 amino acids including a signal peptide and a prodomain, wherein the mature HNP4 peptide comprises amino acids number 64 to 96.

[0025] According to a preferred embodiment of the present application, the peptide used according to the present application consists of between 6 and 27 consecutive amino acids derived from HD-5 and consists of the following sequences from the attached sequence listing:

[0026] Sequence HD-5 1-9 ATCYCRTGR (SEQ ID No. 1) or

[0027] the reverse sequence of SEQ ID No. 1 RGTRCYCTA (SQ ID No. 2),

[0028] Modified HD-5 1-9 : Ac-atcycrtGr-NH2 (SEQ ID No. 5),

[0029] HD-5 1-13 , ATCYCRTGRCATR (SEQ ID No. 34),

[0030] HD-5 1-28 , ATCYCRTGRCATRESLSGVCEISGRLYR (SEQ ID No. 12),

[0031] HD-5 7-32 , TGRCATRESLSGVCEISGRLYRLCCR (SEQ ID No. 14)

[0032] HD-5 10-32 , CATRESLSGVCEISGRLYRLCCR (SEQ ID No. 19),

[0033] HD-5 14-32 , ESLSGVCEISGRLYRLCCR (SEQ ID No. 25)

[0034] HD-5 10-27CATRESLSGVCEISGRLY (SEQ ID No. 28) or

[0035] HD-5 26-32 LYRLCCR (SEQ ID No. 41).

[0036] In general, for amino acid sequences, upper case letters indicate L-amino acids and lower case letters indicate D-amino acids.

[0037] In preferred embodiments, the peptide consists of the sequence:

[0038] ATCYCRTGR (SEQ ID No. 1),

[0039] RGTRCYCTA (SEQ ID No. 2),

[0040] Ac-atcycrtGr-NH2 (SEQ ID No. 5),

[0041] LYRLCCR (SEQ ID No. 41),

[0042] ATCYCRTGRCATR (SEQ ID No. 34),

[0043] ATCYCRTGRCATRESLSGVCEISGRLYR (SEQ ID No. 12) or

[0044] TGRCATRESLSGVCEISGRLYRLCCR (SEQ ID No. 14).

[0045] More preferably, the peptide consists of the sequence:

[0046] ATCYCRTGR (SEQ ID No. 1),

[0047] RGTRCYCTA (SEQ ID No. 2),

[0048] Ac-atcycrtGr-NH2 (SEQ ID No. 5) or

[0049] LYRLCCR (SEQ ID No. 41).

[0050] Preferred peptides include those based on HD-5 1-9 :

[0051] ATCYCRTGR (SEQ ID No. 1),

[0052] RGTRCYCTA (SEQ ID No. 2) or

[0053] Ac-atcycrtGr-NH2(SEQ ID No.5).

[0054] According to a preferred embodiment of the present application, the peptide used according to the present application consists of 7, 9, 11 or 13 consecutive amino acids.

[0055] According to a preferred embodiment of the present application, the peptide used according to the present application consists of 9 consecutive amino acids derived from HD-5, and preferably consists of the sequence ATCYCRTGR (SEQ ID No. 1) of the attached sequence listing or the reverse sequence of SEQ ID No. 1, RGTRCYCTA (SQ ID No. 2).

[0056] According to another preferred embodiment of the present application, the peptide used according to the present application consists of 11 consecutive amino acids derived from HNP4, and preferably consists of the sequence VCSCRLVFCRR (SEQ ID No. 3), the reverse sequence of SEQ ID No. 3, RRCFVLRCSCV (SEQ ID No. 4), or a modified HNP-4 1-11 : Ac-vcscrlvfcrr-NH2(SEQ ID NO.6).

[0057] In one embodiment, the present application relates to a method of manufacturing the peptides of the present application, said method comprising subjecting reduced HD5 or HNP-4 to protease activity (e.g. trypsin or chymotrypsin), followed by purification.

[0058] The peptides derived from HD-5 or HNP4 as disclosed and described herein have been shown to exhibit superior antimicrobial activity against pathogenic bacteria, while not significantly affecting the commensal microbiota, e.g. the gut microbiota.

[0059] According to a preferred embodiment, the peptide used according to the present application comprises L- and / or D-amino acids.

[0060] Currently and as generally understood, an L-amino acid refers to one stereoisomer of a particular amino acid, with the amino group on the left side of its Fisher projection; while a D-amino acid refers to the other stereoisomer of the amino acid, with the amino group on the right side of its Fisher projection. In the sequences herein, L-amino acids are shown in upper case letters, and D-amino acids are shown in lower case letters.

[0061] While most naturally occurring peptides consist of amino acids in the L-configuration, D-amino acids have been shown to exhibit strong resistance to proteolytic degradation.

[0062] According to one preferred embodiment, the peptide used according to the present application consists of D-amino acids.

[0063] According to another embodiment, the peptide according to the present application consists of L-amino acids.

[0064] According to another embodiment, the peptide according to the present application consists of a mixture of D-amino acids and L-amino acids, preferably alternating D-amino acids and L-amino acids, or preferably comprises one L-amino acid, the remaining amino acids being D-amino acids.

[0065] According to one preferred embodiment, the peptide used according to the present application comprises an N-terminal modification and / or a C-terminal modification.

[0066] By N-terminal modification and / or C-terminal modification, it is possible to influence / enhance, for example, the stability or half-life of the peptide according to the present application, in particular in environments that promote the degradation and / or modification of the free N- / C-terminus of the peptide, for example due to the presence of proteases in those environments.

[0067] Currently and as generally understood, the C-terminus (also called carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal or COOH-terminus) is the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH), and the N-terminus (also called amino terminus, NH2-terminus, N-terminus or amine-terminus) is the beginning of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. The convention for writing peptide sequences is to place the C-terminus on the right and to write the sequence from the N-terminus to the C-terminus.

[0068] According to one preferred embodiment, in the peptide used according to the present application, the C-terminal modification is selected from one of the group consisting of: -amide, -acid, -N-alkyl-amide, -aldehyde, -ester, -p-nitroaniline and -7-amino-4-methylcoumarin.

[0069] With these modifications, the C-terminus of the peptide can be protected without largely affecting the antimicrobial activity of the peptide.

[0070] According to one preferred embodiment, in the peptide used according to the present application, the N-terminal modification is selected from one of the group consisting of: acetyl-, formyl-, pyroglutamyl-, fatty acid-, urea-, carbamate- and alkylamine-.

[0071] It is to be understood that either of the two ends, i.e. the N-terminal and the C-terminal end, can be modified with any of the above mentioned modifications, or only one of the ends, i.e. the N-terminal or the C-terminal end, can be modified with any of the above mentioned modifications.

[0072] According to a preferred embodiment, in the peptide used according to the present application, the N-terminal is acetylated -(ac) and the C-terminal is unmodified.

[0073] According to a preferred embodiment, the peptide used according to the present application consists of (or comprises) D-amino acids and the N-terminal is acetylated -(ac) and the C-terminal is unmodified.

[0074] By N-terminal acetylation, the charge of the amino-terminal end of the peptide is removed; furthermore, by the acetyl modification, the peptide is intended to mimic its natural structure in proteins. Moreover, this modification stabilizes the resulting peptide against enzymatic degradation by exopeptidases.

[0075] According to a preferred embodiment, the peptide according to the present application carries an N-terminal acetyl modification and a C-terminal amide-modification.

[0076] By C-terminal amide modification, the peptide is intended to mimic its natural structure in proteins. Moreover, this modification avoids the introduction of an additional charge in the peptide molecule.

[0077] According to a preferred embodiment, the peptide according to the present application comprises 9 amino acids, of which 8 are D-amino acids and 1 is an L amino acid, and further preferably, this peptide comprises an N-terminal acetyl modification and a C-terminal amide modification.

[0078] According to a preferred embodiment, the peptide according to the present application comprises 11 amino acids, of which all are D-amino acids, and further preferably, this peptide comprises an N-terminal acetyl modification and a C-terminal amide modification.

[0079] According to another preferred embodiment, the peptide according to the present application is a chemically synthesized peptide or a biologically expressed peptide.

[0080] Various methods for the chemical synthesis of peptides are known in the art; while chemical synthesis of peptides can be performed using classical solution phase techniques, these solution phase techniques have been replaced by solid phase methods in most research and development environments. The solid phase synthesis of peptides can be performed, for example, as described in Stawikowski et al., ("Introduction to peptide synthesis", Cur. Prot. Prot. Sci., (2012), suppl. 69, 18.1.1-18.1.13) 7An overview of peptide synthesis can be found in the textbook by

[0081] According to one embodiment for the peptides used in the present application, the peptides are in an oxidized or reduced state.

[0082] In this context, "oxidized" refers to the state of a peptide in which disulfide bridges are formed, which occurs in peptides having amino acid residues such as cysteine, methionine, tryptophan, histidine and tyrosine. The "reduced" state denotes the form of a peptide in which no disulfide bonds are formed.

[0083] Within the present application, "biologically expressed" peptides shall encompass the expression of one or more peptides according to the use of the present application by a genetically engineered host cell which has been modified to express said one or more peptides.

[0084] As used herein, the term "host cell" is presently defined as a cell which has been transformed or transfected with, or is capable of being transformed or transfected with, an exogenous polynucleotide sequence encoding a peptide according to the use of the present application.

[0085] A variety of host expression vector systems can be used to express genes encoding peptides according to the use of the present application. Such host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which, upon transformation or transfection with the appropriate nucleotide coding sequences, will exhibit the peptides of the gene products of the present application in situ.

[0086] For recombinant production, the host cell can be genetically engineered to incorporate an expression system or parts thereof or a polynucleotide encoding a peptide according to the use of the present application. The introduction of the polynucleotide into the host cell can be achieved by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (2012) 8 and Sambrook et al., 1989 9 .

[0087] Thus, the polynucleotide encoding a peptide according to the present application can for example be contained in a vector to be stably transformed / transfected into a host cell. In the vector, the polynucleotide(s) encoding one or more peptides of the present application are under the control of e.g. inducible promoters, such that the expression of the gene / polynucleotide can be specifically targeted and, if desired, the gene can be overexpressed in this way.

[0088] A wide variety of expression systems can be used to produce the polypeptides of the present application. Such vectors include, among others, vectors of chromosomal, episomal and virus origins. For example, vectors derived from plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses and combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids. Expression system constructs can contain control regions that regulate as well as control expression. In general, any system or vector that is suitable for maintaining, propagating or expressing polynucleotides and / or expressing polypeptides in a host can be used in connection with expression in this regard. Appropriate DNA sequences can be inserted into the expression system by any of a variety of well-known and routine techniques, such as those set forth in Sambrook et al., supra.

[0089] According to one preferred embodiment, the peptide used according to the present application is selected from at least one of the following:

[0090] HD-5 1-9 : ATCYCRTGR (SEQ ID No. 1)

[0091] HD-5 1-9rev : RGTRCYCTA (SEQ ID No. 2)

[0092] HD-5 1-9mod : Ac-atcycrtGr-NH2 (SEQ ID No. 5)

[0093] HNP-4 1-11 : VCSCRLVFCRR (SEQ ID No. 3)

[0094] HNP-4 1-11rev : RRCFVLRCSCV (SEQ ID No. 4)

[0095] HNP-4 1-11mod : Ac-vcscrlvfcrr-NH2 (SEQ ID No. 6)

[0096] According to another aspect of the present application, the use of the peptide in modulating the microbiome consists of a use for the treatment and / or prevention of the intestine, the lungs, the urogenital apparatus, the oral cavity, the eyes, the ears or the skin or other conditions or diseases associated with dysbiosis conditions.

[0097] As mentioned above, a healthy gut microbiome containing a diverse bacterial microbiota is not only essential for an intact gut, but also for the overall health of a mammal, especially a human. Not only is lower bacterial diversity repeatedly observed in diseases such as inflammatory bowel disease, celiac disease, among others, but also in people with metabolic diseases like obesity and type 2 diabetes, and the efficacy of e.g. checkpoint inhibitor therapy for cancer is highly influenced by the microbiome, and even CNS diseases like schizophrenia are reported to be influenced by the microbiome. The association between reduced diversity and disease suggests that a species-rich gut ecosystem has a stronger robustness against environmental influences, as functionally related microorganisms in an intact ecosystem can compensate for the function of other missing species.

[0098] In addition to genetically influenced intestinal diseases, specific foods and dietary patterns as well as medication can influence the abundance of different types of bacteria in the gut. While positive influences on the gut microbiome are generally associated with changes in nutrition or diet and can also be observed by using prebiotic and probiotic foods, the peptides according to the present application provide a more versatile, more convenient and efficient tool due to their natural origin. Also using the peptides according to the present application, subjects that are highly sensitive to nutritional changes and influences can be treated.

[0099] With the peptides used according to the present application, intestinal diseases as well as diseases of the lung, skin and brain can be effectively prevented and / or treated by positively influencing the natural microbiome of the gut.

[0100] Thus, in a preferred embodiment, the peptides according to the present application are used for the prevention / treatment of a disease selected from the group consisting of inflammatory bowel diseases, in particular Crohn's disease, ulcerative colitis, celiac disease, necrotizing enterocolitis, irritable bowel syndrome, traveler's diarrhea, gastrointestinal cancer and intestinal graft-versus-host disease; and metabolic diseases, preferably diabetes and prediabetes, obesity, NAFLD, NASH, dyslipidemia; and diseases of the lung, preferably asthma and COPD; and brain diseases, preferably schizophrenia, Parkinson's disease, bipolar disorder, autism and depression.

[0101] With the peptides according to the present application, diseases related to the simple microbiome of the skin, mouth, eyes, ears, vagina or circulatory system can be effectively prevented and / or treated.

[0102] Therefore, in a preferred embodiment, the peptide according to the present application is used for the prevention / treatment of a disease selected from the group consisting of: sepsis, atopic dermatitis, rosacea, seborrheic dermatitis, eczema, carbuncle, staphylococcus infection, candidiasis, cellulitis, impetigo, acne, hidradenitis suppurativa, athlete's foot, tinea, molluscum, cutaneous lymphoma, periodontitis, dental caries, dry eye, Sjogren's syndrome, conjunctivitis, blepharitis, hordeolum, chalazion, periorbital cellulitis, dacryocystitis, endophthalmitis, uveitis, iritis, mastoiditis, vestibular neuronitis, bullous myringitis, granular myringitis, otitis externa, otitis media, bacterial vaginosis, trichomonas vaginitis, candidiasis, non-infective vaginitis, inflammatory vaginitis.

[0103] According to another aspect, the peptide as described herein can also be used as an antimicrobial agent against infections induced by multi-resistant bacteria. Within the present application, it has been found that the peptide according to the present application can not only be used to positively influence the natural gut microbiome, but can also be used as a tool to specifically target multi-resistant bacteria, and thus is a highly efficient tool for the treatment / prevention of infections caused by multi-resistant bacteria. According to another object, the present application also relates to a medicament comprising a peptide according to the present application and a pharmaceutically acceptable carrier.

[0104] Currently and as generally understood in the art, a "pharmaceutically acceptable carrier" is understood to mean any excipient, additive or vehicle that is generally used in the field of treating the aforementioned diseases and that simplifies or enables the administration of the product according to the present application to an organism and / or improves its stability and / or activity. The pharmaceutical composition can also be incorporated with a binder, diluent, or lubricant. The choice of pharmaceutical carrier or other additive can be made on the basis of the intended route of administration and standard pharmaceutical practice. As a pharmaceutically acceptable carrier, solvents, extenders or other liquid binding media, such as dispersants or suspending agents, surface-active agents, isotonic agents, spreaders or emulsifiers, preservatives, encapsulating agents, solid binding media, depending on what is most suitable for the respective dosage regimen and likewise compatible with the compound according to the present application, can be used. An overview of such additional ingredients can be found, for example, in Rowe (ed.) et al.: Handbook of Pharmaceutical Excipients, 7thEdition, 2012, Pharmaceutical Press, London, UK. 10

[0105] When formulated for topical administration, the peptide of the present application can be used to treat skin conditions. Methods for topical administration are known in the art.

[0106] ​When formulated for topical administration, the compositions of the present application can contain ingredients typical of topical pharmaceutical or cosmetic compositions, such as carriers, vehicles or media. In particular, the carriers, vehicles or media are compatible with the tissue (e.g., skin, hair, nails, vagina, urethra, ear, mouth, nasal passages, respiratory system, ocular region, and / or mucosa) to which they will be applied. The compositions and components of the present application are suitable for use in contact with infected tissue or for general patient use without undue toxicity, incompatibility, instability, allergic response, and the like. The compositions of the present application can include, as appropriate, any of the ingredients described in the art of formulation.

[0107] In terms of form, the compositions of the present application can include solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or other typical solid or liquid compositions for application to the skin and other tissues where the compositions can be used. Such compositions can contain: additional antimicrobial agents, moisturizers and hydrating agents, penetrants, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents, emollients, antioxidants, fragrances, fillers, thickening agents, waxes, odor absorbers, dyes, colorants, powders, viscosity control agents, and water, and optionally include anesthetics, antipruritic actives, plant extracts, conditioning agents, darkening or lightening agents, glittering agents, humectants, mica, minerals, polyphenols, silicones or derivatives thereof, sunscreens, vitamins, and botanicals. In certain embodiments, the compositions of the present application are formulated with the above ingredients for long-term stability, as can be beneficial where continuous or long-term treatment is intended.

[0108] The compositions of the present application can be in the form of a controlled or sustained release composition, in which the antimicrobial peptides are encapsulated or otherwise contained in a material such that they are released onto the skin or affected area in a controlled manner over time, along with additional active agents. The compositions of the present application can be contained within or on a matrix, liposome, vesicle, microcapsule, microsphere, and the like, or within or on a solid particulate material.

[0109] The compositions of the present application can be applied to any affected or susceptible area, e.g., legs, shoulders, back (including lower back), armpits, palms, feet, neck, groin, back or hand or foot, elbows, upper arms, knees, thighs, hips, torso, pelvis, or any other part of the body that can require treatment or prevention of infection. Such treatment is also contemplated for the treatment and / or dressing of wounds, such as cuts, abrasions, and burns of the skin, in order to treat or prevent infection of the wounded area.

[0110] The compositions of the present invention are suitable for physiological environments with a pH range of about 4.5 to about 6.3, and therefore, the compositions can be formulated at similar or equivalent pH levels. The compositions according to the present invention can be stored at room temperature or under refrigeration. The compositions of the present invention contain a certain amount of antimicrobial peptides effective against microorganisms. Generally, the compositions contain about 0.01% (wt. / vol.) to about 20% of antimicrobial peptides. In some embodiments, the compositions contain about 0.5% to about 10% of antimicrobial peptides, such as about 0.5%, about 1%, about 5%, or about 10%.

[0111] The characteristics, properties, and advantages of the peptides according to the invention also apply to the medicaments according to the invention. Therefore, medicaments containing the peptides according to the invention can also be used to treat and / or prevent diseases selected from: inflammatory bowel disease, particularly Crohn's disease, ulcerative colitis, celiac disease, necrotizing enterocolitis, irritable bowel syndrome, traveler's diarrhea, gastrointestinal cancer, and intestinal graft-versus-host disease; and metabolic diseases, preferably diabetes and prediabetes, obesity, NAFLD, NASH, and dyslipidemia; and lung diseases, preferably asthma and COPD; and skin diseases, such as atopic dermatitis, rosacea, seborrheic dermatitis, eczema, carbuncles, staphylococcal infections, candidiasis, cellulitis, impetigo, acne, and pilonidal follicles. Athlete's foot, tinea, molluscum contagiosum, cutaneous lymphoma; oral diseases, such as periodontitis and dental caries; eye diseases, such as dry eye, Sjögren's syndrome, conjunctivitis, blepharitis, stye, chalazion, periorbital cellulitis, dacryocystitis, endophthalmitis, uveitis, iritis; ear diseases, such as mastoiditis, vestibular neurocytitis, bullous myringitis, granulomatous myringitis, otitis externa, otitis media; vaginal diseases, such as bacterial vaginosis, trichomoniasis, candidiasis, non-infectious vaginitis, inflammatory vaginitis; as well as sepsis and mental illnesses, preferably schizophrenia, Parkinson's disease, bipolar disorder, depression, or autism.

[0112] According to another preferred embodiment, the peptide exists in the form of a dimer (preferably a homodimer). The dimer is preferably linked by covalent bonds (suitably disulfide bonds).

[0113] Peptide dimerization is known in the art. Current chemical properties used for peptide dimerization involve chemoselective reactions between unprotected peptides. Examples include the formation of bonds such as cys-maleimide sulfides, disulfides, or triazoles.

[0114] It should be understood that the above features and the features mentioned below can be used not only in the indicated combinations in the respective circumstances, but also in other combinations or separately without departing from the scope of the invention.

[0115] The application is now explained in further detail by means of embodiments which generate further features, characteristics and advantages of the application. The embodiments are of purely illustrative nature and do not limit the scope of the application.

[0116] Features mentioned in the specific embodiments are also general features of the application, which are not only applicable to the respective embodiments, but can also be applied in isolated fashion in the context of any embodiment of the application.

[0117] The application is further described and explained in detail by reference to the following drawings:

[0118] Figure 1 shows the results of an experiment demonstrating that HD-6 nanoweb formation is not affected by duodenal fluid: (A) shows the chromatogram of HD-6 incubated with duodenal fluid after reduction with 2 mM TCEP. Oxidized and reduced full-length peptides were detected based on retention time and m / z plot and 2, 3, 4, 5 and 6-fold protonated ions and neutral mass. (B) shows reduced beads incubated with 200 pg / ml HD-6 or 0.01 % HAc (control) and duodenal fluid or 0.9 % NaCI (control). Nanoweb formation was not affected as these webs look identical to HD-6 with 0.9 % NaCI. Magnification bar = 0.2 pm.

[0119] Figure 2 shows the results of an experiment, in which incubation of HD-5 and duodenal fluid resulted in a number of different fragments. HD-5 was incubated with duodenal fluid after reduction with 2 mM TCEP. (A) shows an overview of the chromatogram from the incubation of reduced HD-5 with duodenal fluid. In (B) all detectable fragments are labeled in grey (a-m) and listed according to their retention time. The mass-to-charge ratio (m / z) plot of all identified fragments has the detected ions and their neutral mass. Peptides (a), (b), (c), (e), (i), (j), (k), (l) and (m) were selected for synthesis and to investigate their ability in depth. (C) Here the selected fragments (grey) are listed as well as their amino acid sequence and their distribution in the HD-5 sequence.

[0120] Figure 3 shows the results of experiments demonstrating that the HD-5 fragment is an antimicrobial active peptide against symbiotic bacteria: (A) shows a table summarizing tests performed on different symbiotic bacteria based on their sensitivity to the HD-5 fragment. This heatmap lists all bacteria and the activity of the fragment against them in the RDA. In the RDA, 2 μg of the full-length peptide and 4 μg of each fragment were used. Inhibitory regions greater than 5 mm were identified as highly active, those between 2.5 and 5 mm as lowly active, and 2.5 mm being the diameter of the punched orifice and therefore inactive. (B) Here, in the graph, the raw data from (A) are placed together with the mean and standard deviation from at least three independent experiments. (C) shows electron micrographs investigating the modes of action of different peptides: *E. coli* MC1000 was incubated with all the different fragments, and transmission electron microscopy was performed to analyze the resulting phenotypes. Except for the full-length peptide HD-5. fl Except for 1μm, the magnification bar for all images is 0.5μm.

[0121] Figure 4 shows the results of experiments demonstrating the antimicrobial activity of different fragments against pathogenic bacteria: (A) shows a table summarizing the tests of the antimicrobial activity of the HD-5 fragment against pathogenic bacteria. A heatmap system with high activity (inhibition zone >5 mm in RDA), low activity (2.5 to 5 mm), and no activity (2.5 mm) was used. (B) shows a graph of the data in (A) with the mean and standard deviation from at least three independent experiments.

[0122] Figure 5 illustrates HD5 containing cysteine ​​and arginine substitutions. 1-9 The results of experiments showed almost no antimicrobial activity against Escherichia coli and Staphylococcus aureus mutants. After 18 hours, based on optical density (OD)... 600 The measurement results determine HD5 1-9 The minimum inhibitory concentrations (MICs) of the strains against (A) Escherichia coli BW 25113 mutant and (B) Staphylococcus aureus SA113 mutant were determined. Results from three independent experiments were depicted with + / - SEM.

[0123] Figure 6 HD5 is shown 1-9 and synthesized HD5 1-9 The figure shows an experiment in which the reduction of HD5 completely destroyed its antimicrobial activity. Several concentrations of reduced and oxidized HD5 were determined based on optical density measurements after 18 hours. 1-9 The minimum inhibitory concentration (MIC) of the dimer against (A) *Escherichia coli* BW 25113 and (B) *Staphylococcus aureus* SA113 was determined. Bacteria were then plated to confirm the MIC. Results from three independent experiments were plotted with + / - SEM.

[0124] Figure 7 shows a graph of experimental data indicating that there was almost no observed decrease in metabolic activity of cells treated with HD5 1-9 To analyze the metabolic activity of cells treated with HD5 1-9 and dimer, a WST-1 assay was performed. Cell lines were stimulated with HD5 1-9 or HD5 1-9 dimer at concentrations ranging between 3.123-100 μΜ and incubated for 24 hours or 48 hours. Activity was normalized against the negative control. Cells treated with 2% Triton X-100 were used as positive control, while treatment with 0.01% acetic acid was used as negative control. Results show the mean values of three independent experiments (A, B, C) with + / - SEM.

[0125] Figure 8 shows the antimicrobial mode of action of HD5 1-9 Cell wall damage induced by HD5 1-9 varies between E. coli ATCC 25922 and S. aureus SA113. To detect bacterial cell damage caused by HD5 1-9 , flow cytometry analysis was performed. 1.5 x 10 6 E. coli ATCC 25922 and S. aureus SA113 were incubated with different concentrations of HD5 1-9 (6.25 μΜ, 12.5 μΜ and 50 μΜ) for 1 hour. (A) Propidium iodide or (B) membrane sensitive DiBAC3(3) dye were used to stain the bacteria. 12.5 μΜ hBD3 was used as positive control, while untreated cells were used as negative control. Results from three independent experiments are depicted with + / - SEM. (C) Transmission electron microscopy was performed to assess morphological changes of E. coli MC1000 treated with HD5 1-9 (200 μg / ml). For comparison, full-length HD5 (HD5 fl ) was used, while treatment with 0.01% acetic acid (HAc) served as negative control. Bars: top left: 1 μm; top right and bottom left: 0.5 μm; bottom right: 2 μm.

[0126] Figure 9 shows the results of the exploration and experiments on Akkermansia in feces and sensitivity to HD-5 1-9 treatment: (A) The number of Akkermansia in fecal samples collected from mice treated with HD-5 1-9 or PBS for 7 days (days 0, 7, 14) was higher in mice treated with HD-5 1-9increased (linear mixed effects model; p = 0.075). The mean values shown here have 80% confidence intervals from n = 6 per group. In (B), it was tested whether Akkermansia muciniphila affects the growth of HD-5 1-9 treatment sensitive. (B) shows the growth rate (%) after 72 h incubation at 37°C in anaerobic jars compared to untreated controls. No HD-5 1-9 MICs on Akkermansia. The graph shows the mean values with standard deviations of n = 3.

[0127] Figure 10 shows HD5 1-9 Pro- and anti-inflammatory immune responses of stimulated human PBMCs. Human peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with 10 pg / ml LPS (Salmonella typhimurium) and different concentrations of HD5 1-9 for 24 h. Supernatants of PBMCs were used to quantify the amount of cytokines produced by the Multi-Analyte Kit (LEGENDplex). The following cytokines were assessed: pro-inflammatory cytokines (A) TNF-a (B) IFN-g (C) IL-1 b (D) IL-6 (E) IL-8 and anti-inflammatory cytokine (F) IL-10. Untreated cells were used as negative control and cytokine concentrations were normalized to the negative control. Results show the mean values with + / - SEM of three independent experiments. For statistical analysis, a Kruskal-Wallis test was performed. p > 0.05 = ns; p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***; p < 0.0001 = ****.

[0128] Figure 11 shows HD5 1-9 and its dimerized form do not exhibit cytotoxic effects on human cell lines. HD5 1-9 and the dimer (A) metabolic activity and (B) cytotoxicity were determined using the WST-1 assay and the LDH assay. HT29-MTX-E12 cells were stimulated with HD5 1-9 or HD5 1-9 dimer at concentrations between 3.123 - 100 mM and incubated for 48 h. Activity was normalized to the negative or positive control. As positive control, cells were treated with 2% Triton X-100, while treatment with 0.01% acetic acid was used as negative control. Results show the mean values with + / - SEM of three independent experiments. (C) Furthermore, the hemolytic activity of HD5 1-9 and the dimer was analyzed. An erythrocyte suspension was treated with different concentrations of HD5 1-9Incubate together. Hemolytic activity was normalized to that of 0.1% Triton X-100. Experiments were performed in duplicate.

[0129] Figure 12 shows additional toxicity data. Little to no cytotoxic effect on TR146 cells was observed after peptide treatment. LDH assays were performed to assess the cytotoxic effect of HD5 1-9 and dimer. Cell lines were stimulated with HD5 1-9 or HD5 1-9 dimer at concentrations ranging between 3.123-100 μΜ and incubated for 24 hours or 48 hours. Activity was normalized to positive control. As a positive control, cells were treated with 2% Triton X-100, while treatment with 0.01% acetic acid was used as a negative control. Results show the mean of three independent experiments with + / - SEM. Cytotoxic effect on TR146 cells was assessed for (A) HD5 1-9 and (B) HD5 1-9 dimer after 24 hours and 48 hours. In addition, the cytotoxic effect of (C) HD5 1-9 and dimer on HT29-MTX-E12 cells was assayed after 24 hours.

[0130] Figure 13 A and 13B show the overall fecal microbiota community. PCoA of weighted and unweighted UniFrac distances using all mice (n=12 per group) after 1 week of treatment comparing HD5 fl and HD5 1-9 .

[0131] Figure 14 A and 14B show the overall small intestinal microbiota community. PCoA of weighted and unweighted UniFrac distances using all mice (n=6 per group) sacrificed at week 1 comparing HD5fl and HD5 1-9 .

[0132] Figure 15 A and 15B show bacterial genera that were differentially affected by treatment with full-length and fragmented HD-5. Linear mixed models using all mice adjusting for cage at days 0, 7, and 14 of the fecal microbiota (note, n=12 per group at days 0 and 7, and n=6 per group at day 14). Only significantly different genera are presented.

[0133] Figure 16 : HD5 1-9Antimicrobial activity against *Escherichia coli* ATCC 25922 and *Escherichia coli* BW 25113 and its LPS mutants. (A) Cell wall construction of *E. coli* BW 25113 mutants. *E. coli* ATCC 25922 contains full-length LPS, while *E. coli* BW 25113 lacks the O-antigen. The *E. coli* BW 25113 mutant △waaG lacks an outer nucleus, while △waaY lacks some phosphate residues in its inner nucleus. The last mutant △waaP contains an outer nucleus but lacks phosphate residues in its inner nucleus. (B) Determination of HD5 at different peptide concentrations based on optical density after 18 hours. 1-9 Minimum inhibitory concentrations (MICs) against Escherichia coli ATCC25922 and Escherichia coli BW 25113 mutants. Results from at least two independent experiments are presented as mean + / - SEM.

[0134] Figure 17: HD5 1-9 Antimicrobial activity against Staphylococcus aureus SA113 and its cell wall mutants. (A) Staphylococcus aureus SA113 cell wall mutants were used to analyze HD5. 1-9 The charge-dependent antimicrobial activity of Staphylococcus aureus mutant △dltA lacks D-alanine, resulting in a more negatively charged peptidoglycan layer. A similar characteristic is found in mutant △mprF, which lacks L-lysine, resulting in a negatively charged cell membrane. Staphylococcus aureus mutant △tarH contains additional teichoic acid, leading to an enhanced peptidoglycan layer. (B) HD5 was determined at different peptide concentrations based on optical density after 18 hours. 1-9 Minimum inhibitory concentration (MIC) against Staphylococcus aureus SA113 and its mutant. Results from two independent experiments are presented with + / - SEM.

[0135] Figure 18 Compared to the dimer form, HD5 1-9 The antimicrobial activity against Gram-negative bacteria varies. HD5 was determined at different peptide concentrations after 18 hours based on optical density. 1-9 and HD5 1-9 -Minimum inhibitory concentration (MIC) of the dimer against different Salmonella species. Results are presented as mean + / - SEM values ​​from at least two independent experiments.

[0136] Figure 19 Compared to the dimer form, HD5 1-9 The antimicrobial activity against Gram-positive bacteria varies. HD5 was determined at different peptide concentrations after 18 hours based on optical density. 1-9 and HD5 1-9- Minimum inhibitory concentrations (MIC) of dimer against S. aureus ATCC25923 and clinical isolate S. aureus USA300. Results from two independent experiments are presented with + / - SEM.

[0137] Figure 20 : Reduced HNP-4 was digested by trypsin. (A) Overview showing the chromatogram of reduced HNP-4 after incubation with trypsin after reduction with 2 mM TCEP. All detectable fragments are labeled in red or grey (a-j) and listed according to their retention time. Full-length peptide is labeled (i) and fragments HNP-4 1-11 (d).

[0138] Figure 21 : HNP4 derivatives display high antimicrobial activity against commensal and pathogenic bacteria.

[0139] The identified fragments and their modified versions were analyzed for their antimicrobial potential against commensal and pathogenic bacteria using RDA. Heat maps are shown, with inhibition zones larger than 5 mm determined as high activity, between 2.5 and 5 mm as low activity, and a diameter of 2.5 mm (diameter of punched holes) marked as no activity. Heat maps are based on at least three independent experiments.

[0140] Figure 22: HNP-41-11 and HNP-41-11mod show only slight cytotoxicity and hemolytic activity at high concentrations. HNP-4 1-11 and HNP-4 1-11mod were investigated for their cytotoxic activity against (A) CaCo2 / TC7 or (B) HT29 MTX E 29 cells. 1500 cells per well were seeded and treated with different peptide concentrations after 24 hours. Viable cells were determined after 96 hours of treatment using the CellTiterGlo 2.0 assay. (C) Hemolytic activity of the peptides on human red blood cells compared to 0.1 % Triton-X treatment.

[0141] Materials and methods

[0142] Bacterial strains

[0143] Acinetobacter baumannii 4-MRGN, Klebsiella pneumoniae 4-MRGN, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecium 475747, Bifidobacterium longum, Lactobacillus fermentum, Lactobacillus salivarius and Streptococcus salivarius ssp. salivarius were obtained as clinical isolates from Robert-Bosch-Hospital (Stuttgart, Germany). M. mucosa, B. subtilis 168 trpC and S. aureus USA300 were obtained from Institut fur Mikrobiologie und lnfektionsmedizin (Tubingen, Germany). B. adolescentis Ni3,29c, B. breve were provided by Ardeypharm. B. vulgatus DSM 1447 was obtained from DSMZ and Lactobacillus rhamnosus was provided by InfektoPharm (Heppenheim, Germany). E. coli ATCC 25922 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Bonn, Germany). Salmonella species as well as clinical isolates of S. aureus USA300, S. aureus ATCC 25923, S. aureus SA133 and its mutants were provided by Institute of Medical Microbiology and Hygiene Tubingen, Germany. E. coli BW25113 and its mutants were obtained from Interfaculty Institute for Microbiology and Infection Medicine, Tubingen, Germany.

[0144] Peptides For all experiments, oxidized peptides HD-5 and HD-6 (Peptide Institute, Osaka, Japan) were used. All fragments, i.e. HD-5 1-9 and HNP-4 1-11 , HD-5 1-13 , HD-5 1-28 , HD-5 7-32 , HD-5 10-32 , HD-5 14-32 , HD-5 10-27 and HD-5 26-32 (all peptides of the present invention) were synthesized by EMC microcollections GmbH (Tubingen, Germany). All peptides were dissolved in 0.01% acetic acid (HAc) at similar concentrations.

[0145] The following sequences (peptides according to the application) were tested (N -> C-terminus):

[0146] HD-5 1-9 : ATCYCRTGR (SEQ ID No. 1)

[0147] HD-5 1-9rev : RGTRCYCTA (SEQ ID No. 2)

[0148] HD-5 1-9mod : Ac-atcycrtGr-NH2 (SEQ ID No. 5)

[0149] HNP-4 1-11 : VCSCRLVFCRR (SEQ ID No. 3)

[0150] HNP-4 1-11rev : RRCFVLRCSCV (SEQ ID No. 4)

[0151] HNP-4 1-11mod : Ac-vcscrlvfcrr-NH2 (SEQ ID No. 6)

[0152] Duodenal fluid was collected during gastroscopy.

[0153] Human duodenal fluid was collected from three healthy individuals during routine gastroscopy. The duodenum was washed with 0.9% NaCl solution and the NaCl solution was recovered again. The patients gave written informed consent after being informed. The sample collection had previously been approved by the Ethical Committee of the University Hospital of Tuebingen, Germany.

[0154] Screening of fragments of HD-5 and HD-6 using LC / MS

[0155] HD-5 or HD-6 were incubated in 50 mM NH4HCO3buffer (pH 8.0) (Fluka) containing 2 mM tris(2-carboxyethyl)phosphine for 15 min at 37°C. Human duodenal fluid was then added and incubated for another 30 min at 37°C. Finally, formic acid and acetonitrile were added to a final concentration of 0.5% and 10%, respectively, and the samples were analyzed by mass spectrometry. Mass spectrometry analysis was performed with an LC / MS system using an Agilent 1200 series HPLC with an Agilent Advanced Bio Peptide Map (2.1 x 150 mm, 2.7 pm) column at a flow rate of 0.4 ml / min at a column temperature of 55°C and a 6540 UHD Q-TOF LC / MS system (Agilent) for mass analysis. The samples were separated with a gradient of acetonitrile in 0.1% formic acid. The gradient started at 2% acetonitrile for 4 min and then increased to 45% over 35 min. Mass spectrometry analysis was performed in single MS mode from 100 to 3400 m / z in positive ion polarity and analyzed by Agilent MassHunter Quantitative Analysis B 06.00 software.

[0156] Scanning electron microscopy

[0157] Scanning electron microscopy was performed as previously described. Briefly, protein A-coated beads (Spherotech Inc.) were incubated with reduced HD-6 (200 pg / ml) in 10 mM sodium phosphate buffer containing 1% (w / v) TSB for 1.5 h at 37°C to allow net formation. The whole sample was then incubated with duodenal fluid for another 30 min at 37°C. 0.01% acetic acid (HAc) was used as a control. The beads were centrifuged and fixed in Karnovsky’s reagent. The samples were washed with PBS and additionally fixed with 1% OsQ4 in H2O. They were then dehydrated into 100% ethanol and critical point dried from CO2 and analyzed by scanning electron microscopy at the Max Planck Institute for Developmental Biology (Tuebingen, Germany).

[0158] Transmission electron microscopy

[0159] Transmission electron microscopy experiments were performed as previously described 11 . 6 x 10 8Cfu E. coli MC1000 were incubated with 200 pg / ml of each peptide for 2 hours. Bacteria were fixed in Karnovsky fixative, embedded in agarose, solidified, cut into small pieces and fixed again in Karnovsky solution. After post-fixation and embedding in glycidyl ether, the pieces were cut using an ultramicrotome. Sections (30 nm) were mounted on copper grids and analyzed using a Zeiss LIBRA 120 transmission electron microscope.

[0160] Radial diffusion assay

[0161] The antimicrobial activity of all peptides was tested by a modified radial diffusion assay according to Lehrer et al. 12 Briefly, bacteria in the log phase were grown in liquid tryptic soy broth (TSB) (Becton Dickinson) (anaerobic bacteria with AnaeroGen, Oxoid, in an anaerobic jar). After several washing steps with 10 mM sodium phosphate buffer (pH 7.4), 4 x 10 6 cfu / ml were used per assay. To measure the antibacterial effect of the identified peptide fragments, bacteria were incubated in 10 mM sodium phosphate (pH 7.4) containing 0.3 mg / ml TSB powder and 1 % (w / v) low EEO-agarose (Applichem). The peptide fragments were then pipetted into punched-out wells and allowed to diffuse for 3 hours at 37 °C. Afterwards, the nutrient-rich gel was poured on top of the first gel together with 10 mM sodium phosphate buffer containing 6 % TSB (w / v) and 1 % agarose. The inhibition zones were measured after 24 hours. 0.01 % acetic acid was used as a negative control, which did not show an inhibition zone larger than the diameter of the punched-out well. All experiments were performed at least three times.

[0162] Turbidity broth assay

[0163] The bacteria tested were incubated overnight in 1 x TSB broth, centrifuged and washed with 10 mM sodium phosphate buffer containing 1 % (w / v) TSB broth. 5 x 10 5 cfu / ml bacteria were mixed with different concentrations of peptides in 10 mM sodium phosphate buffer containing 1 % (w / v) TSB (final volume 100 μΐ) and incubated for 2 hours at 37 °C. Afterwards, 100 μΐ 2 x TSB broth was added and the optical density at 600 nm was measured (Spark 10M, Tecan, Austria). Bacterial growth was monitored for 12 hours, the bacteria were grown at 37 °C under shaking during the measurements every 30 minutes, except for M. sputenii, which was incubated in an anaerobic jar at 37 °C and the growth was measured after 72 hours.

[0164] The bactericidal activity of E. coli and S. aureus strains in the cell wall target assay as well as in the HD5 dimer assay was assessed as described before. Log phase bacteria were collected by centrifugation (2500 rpm, 10 min, 4°C), washed twice with 10 mM sodium phosphate buffer containing 1 % (w / v) TSB broth and the optical density at OD600 nm (OD600 = 0.1) was determined. Approximately 5 x 10 5 CFU / ml of bacteria were incubated with serial concentrations (1.17 - 150 μΜ) of peptides in a final volume of 100 μΐ in 10 mM sodium phosphate buffer containing 1 % (w / v) TSB broth at 37°C for 2 h. After incubation, 100 μΐ of 6% TSB (w / v) was added and the absorbance at 600 nm was measured (Tecan, Switzerland) and monitored for 18 h. Then, 100 μΐ per well was plated on LB plates to determine the number of viable bacteria microbiologically. The bactericidal activity was expressed as LC99.9, the lowest concentration that kills > 99.9% of the bacteria. The experiments were repeated independently at least three times.

[0165] Cytotoxicity assay of cells

[0166] CaCo2 / TC7 (X,X) and HT29 MTX E29 (X,X) were seeded in 96 well plates in 90 μΐ medium (1500 cells / well) and incubated for 24 h at 37°C. Thereafter the treatment with different concentrations of peptides (10 μΐ volume dissolved in 0.01 % acetic acid) was started and the cells were incubated for 96 h. Untreated and 1 % Triton-X treated cells were used as controls. After incubation, 100 μΐ of CellTiter Glo 2.0 solution was added and the measurement protocol was started. The measurement was performed in a Spark 10M (Tecan) starting with 12 min continuous shaking followed by luminescence measurement with an integration time starting from 1 s per well. The experiments were performed in duplicates.

[0167] Hemolytic assay

[0168] Measurement of hemolytic activity after existing protocol 13Blood was obtained from two voluntary donors (sample collection was previously approved by the Ethical Committee of the University Hospital of Tubingen, Germany) and 1 ml blood was washed twice with PBS. Then blood was centrifuged at 1000 g and a 1% (v / v) blood suspension in PBS was performed. The blood suspension was incubated with different concentrations of the peptide (final concentration 0.5%) for 1 h at 37°C. Then the samples were centrifuged at 1000 g for 10 min and the supernatant was collected and measured at 414 nm. The hemolytic activity is a relative value determined against the hemolytic activity of 0.1% Triton X-100. These experiments were performed in duplicate.

[0169] In vivo microbiota analysis

[0170] To assess the proof of concept for a functional impact on the composition of the microbiota, HD-5 1-9 Nine-week-old healthy chow-fed male mice were housed in groups of 3 per cage and administered. Mice were acclimated to the environment for 3 weeks prior to the start of the experiment and were divided into experimental groups according to the average body weight per cage, ensuring equal body weight distribution between groups. In more detail, wild-type C57BL / 6J mice were administered with 7.19 pg / mouse HD-5 1-9 for 7 days. Control mice were treated with an equal volume of PBS. Initially, a 7-day experiment was performed with 6 mice per group. Based on these results, a new study was designed, including also 6 mice per group, to investigate the time impact of gut microbial modulation. In this study, a 7-day washout period was included after 1 week of oral gavage. Fresh fecal samples were collected from individual mice at 9 AM on days 0, 7, and 14 (n = 6 for each treatment group and control group) while body weight was measured. On day 14, mice were euthanized and small intestinal contents were collected.

[0171] Bacterial DNA was extracted from snap-frozen fecal and necropsy small intestinal contents collected at days 0, 7 and 14 using the NuceloSpin 96 Soil kit (Macherey-Nagel) according to the manufacturer’s instructions. Subsequent library preparation and DNA sequencing were performed by BGI, Europe using in-house standard operating procedures. Briefly, 30 ng of bacterial DNA per sample was PCR amplified using Illumina adapters targeting the V4 16S rDNA region using the following primer pair: 515F: GTGCCAGCMGCCGCGGTAA (SEQ ID No. 7), 806R: GGACTACHVGGGTWTCTAAT (SEQ ID No. 8). PCR products were then purified with Ampure XP beads (AGENCOURT) to remove non-specific products. The average molecular length was determined by an Agilent 2100 Bioanalyzer (Agilent DNA 1000 reagent). DNA quantification was assessed by real-time quantitative PCR (EvaGreen TM ) prior to paired-end sequencing on a HiSeq2500 system.

[0172] Reads processing and quality control were performed using the R package DADA2, version 1.4.0 14 and forward and reverse primers were trimmed from the reads. Next, all reads containing remaining uncalled bases or more than two expected errors were removed. After that, parameters of the DADA2 error model were learned from a random subset of 1 million reads. This error model was then used to denoise all sequences; i.e., to infer ASVs. Denoised reads (ASVs) were then merged and read pairs with one or more conflicting bases between forward and reverse reads were removed. ASVs shorter than 251 bases and longer than 254 bases were discarded. Chimeric sequences were then detected and removed using the function “removeBimeraDenovo”. Finally, reads (ASVs) were classified from phylum to genus level using the Silva reference 16S rRNA gene database, version 132, resulting in an ASV table containing read counts for all ASVs in all samples.

[0173] All animal protocols were conducted according to the guidelines established by the Laval University Animal Care and Handling Committee. C57BL / 6J male mice (Jackson Laboratories, Bar Harbor, ME) were housed in a pathogen-free, temperature-controlled environment under a 12:12 hour light-dark cycle and were given ad libitum access to standard rodent chow (Harlan Teklad T-2018) for 5 weeks at our animal facility (3 weeks of acclimatization and 2 weeks of experimental protocol).

[0174] Statistical analysis

[0175] All data were analyzed using GraphPad Prism 7 except for microbiome analysis. Values of p < 0.05 were considered statistically significant. All results are expressed as mean and its ± standard deviation, and standard error of the mean or 80% confidence interval, as indicated in the legends. Bioinformatics analyses were performed using R Studio (R version 3.4.2 and R Studio version 1.0.136) and packages phyloseq 1.22.3 15 , metagenomeSeq 1.20.0 16 , vegan 2.4-4 17 , Ime4 1.1-15 and ggplot2 2.2.1 18 For the mouse study, cage-adjusted p values were used.

[0176] Software

[0177] For the in silico digestion analysis, the ExPASy Peptide Mass tool from the SIB Bioinformatics Resource Portal (https: / / web.expasy.org / peptide_mass / ) was used.

[0178] Results

[0179] Native human duodenal fluid digests HD-5, while the nanonet forming HD-6 is resistant to proteases

[0180] Since Paneth cell defensins can be reduced by the naturally occurring thioredoxin system, their susceptibility to proteolytic digestion was investigated. Prior to the start of the experimental procedure, the possible fragmentation of HD-5 and HD-6 by intestinal proteases was investigated. Therefore, the PeptideMass module of ExPASy (SIB Bioinformatics Resource Portal) was used to perform in silico digestion of HD-5 and HD-6 using trypsin, chymotrypsin or a combination of both, and was allowed up to five missing cleavages. The possible fragments are listed in Table 1 below according to their respective masses.

[0181] Table 1: In silico (normal letters) digests of Paneth cell HD-5 and HD-6 in the presence of trypsin or chymotrypsin or a combination of both and ex vivo reality (bold letters) digests after incubation with duodenal mucus, up to five missing cleavages and fragments larger than 500 Da. The different sequences were determined using the ExPASy PeptideMass module. Fragments that can be identified by mass spectrometry after incubation of human peptides with human duodenal mucus are in bold. The first row in this table represents the two full-length peptides, which can also be identified.

[0182]

[0183]

[0184]

[0185] In theory, both Paneth cell defensins seem to be susceptible to proteases, while HD-6 shows a tendency to more fragmentation compared to HD-5 (Table 1). In a second step, HD-5 or HD-6 were reduced with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) and the peptides were subjected to the attack of naturally occurring duodenal fluid, which is known to have proteolytic activity. After performing mass spectrometry analysis, it was found that HD-6 was partially reduced in the presence of 2 mM TCEP, which is in agreement with previously published work. In addition to the two full-length forms, i.e. HD-6 ox ( expected: 3705.49 Da) and HD-6 red (3711.54 Da), surprisingly no other fragments were identified, which could be identified by the mass-to-charge ratio (m / z) signals indicative of 2-, 3-, 4-, 5-, 6-fold protonated ions. This surprising observation indicates that HD-6 red can resist proteolytic digestion, although the reasons remain elusive, as proteolytic cleavage sites are predicted based on bioinformatics. It is well known that HD-6 has two forms that are independent of its redox state, HD-6 ox and HD-6red ) were able to form nanonets. Thus, it was hypothesized that the network formation could resist protease degradation and thus could provide a mechanistic explanation for the observed peptide protection. To elucidate whether nanonet formation resulted in a more stable structure, scanning electron microscopy was performed on reduced HD-6 incubated with duodenal fluid (Figure 1). In agreement with this hypothesis, the same nanonets were observed independent of duodenal fluid incubation ( Figure 1B ). Taken together, the formation of nanonets appears to at least contribute to the prevention of proteolytic digestion of HD-6.

[0186] Next, the second and more abundant Paneth cell defensin, HD-5, was investigated in the same experimental setup. After incubation with 2 mM TCEP and duodenal fluid, HD-5 ox was below the limit of detection (LOD), whereas HD-5 red was highly abundant. Although HD-5 was not able to form nanonets, surprisingly, duodenal fluid had a different effect on HD-5 compared to HD-6. In agreement with known protease cleavage sites, different fragments were identified that were not present before the addition of HD-5 ( Figure 2A highlighted in bold letters). These fragments were analyzed and their mass-to-charge ratios were listed, and for each fragment different observed protonated ions were shown with their masses ( Figure 2B ). The identified fragments with their neutral masses and retention times were listed, indicating that the fragmentation of HD-5 in the presence of duodenal fluid resulted in a large number of fragments originating from the entire peptide sequence. However, we still found detectable amounts of full-length HD-5 red , indicating that the proteolytic digestion was not complete. It is known that Zn 2+ can protect HD-5 red from proteolytic digestion, which was indeed confirmed in our setup (data not shown). In summary, the results indicate that duodenal fluid surprisingly has a different effect on HD-5 and HD-6, and that changes in the conditions in the local microenvironment have an impact on defensin fragmentation. It is noteworthy that HD-6 nanonet formation appears to prevent the reduced full-length peptide from being destroyed, and it is known that reduction of HD-6 reveals antimicrobial activity. Thus, reduction changes the activity of both Paneth cell defensins, but HD-6 gains direct antimicrobial activity, whereas HD-5 is digested by intestinal proteases to form biologically active peptide fragments with potentially biologically active antimicrobial activity.

[0187] HD-5 fragments have antimicrobial activity in radial diffusion assays

[0188] To test the antimicrobial activity of HD-5 fragments, selected fragments were chemically synthesized to investigate the antimicrobial function in vitro (Figure 2C ). Several radial diffusion assays were performed using different fragments in amounts of 4 pg and 2 pg of full-length peptide against commensal and pathogenic bacteria. Interestingly and surprisingly, it was observed that most of the HD-5 -derived fragments showed antimicrobial activity against commensal and pathogenic bacteria to different extents. Moreover, different fragments were found to exhibit different activity patterns against the applied bacterial strains Figure 3A and B). As expected, full-length HD-5 showed a broad antimicrobial activity. Still surprisingly, HD-5 1-9 was identified as the most active peptide in terms of antimicrobial activity and in terms of versatility, as it was active against all tested bacteria. In comparison to HD-5 1-9 , HD-5 10-27 did not show measurable activity. Notably, HD-5 1-13 , HD-5 1-28 , HD-5 7-32 and HD-5 26-32 also showed antimicrobial properties, although to a lesser extent than HD-5 1-9 . To further assess the resulting phenotype of the peptide-treated bacteria, E. coli MC1000 was incubated with all fragments and transmission electron microscopy (TEM) was performed Figure 3C ). It was observed that HD-5 fl treatment led to inner membrane detachment and small vesicular structures around the bacterial cell envelope (Figure 8). Interestingly, the bacteria surprisingly showed different typical phenotypes after incubation with different fragments of HD-5, indicating differences in the mode of action. For HD-5 1-9 , inner membrane detachment and additional large vacuolar structures at one pole of the bacteria were observable, while HD-5 1-13 and HD-5 1-28 treatment led to more aggregation inside the bacteria (Figure 8). The results of the study indicate different kinds of bacterial phenotypes and this diversity suggests that small sequence differences, such as HD-5 1-9 and HD-5 1-13 , can lead to different host-microbe interaction mechanisms.

[0189] Notably and surprisingly, these observations indicate that the antimicrobial activity of the different peptides does not apply to a certain class of bacteria. As an example, the Bifidobacterium strains B. adolescentis and B. longum were highly sensitive to the peptides, while B. breve was not. Overall, the results mentioned above indicate that proteolytic digestion of the Paneth cell HD-5 yields small antimicrobial active fragments, which modulate commensal gut bacteria.

[0190] Next, the peptides were investigated against pathogenic Gram-positive and Gram-negative bacteria (Figure 4A and B) were found to have antimicrobial activity, but the antimicrobial effect varied greatly between fragments. Surprisingly, HD-5 1-9 , HD-5 1-13 , HD-5 7-32 and HD-5 fl had a strong impact on the growth of each of the bacteria tested, while other fragments such as HD-5 1-28 , HD-5 10-32 and HD-5 26-32 had only minimal activity or were more selective with respect to different strains. In contrast, HD-5 14-32 and HD-5 10-27 were not active against the bacteria tested under the conditions tested. Interestingly, the activity of HD-5 10-32 was limited to two Gram-positive bacteria. In agreement with the findings described in the first experiment (Figure 3), and with respect to the efficiency of killing the symbiont, HD-5 1-9 was active against all strains tested, regardless of the Gram status, indicating that this particular fragment has a high degree of protection against the bacterial barrier. In contrast, HD-5 1-13 strongly modulated the pathogenic bacteria tested, with the exception of K. pneumoniae 3-MRGN. In summary, these results indicate that proteolytic digestion of the Parnell cell HD-5, but not HD-6, resulted in a large number of short and active antimicrobial fragments. The significance of this unexpected finding can prove to be very important, as these fragments expand the antimicrobial diversity based on a single full-length peptide according to local environmental conditions.

[0191] Minimum inhibitory concentrations of HD-5 fragments and their effect on antibiotic-resistant bacteria

[0192] To further investigate the potential of these fragments for potential antibiotic treatment use, we measured the minimum inhibitory concentrations (MIC) of our peptides against different antibiotic-resistant Gram-negative and Gram-positive bacterial strains. The results of the previously described RDA experiments indicated promising antimicrobial activity against both symbiotic and pathogenic bacteria. To gain a more detailed understanding of the antimicrobial abilities of the different HD-5 fragments, we performed turbidity broth assays to determine the MIC of these peptides. The MIC was determined as the concentration at which no bacterial growth was detected after 12 hours in all experiments. Using this parameter, we were able to detect the antimicrobial activity of HD-5 1-9 , HD-5 1-13 , HD-5 1-28 , HD-5 7-32 and HD-5 10-32 (see Table 2 below).

[0193] Table 2: MIC of HD-5 fragments in μΜ and μg / ml against pathogenic bacteria. Each experiment was performed at least three times. MIC was determined as the concentration at which no bacteria grew after 12 hours of incubation in each experiment.

[0194]

[0195] Comparing the MIC of these fragments in μΜ to the MIC of the full-length peptide in μΜ, surprisingly, most of the defensin fragments were as active as the full-length peptide. However, when comparing the μg / ml MIC concentration of the peptide fragments, they required a lower μg / ml MIC concentration and based on this concentration - especially HD-5 1-9 was more active than the full-length peptide. Notably, in addition to HD-5 1-9 The observed MICs were relatively high except for the MIC against A. baumannii 4-MRGN and E. faecium 475747, indicating that the role of the substance with antimicrobial activity in the intestinal barrier is limited to high concentration areas, such as the crypts. To elucidate their functional capacity in vivo, our aim was to investigate their intestinal microbiome modulating function, especially since Paneth cells are naturally located in the intestinal tract.

[0196] Antimicrobial activity was determined by the minimum inhibitory concentration against gram-negative and gram-positive bacteria and Candida species to explore whether the mode of action of HD5 1-9 was dependent on disulfide bridges and charge. Therefore, several variants of HD5 1-9 were synthesized, including variants obtained by exchanging cysteine with alpha-aminobutyric acid (Abu) and substitution of arginine with citrulline (Cit). In addition, the reverse (RGTRCYCTA) and random (CTRATYCRG) amino acid structure of HD5 1-9 was tested. HD5 1-9 showed a strong bactericidal effect against E. coli BW25113, while HD5 1-9 showed a lower antimicrobial activity against Salmonella enterica enterica serovar var. The reverse variant RGTRCYCTA was observed to have a similar effect on both gram-negative bacteria.

[0197] If the sequence was random and when lacking cysteine or arginine amino acids, HD5 1-9 variants surprisingly lost their antimicrobial activity against the tested gram-negative bacteria. For both Staphylococcus species, no bactericidal effect of HD5 1-9 or its variants was determined. In contrast, HD51-9 showed a strong bacterial activity. However, the cysteine and arginine substitutions of HD5 1-9 led to a complete loss of the bactericidal effect, as did the random sequences RGTRCYCTA and CTRATYCRG. In contrast to E. faecalis, none of HD5 1-9 and its variants showed a bactericidal effect against E. faecium. The antimicrobial activity of HD5 1-9 and the variants against both Candida species was investigated after 24 hours. For C. tropicalis, which showed a low antimicrobial profile, an inhibition of fungal growth could be observed. The cysteine and arginine substitutions of HD5 1-9 had no influence on the inhibition of fungal growth. A similar effect was observed for C. albicans, while HD5 1-9 and the variants did not show an antimicrobial activity. In summary, the results strongly emphasize the importance of the present cysteine and arginine residues and the original amino acid structure of HD5 1-9 for the induction of an antimicrobial effect.

[0198] The following table 3 gives the antimicrobial activity of HD5 1-9 against the tested bacteria and Candida species. The minimum inhibitory concentrations (MIC) of HD5 1-9 at different concentrations against E. coli BW 25113, Salmonella enterica enterica serovar var., S. aureus SA113, S. epidermidis Evans 1916, E. faecalis ATCC 19433, E. faecium ATCC 19434, C. tropicalis ATCC 4563 and C. albicans ATCC 10231 were determined after 18 hours or 24 hours according to the optical density. Results from three independent experiments are given:

[0199]

[0200] The following table 4 also gives the effect of HNP-4 fragments according to the present application. From this table it can be seen that the HNP-4 peptide fragments according to the present application show a similar antimicrobial behavior as the HD-5 1-9 peptide.

[0201] Table 4: The following table 4 gives the antimicrobial activity of HD5 1-9 , HD5 1-9；mod , HNP-4 1-11 and HNP-4 1-11；modAntimicrobial activity against the tested bacteria and C. albicans. The minimum inhibitory concentration (MIC) of different concentrations of the peptide against A. baumannii 4-MRGN, A. baumannii DSM 30007, E. faecium 475747, E. faecium DSM 20477, K. pneumoniae 3-MRGN, K. pneumoniae DSM 301404, P. aeruginosa 4-MRGN, P. aeruginosa ATCC 27853, P. aeruginosa PAOl, P. aeruginosa XPATl, P. aeruginosa XPAT2, S. aureus USA300, S. aureus ATCC 25923, S. enteritidis, E. coli BW 25113, Y. enterocolitica, and C. albicans 525L was determined after 12 hours according to optical density. Results from three independent experiments are given:

[0202]

[0203] HD-5 1-9 Treatment affected certain bacterial genera, but not microbial diversity.

[0204] To explore the microbiome modulatory function of the identified HD-5 fragment, feed- fed mice were treated orally with HD-5 1-9 or PBS for 7 days (7.19 pg / mouse) followed by a 7-day washout period. The microbiome composition of the two groups of mice was indistinguishable at baseline (Adonis PERMANOVA using Bray-Curtis distance, p = 0.22). After 7 days of HD-5 1-9 treatment, there was a borderline difference in the overall microbiome composition in the fecal samples compared to controls (Adonis PERMANOVA using Bray-Curtis distance, p = 0.08), but not compared to the baseline microbiome of the group (Adonis PERMANOVA using Bray-Curtis distance, p = 0.38, data not shown). Since HD-5 is naturally secreted by the Paneth cells of the small intestine, we explored the microbiome of the small intestine at necropsy. After the 7-day washout period, there was no statistical change in the microbiome community structure in the small intestine of HD-5 1-9 treated mice compared to controls (Adonis PERMANOVA using Bray-Curtis distance, p = 0.09, data not shown). This result is consistent with the initial experiment, suggesting that HD-5 1-9 Despite having significant antimicrobial efficacy, HD-5 did not surprisingly alter the overall fecal microbiome composition of healthy, feed-fed mice (Figure 12).

[0205] Shannon diversity in fecal microbiota composition was equal among the groups at baseline (Wilcoxon test, p = 1), and in HD-5 1-9 The differences remained similar after 7 days of treatment (Wilcoxon test, p = 0.18) and after the 14-day clearance period (Wilcoxon test, p = 0.07) (data not shown). At day 14, the diversity of the small intestinal microbiota was also similar between the two groups (Wilcoxon test, p = 0.45). Figure 5B (Right), but not on day 7, when HD-5 mice were compared to control mice fed with vector via tube feeding. 1-9 The treated mice unexpectedly showed increased bacterial diversity (p = 0.004, data not shown).

[0206] The experimental design further allows for paired analysis of fecal samples using a stratified linear mixture model that addresses potential co-cage effects and repeated sampling from the same mouse. HD-5 was identified. 1-9 It had a significant impact on some low-abundance microbial genera in fecal samples. More specifically, an increase in the relative abundance of *Parasartella* and *Candidatus Stoquefichus* was observed, while the relative abundance of *GCA-900066575* from the *Trichophyceae* family and *Hydrogenogenic Anae* genus was affected by HD-5. 1-9 Treatment reduces the incidence of Akkermansia in fecal samples due to HD-5. 1-9 The marginal increase in treatment (linear mixture model, p = 0.0754, (data not shown)) confirms that *Akermansia* spp. due to HD-5 1-9 Preliminary experimental findings showed that the treatment specifically increased (linear mixture model, p = 0.0748). Despite HD-5 1-9 The relative abundance of *Akermansia* increased (linear mixture model, p = 0.0085), but at day 14, the small intestinal microbiota was roughly similar between the two groups, consistent with results from previous experiments (linear mixture model, p = 0.017). Furthermore, the relative abundance of *Ruminococcus* _1 (Ruminococciaceae) increased, while the relative abundance of *Enteromonococcus*, *Clostridium* ASF356, and *Ruminococciaceae* UCG-013 decreased (data not shown).

[0207] Taken together, these results indicate that HD-5 1-9 It altered the amount of certain low-abundance bacteria in the fecal microbiota, surprisingly without affecting the overall community structure or diversity of healthy, feed-fed mice.

[0208] In addition, the presence of *Akermansia myxophilus* in the determination of turbidity broth was tested to determine whether it affected HD-5. 1-9Sensitive. The studies showed that even small concentrations of HD-5 1-9 slightly reduced the growth of Akkermansia muciniphila, but did not kill the bacteria Figure 9B

[0209] HD5 1-9 was tested for pro- and anti-inflammatory effects, and although the fact that the anti-inflammatory effects were low and had a dose-dependent trend to decrease IFN-γ and IL-8, this was not statistically significant, and the dose-dependent trend to increase IL-10 was not statistically significant either Figure 10

[0210] Toxic effects of HD5 1-9 were also tested in a number of in vitro experiments (Figures 11 and 12), but surprisingly no toxicity was observed, although HD5 1-9 is a short linear peptide.

[0211] Identification of cell wall targets in mutants of E. coli BW25113 and S. aureus SA113

[0212] The mode of action of HD5 1-9 was identified using cell wall mutants of E. coli BW 25113 and S. aureus SA113. The aim was to analyze which components of the cell wall are important for the binding of HD5 1-9 and whether the charge plays a key role. Turbidity assays were performed as described above. E. coli strains with different LPS structures were investigated by determining the MIC Figure 16 . Wild-type E. coli BW 25113 as well as E. coli ATCC 25922 were used as controls. The last one contains a similar cell wall composition as E. coli BW 25113, but additionally O antigens and thus full-length LPS. The mutant E. coli BW 25113△waaG contains the same number of phosphate residues in the inner membrane as the wild type and thus a similar charge, but lacks the outer core. The mutant△waaY contains the outer core, while some of the phosphate residues in the inner core are missing. The last E. coli mutant△waaP also has the outer membrane, but no phosphate residues in the inner membrane, resulting in a more positively charged inner membrane 19 .

[0213] The antimicrobial activity of HD5 1-9 was analyzed on different E. coli strains and mutants. E. coli ATCC 25922 with full-length LPS was as highly sensitive to HD5 1-9 as the tested E. coli BW25113, which lacks O antigens. To elucidate the importance of the charge for the activity of HD5 1-9 ​​The function of the outer core in the binding process to the bacterial cell wall was investigated using the mutant ΔwaaG. MIC determination showed that HD5 1-9 was not more resistant to ΔwaaG than to E. coli ATCC 25922 and E. coli BW25113. Surprisingly, E. coli ΔwaaY, which lacks one inner core phosphate, and E. coli ΔwaaP, which lacks two inner core phosphates, were not more resistant to HD5 1-9 . This observation was surprising, as cationic antimicrobial peptides are known to interact with the anionic phospholipids in the cell wall due to electrostatic interactions. The absence of this negative charge in the inner core of the cell wall would normally decrease the ability of the peptide to bind to the bacterial cell wall. However, HD5 1-9 surprisingly showed bactericidal effects at a concentration of approximately 12.5 μΜ against all described E. coli mutants. One explanation could be that the antimicrobial activity does not only depend on the negative charge of the cell wall, but that additional binding sites must be present in Gram-negative bacteria.

[0214] The antimicrobial activity of HD5 1-9 and variants against various S. aureus SA113 strains with different cell wall mutations was investigated (Figure 17). The aim was to study how HD5 1-9 is able to bind to the cell wall of Gram-positive bacteria and whether the charge plays a decisive role for the antimicrobial activity of HD5 1-9 .

[0215] The first mutant, ΔdltA, does not contain D-alanine in the peptidoglycan layer, resulting in a more negatively charged peptidoglycan layer 20 . The mutant ΔmprF lacks L-lysine, resulting in a more negatively charged cell membrane 21 . The last mutant, ΔtarH, additionally contains wall teichoic acids, resulting in an enhanced peptidoglycan 22 .

[0216] HD5 1-9 did not show growth inhibition against the wild-type S. aureus SA113, while the S. aureus ΔdltA mutant, which contains a peptidoglycan layer with more negative charge, was much more susceptible to HD5 1-9 , showing a MIC of 6.25 μΜ Figure 15 . HD5 1-9 also showed bactericidal effects against the ΔmprF mutant, which lacks L-lysine, resulting in a more negatively charged bacterial cell membrane. However, the enhanced peptidoglycan layer due to the additional teichoic acids in the S. aureus ΔtarH mutant surprisingly decreased the antimicrobial effect of HD5 1-9 .

[0217] These results highlight the importance of cell wall charge for HD5. 1-9 The importance of binding to Gram-positive bacteria is emphasized, while cell wall charge appears to be less important for binding to Gram-negative bacteria.

[0218] For HD5 1-9 and HD5 1-9 Characterization of the antimicrobial activity of the dimer against different bacteria

[0219] HD5 contains leucine residues that enable it to form dimers. Substitution of leucine residues in HD5 leads to a decrease in its antimicrobial activity and ability to kill microorganisms, indicating that dimerization of HD5 is important for its function (Rajabi et al., 2008, 2012; Szyk et al., 2006). 23-25 Furthermore, cysteine ​​residues in HD5 can form dimers due to disulfide or hydrogen bonds. Studies have shown that cysteine ​​mutations in HD5 affect oxidative folding, antibacterial activity, Gram-negative bacterial membrane permeability, and proteolytic stability (Wanniarachchi et al., 2011). 26 .

[0220] Therefore, we can reasonably assume that HD5 is due to the presence of cysteine ​​residues. 1-9 It can also form dimers. HD5 1-9 The two monomers are linked by disulfide bonds, resulting in dimerization. The aim is to investigate the formation of HD5 in dimer form. 1-9 Compared to the HD5 built in a monolithic form 1-9 Compared to antimicrobial activity against selected bacteria, HLPC and mass spectrometry identified the dimer as having a microwave range of 2058, consistent with a disulfide bond between the two monomers.

[0221] The turbidity was measured for HD5. 1-9 The minimum inhibitory concentration for Gram-positive bacteria (Staphylococcus aureus species) and Gram-negative bacteria (Salmonella species) treated at the same concentration.

[0222] The experiments conducted confirmed that HD5 1-9 The antimicrobial activity against the tested bacteria was surprisingly similar to, or even better than, its dimer form. HD5 1-9 And its dimer form has almost the same antimicrobial activity against different Salmonella species. Figure 18 However, surprisingly, the HD5 in its monomer form... 1-9 In comparison, the dimerized HD5 1-9 It exhibits much better bactericidal activity against Staphylococcus aureus species. Figure 19 This implies two forms of HD5. 1-9surprisingly different modes of action.

[0223] Identification of new HNP-4 fragments after trypsin digestion

[0224] HNP-4 was incubated with 2 mM TCEP to open disulfide bridges, thus forming a linear structure more susceptible to proteolytic digestion. We analyzed the reduced HNP-4 incubated with trypsin by LC / MS method and were able to detect several fragments Figure 20 A). Based on the observed ions and their mass-to-charge ratios, we were able to clearly identify the fragments, which were mainly located in the N-terminal region based on the trypsin cleavage sites. Since it is generally accepted that the net charge of AMPs can play an important role in their antimicrobial activity, we focused on the HNP-4 1-11 fragment with a +3 positive net charge.

[0225] Antimicrobial efficacy of HNP-4 1-11

[0226] Since short linear peptides are naturally less stable, we used an additional modified form of HNP-4 1-11 (HNP-4 1-11mod ). Here, we exchanged L-amino acids with D-amino acids and modified the N-terminus (acetylation) and the C-terminus (amidation). Both modifications should lead to an increased stability 27,28 and thus possibly to a stronger antimicrobial activity. To analyze the antimicrobial activity of HNP-4 fl , HNP-4 1-11 and HNP-4 1-11mod , we used RDA against different subgroups of commensal and pathogenic bacteria. All tested peptides showed strong antimicrobial activity against most of the tested bacteria Figure 21 ). While RDA is a suitable assay for determining the general antimicrobial activity of different peptides, it is not possible to compare them based on their different abilities (e.g. spreading) in agarose gels. We therefore next used turbidity broth assays to determine the MIC of HNP-4 fl , HNP-4 1-11 and HNP-4 1-11mod against pathogenic (some multi-drug resistant) Gram-negative and Gram-positive bacteria and one fungal strain (Table 5). While all peptides exhibited antimicrobial activity against the tested bacteria (only exception: HNP-4 fl exhibited antimicrobial activity against K. pneumoniae DSM 30104), the molar concentrations of HNP-4 1-11 and HNP-4 fl were surprisingly equal, indicating that the generation of HNP-4​fl antimicrobial efficacy of the native complex relies only on the first 11 amino acids (HNP-4 1-11 ). It is further noted that such linear fragments, HNP-4 1-11mod , are expected to exhibit increased stability compared to the unmodified version. HNP-4 fl and HNP-4 1-11 both have enhanced bactericidal efficacy over HNP-4 1-11 , with MICs several-fold lower than observed for the naturally occurring full-length peptide. Thus, by releasing the antimicrobial activity of the full-length peptide by trypsin digestion, we identified individual fragments with significant antimicrobial potential that exceeded the antimicrobial potential of the full-length peptide at the molar level. Surprisingly, we observed that the antimicrobial efficacy of the peptides was equally effective between multi-drug resistant and non-drug resistant strains. Thus, proteolytic digestion of AMPs can be used to generate new active sequences that can lead to new strategies to overcome antibiotic resistant bacteria.

[0227]

[0228] HNP-4 1-11 and HNP-4 1-11mod cytotoxicity and hemolytic effects

[0229] To determine the potential of HNP-4 1-11 and HNP-4 1-11mod as therapeutic agents for in vivo applications, we used two different cell lines to investigate their cytotoxic abilities. While we only observed a slight cytotoxic effect on CaCo2 / TC7 cells at higher peptide concentrations Figure 22A ), HT29 MTX E29 cells were more sensitive to both of the tested peptide derivatives Figure 22B ). Importantly, at lower concentrations (e.g. 12.5 μΜ, at which HNP-4 1-11mod has strong antimicrobial effects), these fragments only exhibited modest cytotoxicity. We additionally examined the hemolytic activity of the peptides Figure 22C . While HNP-4 1-11mod had 20% hemolysis at 150 μΜ (far exceeding the highest concentration required for bactericidal efficacy), the toxicity at < 18.75 μΜ (i.e. the highest biologically relevant concentration) was negligible. Thus, both HNP-4 1-11 and HNP-4 1-11mod have low hemolytic activity compared to melittin, which showed 80% hemolysis at 1.25 μΜ. In summary, the magnitude of the cytotoxic concentrations identified are much higher than the corresponding bactericidal concentrations.

[0230] SUMMARY

[0231] In the research and invention as presented above, several new findings were made regarding a-defensins and their susceptibility to proteases. The in silico digestion of HD-6 showed, quite surprisingly, that HD-6 is not affected by the naturally occurring proteases in duodenal juice. Unlike HD-6, HD-5 is degraded and its fragments surprisingly contain antimicrobial activity against both commensal and pathogenic bacteria.

[0232] The determination of the antimicrobial profile of the HD-5 fragments revealed that it has high antimicrobial activity against both commensal and pathogenic bacteria. The antimicrobial profile is different from that of the parent peptide and also differs between the HD5 fragments, thus these HD-5 fragments seem to add additional bacterial killing or microbiota modulating capacity. This interesting and surprising phenomenon also contributes to the understanding of how some intestinal defensins can modulate or support very different commensal colonization of different parts of the intestine.

[0233] Within the present research and invention, the results show that HD5 1-9 , HD5 1-13 , HD5 1-28 , HD5 7-32 , HD5 10-32 , HD5 14-32 , HD5 10-27 and HD5 26-32 have microbiota modulating effects. In addition to the effects on some low abundance bacterial strains, the current results show, surprisingly, that oral treatment of mice with HD5 1-9 increases the amount of Akkermansia muciniphila in mice compared to untreated mice, in addition to other bacteria, Akkermansia muciniphila is not susceptible to HD5 1-9 in turbidity broth assays, in line with the increased finding of Akkermansia in the microbiome analysis. This surprising effect on Akkermansia muciniphila has not been previously described for the full length of HD-5 peptide. This highlights the different spectra between the full length peptide and its fragments.

[0234] The present research and invention highlight the importance of the cell wall charge for HD5 1-9 binding to gram-positive bacteria, while the cell wall charge seems to be less important for binding to gram-negative bacteria. The experiments performed further surprisingly demonstrate that dimerized HD5 1-9 exhibits much better bactericidal activity against Staphylococcus aureus species compared to HD5 1-9 in monomeric form. This implies that the two forms of HD5 1-9 have different modes of action. HNP-4 1-11 is more effective in killing bacteria than HNP-4 flsurprisingly equal, indicating that the antimicrobial efficacy of the native complex of HNP-4 fl is dependent only on the first 11 amino acids (HNP-4 1-11 ). Surprisingly and further to note, the linear fragment HNP-4 1-11mod was expected to exhibit increased stability compared to the unmodified version, had enhanced bactericidal efficacy, superior to both HNP-4 f1 and HNP-4 1-11 and a MIC several fold lower than observed for the naturally occurring full length peptide. We thus released the antimicrobial activity of the full length HNP-4 peptide by trypsin digestion, whereby we identified a single fragment with significant antimicrobial potential that exceeded the antimicrobial potential of the full length peptide at the molar level. Surprisingly, we observed that the antimicrobial efficacy of the peptide was equally effective between multi-drug resistant strains and non-drug resistant strains.

[0235] In addition to the microbiota modulating ability, another important area for antimicrobial active peptides is the rapid increase in the number of antibiotic resistant bacteria. The antimicrobial profile of the peptide fragments identified herein allows for the use of these peptides as a source of new antibiotics against multi-drug resistant bacteria. Furthermore, the discovery of these easily produced and inexpensive peptide fragments is a new alternative approach for therapeutically manipulating the microbiome composition and treating Paneth cell related diseases such as Crohn's disease of the small intestine.

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[0262] 26. Wanniarachchi, Y. A., Kaczmarek, P, Wan, A & Nolan, E, M. Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity Against Staphylococcus aureus. Biochemistry 50, 8005-8017 (2011).

[0263] 27. Hong, S. Y., Oh, J. E. & Lee, K. H. Effect of D-amino acid substitution on the stability, the secondary structure, and the activity of membrane-active peptide. Biochem Pharmacol. 58, 1775-1780 (1999).

[0264] 28. Brinckerhoff, L. H. et al. Terminal modifications inhibit proteolytic degradation of an Immunogenic MART-1 (27-35) peptide: implications for peptide vaccines. Int. J. Cancer 83, 326-334 (1999). SEQUENCE LISTING <110> ESCALCES BIOLOGICS LLC <120> Defensin fragments for use in therapy or prophylaxis <130> P5503PC00 <160> 98 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 1 Ala Thr Cys Tyr Cys Arg Thr Gly Arg 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 2 Arg Gly Thr Arg Cys Tyr Cys Thr Ala 1 5 <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 3 Val Cys Ser Cys Arg Leu Val Phe Cys Arg Arg 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 4 Arg Arg Cys Phe Val Leu Arg Cys Ser Cys Val 1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> Acetylation <220> <221> MISC_FEATURE <222> (1)..(7) <223> D-amino acid <220> <221> MOD_RES <222> (9)..(9) <223> Amidation <220> <221> MISC_FEATURE <222> (9)..(9) <223> D-amino acid <400> 5 Ala Thr Cys Tyr Cys Arg Thr Gly Arg 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> Acetylation <220> <221> MISC_FEATURE <222> (1)..(11) <223> D-amino acid <220> <221> MOD_RES <222> (11)..(11) <223> Amidation <400> 6 Val Cys Ser Cys Arg Leu Val Phe Cys Arg Arg 1 5 10 <210> 7 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 7 gtgccagcmg ccgcggtaa 19 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 8 ggactachvg ggtwtctaat 20 <210> 9 <211> 32 <212> PRT <213> Homo sapiens <400> 9 Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu 1 5 10 15 Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg 20 25 30 <210> 10 <211> 29 <212> PRT <213> Homo sapiens <400> 10 Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu 1 5 10 15 Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg Leu 20 25 <210> 11 <211> 28 <212> PRT <213> Homo sapiens <400> 11 Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys 1 5 10 15 Glu Ile Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg 20 25 <210> 12 <211> 28 <212> PRT <213> Homo sapiens <400> 12 Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu 1 5 10 15 Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg 20 25 <210> 13 <211> 27 <212> PRT <213> Homo sapiens <400> 13 Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu 1 5 10 15 Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr 20 25 <210> 14 <211> 26 <212> PRT <213> Homo sapiens <400> 14 Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile 1 5 10 15 Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg 20 25 <210> 15 <211> 26 <212> PRT <213> Homo sapiens <400> 15 Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu 1 5 10 15 Ser Gly Val Cys Glu Ile Ser Gly Arg Leu 20 25 <210> 16 <211> 25 <212> PRT<000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 20 <210> 19 <211> 23 <212> PRT <213> Homo sapiens <400> 19 Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg 1 5 10 15 Leu Tyr Arg Leu Cys Cys Arg 20 <210> 20 <211> 23 <212> PRT <213> Homo sapiens <400> 20 Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys 1 5 10 15 Glu Ile Ser Gly Arg Leu Tyr 20 <210> 21 <211> 22 <212> PRT <213> Homo sapiens <400> 21 Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile 1 5 10 15 Ser Gly Arg Leu Tyr Arg 20 <210> 22 <211> 22 <212> PRT <213> Homo sapiens <400> 22 Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg 1 5 10 15 Glu Ile Ser Gly Arg Leu 20 <210> 23 <211> 21 <212> PRT <213> Homo sapiens <400> 23 Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg 1 5 10 15 Ser Gly Arg Leu Tyr 20 <210> 24 <211> 21 <212> PRT <213> Homo sapiens <400> 24 Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg 1 5 10 15 Glu Ile Ser Gly Arg 20 <210> 25 <211> 19 <212> PRT <213> Homo sapiens <400> 25 Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg 1 5 10 15 Cys Cys Arg <210> 26 <211> 19 <212> PRT <213> Homo sapiens <400> 26 Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg 1 5 10 15 Leu Tyr Arg <210> 27 <211> 19 <212> PRT <213> Homo sapiens <400> 27 Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile 1 5 10 15 Ser Gly Arg <210> 28 <211> 18 <212> PRT <213> Homo sapiens <400> 28 Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg 1 5 10 15 Leu Tyr <210> 29 <211> 16 <212> PRT <213> Homo sapiens <400> 29 Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg 1 5 10 15 <210> 30 <211> 16 <212> PRT <213> Homo sapiens <400> 30 Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu 1 5 10 15 <210> 31 <211> 15 <212> PRT <213> Homo sapiens <400> 31 Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg 1 5 10 15 <210> 32 <211> 16 <212> PRT <213> Homo sapiens <400> 32 Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg 1 5 10 15 <210> 33 <211> 14 <212> PRT <213> Homo sapiens <400> 33 Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr 1 5 10 <210> 34 <211> 13 <212> PRT <213> Homo sapiens <400> 34 Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg 1 5 10 <210> 35 <211> 13 <212> PRT <213> Homo sapiens <400> 35 Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg Leu 1 5 10 <210> 36 <211> 12 <212> PRT <213> Homo sapiens <400> 36 Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu 1 5 10 <210> 37 <211> 12 <212> PRT <213> Homo sapiens <400> 37 Glu Ser Leu Ser Gly Val Cys Glu Ile Ser Gly Arg 1 5 10 <210> 38 <211> 11 <212> PRT <213> Homo sapiens <400> 38 Ser Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr 1 5 10 <210> 39 <211> 9 <212> PRT <213> Homo sapiens <400> 39 Cys Arg Thr Gly Arg Cys Ala Thr Arg 1 5 <210> 40 <211> 10 <212> PRT <213> Homo sapiens <400> 40 Ser Gly Val Cys Glu Ile Ser Gly Arg Leu 1 5 10 <210> 41 <211> 7 <212> PRT <213> Homo sapiens <400> 41 Leu Tyr Arg Leu Cys Cys Arg 1 5 <210> 42 <211> 6 <212> PRT <213> Homo sapiens <400> 42 Tyr Arg Leu Cys Cys Arg 1 5 <210> 43 <211> 7 <212> PRT <213> Homo sapiens <400> 43 Thr Gly Arg Cys Ala Thr Arg 1 5 <210> 44 <211> 6 <212> PRT <213> Homo sapiens <400> 44 Ala Thr Cys Tyr Cys Arg 1 5 <210> 45 <211> 5 <212> PRT <213> Homo sapiens <400> 45 Arg Leu Cys Cys Arg 1 5 <210> 46 <211> 5 <212> PRT <213> Homo sapiens <400> 46 Cys Arg Thr Gly Arg 1 5 <210> 47 <211> 32 <212> PRT <213> Homo sapiens <400> 47 Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser 1 5 10 15 Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg Phe Cys Cys Leu 20 25 30 <210> 48 <211> 30 <212> PRT <213> Homo sapiens <400> 48 Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly 1 5 10 15 Thr Cys Thr Val Met Gly Ile Asn His Arg Phe Cys Cys Leu 20 25 30 <210> 49 <211> 29 <212> PRT <213> Homo sapiens <400> 49 Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser 1 5 10 15 Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg Phe 20 25 <210> 50 <211> 28 <212> PRT <213> Homo sapiens <400> 50 Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser 1 5 10 15 Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg 20 25 <210> 51 <211> 27 <212> PRT <213> Homo sapiens <400> 51 Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly 1 5 10 15 Thr Cys Thr Val Met Gly Ile Asn His Arg Phe 20 25 <210> 52 <211> 26 <212> PRT <213> Homo sapiens <400> 52 Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly 1 5 10 15 Thr Cys Thr Val Met Gly Ile Asn His Arg 20 25 <210> 53 <211> 25 <212> PRT <213> Homo sapiens <400> 53 Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met 1 5 10 15 Gly Ile Asn His Arg Phe Cys Cys Leu 20 25 <210> 54 <211> 24 <212> PRT <213> Homo sapiens <400> 54 Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met Gly 1 5 10 15 Ile Asn His Arg Phe Cys Cys Leu 20 <210> 55 <211> 23 <212> PRT <213> Homo sapiens <400> 55 Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser 1 5 10 15 Tyr Gly Thr Cys Thr Val Met 20 <210> 56 <211> 22 <212> PRT <213> Homo sapiens <400> 56 Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met 1 5 10 15 Gly Ile Asn His Arg Phe 20 <210> 57 <211> 21 <212> PRT <213> Homo sapiens <400> 57 Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly 1 5 10 15 Thr Cys Thr Val Met 20 <210> 58 <211> 21 <212> PRT <213> Homo sapiens <400> 58 Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met 1 5 10 15 Gly Ile Asn His Arg 20 <210> 59 <211> 21 <212> PRT <213> Homo sapiens <400> 59 Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met Gly 1 5 10 15 Ile Asn His Arg Phe 20 <210> 60 <211> 21 <212> PRT <213> Homo sapiens <400> 60 Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg Phe Cys Cys Leu 1 5 10 15 Arg Phe Cys Cys Leu 20 <210> 61 <211> 20 <212> PRT <213> Homo sapiens <400> 61 Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg Phe Cys Cys Leu 1 5 10 15 Ile Asn His Arg 20 <210> 62 <211> 17 <212> PRT <213> Homo sapiens <400> 62 Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr 1 5 10 15 Tyr <210> 63 <211> 18 <212> PRT <213> Homo sapiens <400> 63 Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg Phe 1 5 10 15 Arg Phe <210> 64 <211> 17 <212> PRT <213> Homo sapiens <400> 64 Ser Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg Phe Cys Cys Leu 1 5 10 15 Arg <210> 65 <211> 17 <212> PRT <213> Homo sapiens <400> 65 Ser Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg Phe Cys Cys 1 5 10 15 Leu <210> 66 <211> 15 <212> PRT <213> Homo sapiens <400> 66 Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr 1 5 10 15 <210> 67 <211> 15 <212> PRT <213> Homo sapiens <400> 67 Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr 1 5 10 15 <210> 68 <211> 15 <212> PRT <213> Homo sapiens <400> 68 Gly Thr Cys Thr Val Met Gly Ile Asn His Arg Phe Cys Cys Leu 1 5 10 15 <210> 69 <211> 13 <212> PRT <213> Homo sapiens <400> 69 Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr 1 5 10 <210> 70 <211> 14 <212> PRT <213> Homo sapiens <400> 70 Ser Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg Phe 1 5 10 <210> 71 <211> 13 <212> PRT <213> Homo sapiens <400> 71 Ser Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg 1 5 10 <210> 72 <211> 11 <212> PRT <213> Homo sapiens <400> 72 Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr 1 5 10 <210> 73 <211> 12 <212> PRT <213> Homo sapiens <400> 73 Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met 1 5 10 <210> 74 <211> 12 <212> PRT <213> Homo sapiens <400> 74 Gly Thr Cys Thr Val Met Gly lie Asn His Arg Phe 1 5 10 <210> 75 <211> 10 <212> PRT <213> Homo sapiens <400> 75 Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr 1 5 10 <210> 76 <211> 11 <212> PRT <213> Homo sapiens <400> 76 Gly Thr Cys Thr Val Met Gly lie Asn His Arg 1 5 10 <210> 77 <211> 9 <212> PRT <213> Homo sapiens <400> 77 Thr Cys His Cys Arg Arg Ser Cys Tyr 1 5 <210> 78 <211> 9 <212> PRT <213> Homo sapiens <400> 78 Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr 1 5 <210> 79 <211> 9 <212> PRT <213> Homo sapiens <400> 79 Gly lie Asn His Arg Phe Cys Cys Leu 1 5 <210> 80 <211> 8 <212> PRT <213> Homo sapiens <400> 80 Arg Ser Cys Tyr Ser Thr Glu Tyr 1 5 <210> 81 <211> 8 <212> PRT <213> Homo sapiens <400> 81 Ala Phe Thr Cys His Cys Arg Arg 1 5 <210> 82 <211> 8 <212> PRT <213> Homo sapiens <400> 82 Ser Tyr Gly Thr Cys Thr Val Met 1 5 <210> 83 <211> 7 <212> PRT <213> Homo sapiens <400> 83 Ser Cys Tyr Ser Thr Glu Tyr 1 5 <210> 84 <211> 7 <212> PRT <213> Homo sapiens <400> 84 Ala Phe Thr Cys His Cys Arg 1 5 <210> 85 <211> 6 <212> PRT <213> Homo sapiens <400> 85 Thr Cys His Cys Arg Arg 1 5 <210> 86 <211> 6 <212> PRT <213> Homo sapiens <400> 86 Ser Thr Glu Tyr Ser Tyr 1 5 <210> 87 <211> 6 <212> PRT <213> Homo sapiens <400> 87 Gly Ile Asn His Arg Phe 1 5 <210> 88 <211> 5 <212> PRT <213> Homo sapiens <400> 88 Thr Cys His Cys Arg 1 5 <210> 89 <211> 6 <212> PRT <213> Homo sapiens <400> 89 Gly Thr Cys Thr Val Met 1 5 <210> 90 <211> 4 <212> PRT <213> Homo sapiens <400> 90 Arg Ser Cys Tyr 1 <210> 91 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (3)..(3) <223> X is (2S)-2-aminobutyric acid (Abu) <400> 91 Ala Thr Xaa Tyr Cys Arg Thr Gly Arg 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> X is (2S)-2-aminobutyric acid (Abu) <400> 92 Ala Thr Cys Tyr Xaa Arg Thr Gly Arg 1 5 <210> 93 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (3)..(3) <223> X is (2S)-2-aminobutyric acid (Abu) <220> <221> MISC_FEATURE <222> (5)..(5) <223> X is (2S)-2-aminobutyric acid (Abu) <400> 93 Ala Thr Xaa Tyr Xaa Arg Thr Gly Arg 1 5 <210> 94 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (6)..(6) <223> X is Citrulline (Cit) <400> 94 Ala Thr Cys Tyr Cys Xaa Thr Gly Arg 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (9)..(9) <223> X is Citrulline (Cit) <400> 95 Ala Thr Cys Tyr Cys Arg Thr Gly Xaa 1 5 <210> 96 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (6)..(6) <223> X is citrulline (Cit) <220> <221> MISC_FEATURE <222> (9)..(9) <223> X is citrulline (Cit) <400> 96 Ala Thr Cys Tyr Cys Xaa Thr Gly Xaa 1 5 <210> 97 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 97 Arg Gly Thr Arg Cys Tyr Cys Thr Ala 1 5 <210> 98 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 98 Cys Thr Arg Ala Thr Tyr Cys Arg Gly 1 5

Claims

1. A peptide having antimicrobial activity, wherein said peptide consists of the sequence of: ATCYCRTGR (SEQ ID No. 1), RGTRCYCTA (SEQ ID No. 2) or Ac-atcycrtGr-NH2 (SEQ ID No. 5).

2. The peptide of claim 1, wherein said peptide consists of the sequence of ATCYCRTGR (SEQ ID No. 1) or RGTRCYCTA (SEQ ID No. 2).

3. The peptide of claim 1, wherein said peptide consists of the sequence of ATCYCRTGR (SEQ ID No. 1).

4. The peptide of claim 1, wherein said peptide consists of the sequence of RGTRCYCTA (SEQ ID No. 2).

5. The peptide of claim 1, wherein said peptide consists of the sequence of Ac-atcycrtGr-NH2 (SEQ ID No. 5).

6. The peptide of claim 1, wherein said peptide is a homodimer of SEQ ID No. 1 linked by a disulfide bond.

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