An antimicrobial daptide from human skin commensal Staphylococcus hominis protects against skin pathogens

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: major_revision

Abstract
Coagulase-negative staphylococci are dominant human skin colonizers, producing natural products that shape the community and prevent pathogen colonization. The molecular mechanisms by which these natural products mediate interbacterial competition are not fully understood. Here, we identify a plasmid-borne daptide bacteriocin (hominicin) from a human skin isolate of Staphylococcus hominis, which features an unusual N,N-dimethyl-1,2-propanediamine C-terminus. Heterologous expression of the reconstituted biosynthetic loci yields a daptide product of the same molecular mass that exhibits antimicrobial activity against the skin pathogen Staphylococcus aureus, with amino-modified termini being essential for activity. Membrane permeability and voltage-clamp lipid bilayer experiments support a mechanism by which the daptide rapidly dissipates the transmembrane potential by forming peptidic channels. Additionally, we identify a cognate homI gene that confers resistance against membrane damage. Finally, the purified daptide effectively protects mouse skin from S. aureus-induced epicutaneous injury, as evidenced by reduced bacterial burden, inflammation, and transepithelial water loss, highlighting its therapeutic potential for treating bacterial skin infections. Our findings elucidate a mechanism of action, biosynthesis, and resistance for a staphylococcal bacteriocin belonging to a class of natural products called daptides.

Introduction
Human skin serves as both a physical and immunologic barrier between the body and the environment; a hostile landscape characterized by dryness, low pH, and high levels of salt1. Yet, the skin microbiota stably colonizes in this niche and plays an integral role in shaping the diversity of the community, preventing pathogen colonization, fortifying the epithelial barrier, and priming immune responses2. Coagulase-negative staphylococci (CoNS) are prominent members of the skin microbiome, comprising a heterogenous group of 38 known species3. CoNS maintain skin homeostasis by secreting natural products that antagonize microbial competitors and pathogens4. While the roles of CoNS in the microbiome have primarily focused on Staphylococcus epidermidis, emerging evidence underscores the beneficial roles of Staphylococcus hominis, a common CoNS on human skin, in defending against bacterial pathogens. S. hominis secretes autoinducing peptides that inhibit the accessory gene regulator (agr) system of the opportunistic skin pathogen Staphylococcus aureus, thereby preventing S. aureus-mediated inflammation and cutaneous damage5,6. Furthermore, S. hominis produces an arsenal of bacteriocins, including lantibiotics and other peptidic antibiotics, which inhibit S. aureus in vitro and in vivo7–9.

Bacteriocins are often ribosomally synthesized and post-translationally modified peptides (RiPPs) with targeted antibacterial activity against specific genera and species10. These RiPPs are generally encoded by complex and variable biosynthetic gene clusters (BGCs) that include the precursor bacteriocin, post-translationally modifying enzymes, and accessory proteins involved in export and producer immunity. Bacteriocin production is traditionally regarded as a probiotic trait, as it facilitates the producer strain in colonizing a niche, eliminating competing microbes, and potentially modulating the immune responses11. Contextually, the loss of bacteriocin-producing CoNS is associated with increased burden of S. aureus on the skin of subjects with atopic dermatitis (AD), underscoring the importance of commensal bacteria in fortifying the skin’s antimicrobial barrier7. In a post-antibiotic era, bacteriocins present a promising source of potent antimicrobials that have the potential to inhibit pathogens. Human clinical trials have demonstrated the safe delivery and effectiveness of lantibiotic-producing S. hominis as a bacteriotherapy treatment for S. aureus-colonized AD skin, highlighting the effect of a bacteriocin producer in reducing S. aureus colonization and disease severity12.

(Meta)genome-based and activity-based screening approaches continue to advance the discovery of RiPPs, which are categorized by class-defining chemical features13. Daptides emerged as a new class of RiPPs, characterized by an unusual (S)-N,N-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus and defined in Microbacterium paraoxydans14–16. Daptide BGCs are widely distributed across the Actinomycetota, Bacill, Bacillota, and Pseudomonadota phyla14, implying a potential biological role for these small peptides. Characterization of M. paraoxydans (Mpa) daptides revealed hemolytic activity against bovine erythrocytes, but no antibacterial properties were observed14. Since then, no other daptides have been isolated; however, an earlier report described hominicin17, a Dmp-carrying peptide, as antimicrobial, although its genetic and biosynthetic origin as a RiPP remains undetermined. Thus, the target specificity, mode of action, and mechanism of resistance for daptides have not been fully elucidated. Such knowledge is critical to the field of drug discovery for developing novel and rational interventions that reduce morbidity and mortality from bacterial infections.

Herein, we identify a daptide bacteriocin produced by a human skin isolate of S. hominis. The expression of the reconstituted biosynthesis genes sufficiently confers antimicrobial activity. We isolate the daptide from culture supernatant and solve its structure using mass spectrometry and nuclear magnetic resonance (NMR). Mechanistically, the daptide induces membrane damage by dissipating the transmembrane potential of susceptible bacteria through the formation of peptidic channels. Genetic studies identify a cognate protein, HomI, as the determinant for resistance against daptide-induced membrane damage. Finally, we demonstrate that topical treatment with the purified daptide reduces S. aureus colonization and cutaneous injury in a murine model of epicutaneous infection. This study elucidates a mechanism of action, biosynthesis, and resistance for a staphylococcal bacteriocin belonging to an understudied class of natural products called daptides.

Results
Antimicrobial activity of a skin commensal S. hominis strain AH5011
We screened the cell-free, conditioned media (CM) from a human skin isolate collection of S. hominis for antimicrobial activity and determined that the strain AH5011 (D11), an isolate from non-lesional skin of a subject with atopic dermatitis, suppressed the growth of S. aureus strain AH6350 (Fig. 1a, c). To determine the antimicrobial specificity of AH5011, we tested the susceptibility of skin commensals and pathogenic strains of Staphylococcus, including S. aureus, S. hominis, S. epidermidis, S. capitis, S. haemolyticus, S. lugdunensis, S. simulans, S. warneri, and S. pseudintermedius, as well as Micrococcus luteus and Streptococcus pyogenes (Fig. 1b, Supplementary Data 3). Streptococcus agalactiae and Enterococcus faecalis were included as non-skin-associated bacteria. Antimicrobial susceptibility was assessed by calculating the ratio of optical density of the test condition (40% v/v CM) to untreated cells grown in tryptic soy broth (TSB). We defined inhibition as a ratio of <0.5. This analysis revealed that AH5011 is inhibitory towards staphylococci, M. luteus, and S. pyogenes, but not S. agalactiae or E. faecalis. However, the inhibitory activity was strain dependent, as evident by a bimodal distribution of susceptibility for S. aureus, S. hominis, S. epidermidis, S. lugdunensis, and S. warneri. Intriguingly, this observation was not seen for S. agalactiae and E. faecalis. Therefore, we hypothesize that S. hominis AH5011 targets specific members of the skin community, largely the Staphylococcus genus. We observed that the antimicrobial function inhibits the S. hominis reference strain ATCC 27844 (AH4553) from the American Type Culture Collection in a concentration-dependent manner (Fig. 1c–e). This strain was adopted as a sensitive control strain for subsequent experiments. The antimicrobial activity is thermally stable and can be inactivated by the serine protease Proteinase K (Supplementary Fig. 1). Additionally, AH5011 is resistant to the inhibitory effect from its own CM (Fig. 1f, g), which indicates a self-protection mechanism.

S. hominis AH5011 bacteriocin is a member of the daptide family of RiPPs
The whole-genome sequence of AH5011 revealed two plasmids, p1 and p2, that are 19.8 kilo bases (kb) and 22 kb, respectively. Bacteriocin biosynthesis machineries are often found on genetically mobile plasmids18. To assess whether the inhibitory function was plasmid-encoded, we generated plasmid-cured mutants of AH5011 upon exposure to acriflavine and tested for loss of activity. The absence of p1, but not p2, led to a complete loss of activity, as evidenced by the lack of an inhibitory zone and detectable activity in the CM from the Δp1 and Δp1Δp2 mutants against the sensitive control strain AH4553 (Fig. 2a, Supplementary Fig. 2a). Furthermore, the growth of Δp1 and Δp1Δp2 mutants was attenuated in AH5011’s CM, but not that of the Δp2 mutant (Supplementary Fig. 2b–d). Thus, we inferred that the p1 plasmid is the main contributor to the antimicrobial activity and resistance.

We analyzed the AH5011 plasmids using the BAGEL4 database19 and identified a gene cluster containing a lanthipeptide dehydratase and serine peptidase in the p1 plasmid. The 9.6-kb gene cluster consists of ten genes encoding the precursor peptide, seven biosynthesis enzymes, a peptidase, and a hypothetical protein (Fig. 2b, Supplementary Table 5). Based on sequence similarity with proteins of known functions and/or domains, homB encodes a domain of unknown function (DUF)-fused RiPP recognition element (RRE) protein; homC encodes an enoyl-(acyl carrier protein) reductase family member; homD encodes a class-III pyridoxal phosphate-dependent aminotransferase; homJ encodes a NAD(P)H-dependent oxidoreductase, belonging to the flavoprotein-like superfamily; homM1 and homM2 both encode S-adenosyl-L-methionine-dependent methyltransferases; homP encodes a serine peptidase, belonging to the subtilisin family; homM encodes a DUF4135 domain-containing protein that represents only the dehydratase domain of class II lanthipeptide synthetases (LanM)20. Therefore, we proposed to designate this single domain enzyme as LanMD to indicate its proposed dehydration activity21,22.

RiPP precursor peptides consist of an N-terminal leader sequence, which serves as the binding element for modifying enzymes, and a C-terminal core sequence, which receives the post-translational modifications. The homA gene encodes a 65-residue precursor bacteriocin (Fig. 2c), with the core peptide mapping to the reported final structure of hominicin, a peptidic natural product that features the Dmp-modified C-terminus17. We detected differences at the 10th and 17th residues between the core peptide of the AH5011 bacteriocin and the final structure of hominicin (Supplementary Fig. 14d). Although the genetic and biosynthetic information of the hominicin-producing strain MBBL2-917 are not publicly available, we infer that the AH5011 bacteriocin is likely hominicin. The Dmp-modified C-terminus is a class-defining feature of daptides, which has been characterized in M. paraoxydans14 (Supplementary Fig. 14a, b). The Dmp moiety arises from enzymatic modifications of a C-terminal threonine residue, beginning with the oxidative decarboxylation of the hydroxyl group to a ketone, catalyzed by the DUF-RRE and alcohol dehydrogenase DapBC14. This is followed by transamination via transaminase DapD and a dimethylation step by the methyltransferase DapM14. We propose that the hom BGC contains the necessary tailoring enzymes, HomBCDM2, that modify the C-terminal Thr into Dmp (Fig. 2d, Supplementary Fig. 14a). Additionally, the LanMD likely functions as a dehydratase to convert the core threonines into dehydrobutyrines and serines into dehydroalanines20–22. We anticipate that the flavin-dependent oxidoreductase HomJ likely catalyzes the reduction of dehydroalanines to D-alanines, as previously reported in the biosynthesis of lanthipeptides23–25. The modified core is proteolytically cleaved from the leader peptide by the serine peptidase HomP26 while the second methyltransferase HomM2 is likely responsible for the dimethylation of the free N-terminal isoleucine, which results in a fully modified and bioactive daptide.

Detection and structural characterization of AH5011 daptide
Using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS), we analyzed the CM from daptide-positive AH5011 and daptide-negative Δp1Δp2 strains. We detected a distinct chromatographic peak at retention time 5.32 min with a measured m/z value of 1020.1114 in the CM from AH5011 but absent from Δp1Δp2. The measured mass was within 2 ppm of the predicted m/z (1020.1103) for a doubly-charged protonated ion of the daptide (Fig. 3a, Supplementary Fig. 3a). The observed MS-MS fragmentation pattern of this m/z in combination with the genetic data allowed us to propose the bacteriocin sequence as DmIle–Dhb–Pro–Ala–Dhb–Pro–Phe–Dhb–Pro–Ala–Ile–Thr–Glu–Ile–Dhb–Ala–Ala–Val–Ile–Ala–Dmp (Fig. 3c, d, Supplementary Fig. 3b). The initial hominicin investigation lacked genetic information for S. hominis strain MBBL2-917, so the precursor peptide sequence was not proposed. However, the final modified bacteriocin structure remains consistent with the previously proposed structure of hominicin. The determination of the accurate mass of hominicin from S. hominis AH5011 confirmed its molecular weight to be 2038.5110 Da. 1D and 2D NMR experiments were carried out to confirm the proposed structure of AH5011 hominicin (Fig. 3e, Supplementary Fig. 6 and Supplementary Data 1).

To characterize the configurations of hominicin’s amino acids, we performed a full hydrolysis of hominicin followed by Marfey’s derivatization27. The results (Supplementary Fig. 7) indicate that all hominicin amino acids are of L-configuration, with the exception of alanine, which was detected in both the L- and D-configurations with a ratio of 1.2:1 L:D (calculated from UPLC-MS peak area). These findings are consistent with the biosynthetic model that indicates a product containing three ribosomally derived L-alanines and two D-alanines derived from serines (L:D ratio of 1.5:1). The configuration of the stereocenter of the Dmp group on the C-terminus was not confirmed for hominicin, although previous work has shown this functional group to have an (S) configuration in other daptides14.

To determine whether the hom BGC from AH5011 is sufficient to produce the daptide, we cloned the 9.6-kb BGC into the plasmid pCM28 and heterologously expressed the construct in a restriction-deficient cloning host, S. aureus strain RN4220. From the CM of the hom BGC expressing S. aureus, but not the empty vector, an ion was detected with the same retention time (5.33 mins), accurate mass (1020.1115 m/z), and MS-MS fragmentation pattern as the ion detected in the AH5011 strain (Fig. 3b, Supplementary Fig. 3c, d). Thus, we inferred that the expression of the hom BGC was sufficient for the biosynthesis and secretion of hominicin.

Given the antimicrobial potency from CM and optimal aerobic growth conditions, we enriched hominicin through chloroform extraction, then subjected the material to flash chromatography, followed by preparative high-performance liquid chromatography (HPLC) (Supplementary Fig. 4). The peptide product (6.4 mg) was isolated from 892 mg of the chloroform extract with >95% purity, corresponding to a yield of 0.8%. UPLC-MS analysis of the product yielded an ion with mass and fragmentation pattern consistent with hominicin (Supplementary Fig. 4), and this product was further subjected to NMR and Marfey’s analysis (Supplementary Fig. 6 and 7) confirming the hominicin structure. A simple extraction by n-butanol also provided a partially purified and enriched antimicrobial fraction from conditioned media (Supplementary Fig. 5). Therefore, the AH5011 extract obtained via n-butanol extraction was adopted for further experiments.

Activity of AH5011 hominicin against skin commensals and pathogens
Based on the inhibitory effect of the CM against a wide range of Gram-positive bacteria, as shown in Fig. 1, we next measured the minimum inhibitory concentration (MIC) of the purified daptide against a selection of skin commensal and pathogenic bacteria (Table 1) using a broth microdilution assay adapted from the Clinical Laboratory Standards Institute (CLSI) guidelines28. Hominicin demonstrated antimicrobial potency comparable to that of the clinically relevant antibiotic vancomycin across all tested strains (Table 1). Full MIC curves are included as supporting information (Supplementary Fig. 8).

The amino-modified termini are essential to hominicin’s activity
Given that the hom BGC represents a fully assembled gene cluster for a staphylococcal daptide, we next investigated the minimal genes required for antimicrobial activity. We generated plasmid constructs lacking homBDJM2 (i.e., predicted involvement in the Dmp biosynthesis and oxidoreduction of dehydroalanines), homD (i.e., predicted transamination in the Dmp biosynthesis), homM1 (i.e., predicted dimethylation of the N-terminus), lanMD (i.e., predicted dehydratase function), and homI, a gene of unknown function (Fig. 4a). Each construct was heterologously expressed into RN4220 and spot plated against AH4553. The expression of the full-length hom BGC confers antimicrobial activity, as evidenced by an inhibitory zone with a mean diameter of 17.3 ± 0.6 mm, compared to the empty vector (10.0 ± 0.2 mm), whereas the omission of homBDJM2, homD, homM1, and lanMD genes abolished antimicrobial activity (Fig. 4b). Interestingly, the omission of homI gene had no apparent impact on the antimicrobial activity, suggesting that HomI is not essential for hominicin biosynthesis.

We next tested whether the activity, or lack thereof, could be detected in the CM of each mutant. The CM from strains expressing the full-length hom gene cluster and HomABCDJMM1M2PLanMD (i.e., omission of homI) were growth suppressive; however, CM from strains lacking homBDJM2, homD, homM1, and lanMD were not inhibitory (Fig. 4c). To corroborate the activity results, we performed mass spectrometric analysis of each CM to confirm the presence or absence of hominicin. The omission of homI resulted in a 21-residue product with an m/z value for the [M+2H]2+ ion of 1020.1095 [Δ = 1 ppm] (Supplementary Fig. 9), corresponding to hominicin. However, the calculated daptide mass was not detected in the CM from mutants lacking homBDJM2; instead, multiple peptide species of similar mass to hominicin were detected with the most abundant ion displayed an [M+2H]2+ of 1026.5679 (Supplementary Fig. 10a). The MS-MS spectra displayed a fragmentation pattern that is distinct from hominicin (Supplementary Fig. 10b). This structure likely differs from hominicin in the lack of C-terminal modification and oxidoreduction of dehydroalanine residues, matching the predicted m/z of 1026.5659 [Δ = 2 ppm] (Supplementary Fig. 10c). No peptide product was detected in the expression strain lacking the lanMD gene, suggesting this biosynthetic step is needed for stability of the peptide (Supplementary Fig. 11a). For the homM1 omission, mass spectrometric analysis indicated the production of a peptide lacking the N-terminal methylations while retaining formation of D-Ala from L-Ser (Supplementary Fig. 11a, b). Surprisingly, the observed product also lacked the Dmp modification on the C-terminus. The biosynthetic rationale for this is unclear as the installation of the C-terminal modifications should precede removal of the leader peptide needed for N-terminal methylation14. Whether this observation is due to polar effects on transcription or a requirement for these N-methylations to form the Dmp motif remains to be elucidated. For the homD omission, mass spectrometric analysis indicated the formation of a hominicin analogue with the expected N-terminal methylations and D-Alas, but a C-terminal ketone instead of the fully modified Dmp moiety (Supplementary Fig. 11c). This indicates that the N-terminal methylations can be installed even without formation of the Dmp motif.

AH5011 hominicin is a membrane-disruptive, pore-forming antimicrobial
The structural characteristics of daptides include a hydrophobic core and positively charged amino termini14. These features are found in many membrane-active, pore-forming antimicrobial peptides29. Whether daptides also form membrane pores has not been demonstrated although previous work observed that daptides from M. paraoxydans exhibit hemolytic activity14. We first asked whether hominicin could permeabilize the cytoplasmic membranes of S. aureus by measuring the uptake of the impermeant SYTOX Green fluorescent probe into bacterial cells. Upon cell entry, the fluorescence intensity of SYTOX Green is enhanced after binding to nucleic acids30. S. aureus cells treated with 2x or 4x conditioned media from AH5011, prepared from n-butanol extraction, which contained approximately 8.2 µg and 16.3 µg of hominicin, respectively, were significantly permeabilized compared to Δp1Δp2-treated cells (Fig. 5a). We next examined the effects of hominicin on the membrane potential by measuring the release of potentiometric, cationic 3,3’-dipropylthiadicarbocyanine iodide [DiSC3(5)] dye from bacterial cells. DiSC3(5) accumulates on polarized membranes and translocates into lipid bilayers, resulting in fluorescence self-quenching31. S. aureus cells treated with AH5011 extract were depolarized in a dose-dependent manner, as indicated by the increased fluorescence intensity in the culture medium due to the release of DiSC3(5) (Fig. 5b).

To test whether hominicin exhibits ion channel activity, we reconstituted the pure peptide in artificial lipid bilayers bathed in a solution of 1 M KCl and 10 mM HEPES and measured ion flux across the membrane using voltage-clamp electrophysiology (Supplementary Fig. 12a). The current/voltage relationship of hominicin displayed an ohmic relationship with a calculated channel conductance of 692.4 ± 70.4 pS while the synthetic unmodified core peptide and buffer alone exhibited no channel activity (Fig. 5c). As a control, we added alpha-toxin32, a known pore-forming protein produced by S. aureus, and observed a channel conductance of 489.9 ± 82.8 pS. At both positive and negative holding potentials, hominicin exhibited ion-conducting activity, as evidenced by observable openings and closings of single channels (Fig. 5d, e). While hominicin exhibited antimicrobial activity, its unmodified form was inactive against S. aureus (Supplementary Fig. 12b). This suggests that the post-translational modifications of hominicin are crucial for its membrane insertion and channel activity. The unmodified peptide did not alter the current trace from baseline, whereas hominicin caused stepwise increases in the current trace at +50 mV and decreases at −50 mV and −100 mV holding potentials (Supplementary Fig. 12c–e). The changes in current traces are indicative of ion flux across the bilayer through the opening of membrane pores.

To test whether hominicin disrupts host membranes, we investigated the hemolytic activity induced by AH5011 using whole human blood. Alpha-toxin-producing S. aureus strains AH1263 and AH6350 were included, and AH1263 Δagr mutant served as a non-hemolytic control33. AH1263 and AH6350 exhibited significant hemolysis when compared to uninfected (PBS) control while AH5011 did not induced hemolysis (Supplementary Fig. 13a). We did not observe differences in hemolysis of the AH5011 extract when compared to treatment with the Δp1Δp2 extract (Supplementary Fig. 13b). In confirmation, we did not observe zones of hemolysis for AH5011, Δp1Δp2 mutant, and AH1263 Δagr mutant when plated on sheep’s blood agar (Supplementary Fig. 13c). We next measured cytotoxicity in human N/TERT keratinocytes using lactate dehydrogenase (LDH) release assays. AH1263 and AH6350 induced significant cytotoxicity on host cells whereas cytotoxicity caused by AH5011 and Δp1Δp2 mutant was undetectable compared to untreated cells (Supplementary Fig. 13d). Altogether, these data indicate the mode of action of AH5011 hominicin involves impairing the membrane integrity by depolarizing the bacterial transmembrane potential through the formation of pores.

The genetic diversity and conservation of daptide BGC in Staphylococcus
To assess the diversity and conservation of the hom BGC in Staphylococcus, we performed a gene cluster comparison using the CAGECAT pipeline34, which enables detailed homology search, filtering, gene neighborhood estimation, and visualization of resulting variant BGCs. Fourteen assemblies were identified, carrying homologs of the precursor peptide along with at least one or two associated biosynthesis genes (Supplementary Table 3). These assemblies were sourced from shotgun sequencing data of various staphylococci, including S. aureus, S. pseudintermedius, S. felis, S. epidermidis, S. warneri, S. agnetis, and S. cohnii. Some of the BGCs exhibited variations in the composition of tailoring enzymes and the number of precursor peptides (Supplementary Fig. 14c). Seven assemblies lack the genes encoding for the dehydratase LanMD and oxidoreductase HomJ, suggesting that the installation of dehydroamino acids and the conversion of serines into D-alanines may have evolved independently from the Dmp biosynthesis. Additionally, we identified accessory genes likely involved in bacteriocin export and immunity. These were predicted to encode ATP-binding cassette (ABC) transporters, previously reported in the export of bacteriocins across the cytoplasmic membrane as a resistance mechanism35. Intriguingly, these were not found in S. hominis AH5011; however, the gene of unknown function, homI, was present in seven BGCs, including AH5011 and seems to correlate with hominicin-like molecules. We also observed that these daptide BGCs are located on or near mobile genetic elements, such as plasmids, recombinases, and transposases. This suggests the acquisition of daptides via horizontal gene transfer. Alignment of the precursor peptides revealed diversity in sequence and length while conservation of the C-terminal threonine was observed (Supplementary Fig. 14d).

HomI is the immunity protein that provides resistance to AH5011 hominicin
Producer strains are often protected from the activity of their bacteriocin by self-immunity systems36–38. The mechanisms of producer immunity to daptides have never been investigated. We ascertained the role of homI given that 6 out of the 14 daptide BGCs encode this gene (Supplementary Fig. 14c) and previous work observed the co-occurrence of HomI in staphylococcal daptide BGCs14. We hypothesized that HomI is the immunity protein that provides cognate resistance to hominicin. Using a genetically tractable and hominicin-susceptible S. aureus strain Newman, we cloned homI on a multicopy plasmid and observed no growth defects between the wild-type and homI-expressing mutant in TSB (Fig. 6a). Under growth conditions with AH5011 CM, the mutant exhibited significant resistance while the growth of wild-type and empty vector were attenuated (Fig. 6b). HomI-mediated resistance was confirmed by the lack of inhibitory zones in the presence of hominicin-producing AH5011 and S. aureus strain RN4220 expressing hom BGC (Fig. 6c).

HomI is predicted to be a 32.8 kDa membrane-associated protein with five transmembrane helices39 (Supplementary Fig. 15a). To identify structural homologs, we predicted the structure of S. hominis HomI using AlphaFold40 and submitted the structure as a query in Foldseek41. This analysis revealed that HomI shares structural homology with the YidC family of membrane protein insertases (Supplementary Fig. 15b, c), suggesting that HomI may function at the membrane interface. Alignments of the HomI model with an AlphaFold-predicted structure of S. aureus YidC and a crystal structure of Bacillus halodurans YidC (PDB ID 3WO7_A) yielded RMSD values of 1.41 Å and 1.76 Å, respectively, indicating high structural similarity. To investigate the potential mechanism behind HomI-mediated resistance against hominicin, we used Boltz-1 to predict a HomI-hominicin complex structure40,42. For comparison, the AlphaFold-predicted HomI structure was superimposed onto the HomI-hominicin complex. Hominicin docked within the elongated positively charged pocket in the HomI core, with minimal structural changes between HomI modeled with and without hominicin. Intriguingly, the N-terminal α1 helix exhibited a conformational shift, suggesting a potential binding interaction between HomI and hominicin (Fig. 6d, Supplementary Fig. 15d, e).

We next examined the potential function of HomI in protecting against daptide-induced membrane damage by measuring the release of DiSC3(5) in the culture medium. Under Δp1Δp2-treated and untreated conditions, the wild-type strain Newman, homI-expressing mutant, and empty vector strains exhibited fluorescence self-quenching, which reflects the uptake of DiSC3(5) and the energized and/or polarized state of the cells. When exposed to AH5011 extract, the wild-type (Fig. 6e) and empty vector (Fig. 6f) strains displayed membrane depolarization. However, the homI-expressing mutant retained its membrane potential, comparable to the untreated control (Fig. 6g). Altogether, we identified homI as the genetic determinant that confers resistance against membrane damage caused by hominicin.

AH5011 hominicin restricts S. aureus skin infection in vivo
The translational potential of daptides to protect against invading pathogens has never been tested. Therefore, we investigated hominicin-mediated skin protection from S. aureus infection. S. aureus is the most common cause of skin and soft tissue infections in the United States and frequently associated with high morbidity, mortality, and healthcare costs43,44. As an opportunistic pathogen, S. aureus can exacerbate diseases, such as atopic dermatitis, an inflammatory skin disease with a complex pathogenesis marked by impaired skin barrier function and structure, immunological abnormalities, and dysbiosis45. Nasal carriage is a known risk factor for invasive S. aureus infections46. Therefore, we selected the strain AH6350, a high-toxin producing isolate from an acute lesion of pediatric AD, as a clinical organism to study in vivo (Fig. 7a, Supplementary Fig. 13). AH6350 is highly susceptible to hominicin, with a minimum inhibitory concentration of 1 µg/mL (Table 1). Genomic analysis revealed that it is closely related to strains from sequence type (ST) 15 lineage with an accessory gene regulator (agr) quorum-sensing system of type II (Fig. 7b). ST15 is commonly associated as nasal carriage lineage47 and often expresses surface proteins with skin-binding capacity48,49 and has the potential pathogenicity to cause skin-related diseases50,51.

Epicutaneous infection of female and male C57BL6/J mice with AH6350 alone resulted in severe skin scaling, erosion, and erythema (Fig. 7c). Infection with S. aureus was attenuated by the topical addition of hominicin to the skin surface, as evidenced by a reduced bacterial burden compared to the untreated group (Fig. 7d). Additionally, the infected mice treated with hominicin retained skin barrier function and integrity, as measured by transepithelial water loss (Fig. 7e). The severity of local inflammation on mouse skin at 72 h post-infection was assessed using a skin disease score, which represents the sum of individual grades for erythema, edema, erosion and scaling52. We observed minimal skin damage with hominicin alone in the absence of infection (Fig. 7f, Supplementary Fig. 16). Topical treatment with hominicin on infected skin resulted in significantly less skin inflammation and cutaneous injury, which correlates to the reduction of S. aureus on the skin.

Methods
Collection of bacteria from human subjects
Bacterial isolates from human skin swabs were used from a previously published collection5,72. Swabs from a 5-cm2 area of the antecubital fossa skin were collected from both the left and right arms from healthy subjects and patients with AD. Skin swab samples were suspended in 1 mL TSB containing 15% v/v glycerol and plated on mannitol salt agar with egg yolk for the selective growth of Staphylococcus species. Participants included individuals of both sexes who had not used an antibiotic in the preceding month. Age and sex were self-reported. Skin swab collection was done in accordance with protocols approved by the University of California, San Diego (UCSD) Institutional Review Board under project number 140144. Bacterial isolates from skin swabs of healthy subjects were collected in accordance with protocols approved by COMIRB under protocol number 19-2218 and UCSD Institutional Review Board under project number 071032. Informed consent was obtained from all subjects. Isolates used in this study are listed in Supplementary Data 3.

Growth conditions and reagents
The bacterial strains and plasmids used in this study are listed in Supplementary Table 1. S. hominis isolates were confirmed to be S. hominis by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry prior to experimentation. All staphylococcal strains and Micrococcus luteus were grown in TSB growth medium at 37°C with shaking at 250 rpm. Escherichia coli was grown in Luria broth (LB) at 37°C with shaking. Enterococcus faecalis was grown in Brain Heart Infusion at 37°C with shaking. Streptococcus agalactiae was grown statically in Todd Hewitt broth at 37°C. Streptococcus pyogenes was grown statically in Todd Hewitt broth supplemented with yeast extract at 37°C + 5% CO2. For strains with pCM28, chloramphenicol was added to a final concentration of 10 µg/mL. The unmodified core peptide was custom synthesized by AnaSpec (Fremont, CA).

Bacteriocin antimicrobial activity assays
For conditioned medium (CM) assays, overnight bacterial cultures of S. hominis or S. aureus strains were pelleted, and the spent media was filtered through a 0.22-µm cellulose acetate Spin-X filter (Costar). 100 µL of 80% (vol/vol) CM was added to a 96-well culture plate unless otherwise indicated. The indicator strains were prepared by diluting overnight cultures 1:50 in fresh TSB. 100 µL of the indicator strain was added to the culture plate to achieve a final volume of 200 µL per well. Cultures were grown in a Stuart humidified incubator at 37°C with shaking at 800 rpm. Cell density (OD600) was measured on a Tecan Group Ltd. Infinite Pro plate reader. At specified time-points, 20 µL of culture was removed, serially diluted, and plated on TSA for colony counting.

For the spot-on-lawn assays, S. hominis AH4553 was used as the sensitive strain for all experiments, unless otherwise stated. 100 µL of the 1:10 diluted suspension of AH4553 was spread on TSA using sterile glass beads and air dried. 10 µL of the daptide-producing or non-producing strains were inoculated onto the bacterial lawn. Plates were incubated at 37°C for 16–20 h, and images of the zones of inhibition were collected and analyzed using ImageJ software (version 1.53).

Isolation of plasmid-cured mutants
S. hominis AH5011 was subcultured 1:100 in fresh TSB and allowed to reach an OD600 of 0.50. Acriflavine was added to the bacterial culture at a final concentration of 25 µg/mL and incubated for 1 h. The culture was diluted and plated on TSA to obtain single colonies, which were then picked and patched on fresh TSA. Bacterial strains cured of p1 failed to produce inhibition zones when spotted on a lawn of AH4553. Strains cured of p2 failed to grow on agar containing 1 µg/mL mupirocin. To confirm the loss of plasmids, polymerase chain reaction (PCR) was performed using primers for the p1- and p2-specific genes, homA and mupA, respectively.

Cloning of the hom biosynthetic gene cluster
All primers used in this study are listed in Supplementary Table 2 and purchased from Integrated DNA Technologies (Coralville, Iowa). The hom gene cluster was cloned into pCM28 by a Gibson assembly strategy. The construction of the S. aureus shuttle vector pCM28 has been previously described73. The pCM28 vector was linearized by restriction digestion using BamHI-HF and EcoRI-HF (New England Biolabs). The hom gene cluster was amplified in four parts from the genomic DNA of S. hominis AH5011 using primers homBGC_p1_Frw/_Rev, homBGC_p2_Frw/_Rev, homBGC_p3_Frw/_Rev, and homBGC_p4_Frw/_Rev. The resulting 15-kb construct, pAN1, was introduced into chemically competent E. coli DH5-alpha cells and selected on LB agar with 100 µg/mL ampicillin. The plasmid was verified by DNA sequencing. The pAN1 and empty vector pCM28 were electroporated into competent S. aureus RN4220, as previously described74. Transformants were selected on TSA with 10 µg/mL chloramphenicol.

For gene omission studies, expression constructs were generated as mentioned above. To construct pCM28-homABCDJMM1M2PLanMD (i.e., omission of homI), the gene cluster was amplified in three parts using primers homBGC_p3_Fwd/_Rev, homBGC_p4_Fwd/_Rev, and homBGC_p7_Fwd/_Rev. The resulting 14-kb construct was designated as pAN2. To construct pCM28-homACIM1M2PLanMD (i.e., omission of BDJM2), the gene cluster was amplified in three parts using primers homBGC_p4_Fwd/_Rev, homBGC_p5_Fwd/_Rev, and homBGC_p6_Fwd/_Rev. The resulting 11-kb construct was designated as pAN3. To construct pCM28-homABCDIJM2PLanMD (i.e., omission of homM1), the gene cluster was amplified in four parts using primers homBGC_p1_Fwd/_Rev, homBGC_p2_Fwd/_Rev, homBGC_p14_Fwd/_Rev, and homBGC_p15_Fwd/_Rev. The resulting 14-kb construct was designated as pAN4. To construct pCM28-homABCIJM1M2PLanMD (i.e., omission of homD), the gene cluster was amplified in four parts using primers homBGC_p3_Fwd/_Rev, homBGC_p4_Fwd/_Rev, homBGC_p8_Fwd/_Rev, and homBGC_p9_Fwd/_Rev. The resulting 14-kb construct was designated as pAN6. To construct pCM28-homABCDIJM1M2P (i.e., omission of lanMD), the gene cluster was amplified in four parts using primers homBGC_p1_Fwd/_Rev, homBGC_p2_Fwd/_Rev, homBGC_p10_Fwd/_Rev, and homBGC_p11_Fwd/_Rev. The resulting 13-kb construct was designated as pAN7.

Cloning of the immunity gene, homI, was performed as follows: The pCM28 vector was linearized by restriction digestion using BamHI-HF and PstI-HF. The insert was PCR-amplified using primers homI_Frw/_Rev. Purified vector and insert were then ligated using T4 ligase. The resulting 6.6-kb construct, designated as pAN5, was introduced into DH5-alpha cells and verified by DNA sequencing. pAN4 was passaged through RN4220 and subsequently introduced into S. aureus strain Newman by electroporation.

Mass spectrometric identification of the purified daptide and in conditioned media
Samples were analyzed on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with a heated electrospray ionization source coupled to an Acquity ultra high-performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA). Conditioned media samples, obtained from three independent biological replicates, were centrifuged to pellet bacterial cells and the resulting supernatants were passed through 0.22 µm PVDF syringe filters prior to analysis. A culture medium blank processed under identical conditions was also included. Purified daptide samples were dissolved in LC-MS grade methanol and injected in triplicate as technical replicates. All samples were injected with a 3-7 µL volume and eluted from an Acquity UPLC BEH C18 1.7 µm 2.1 × 50 mm column (Waters Corporation) at a 0.3 mL/min flow rate using a binary solvent system consisting of 0.1% formic acid (A) and acetonitrile (CH3CN) with 0.1% formic acid (B). Mass spectra were collected using positive ion mode electrospray ionization with two scan events, a full-scan event over a mass range of 300 to 2000 at a resolving power of 35,000, and a data-dependent tandem mass spectrometry (MS/MS) scan event selecting the calculated m/z or a data-independent scan event using all ion fragmentation. The solvent gradient began with a 1.5 min isocratic hold at 20% B which was diverted to waste. Subsequently, the flow was diverted to the mass spectrometer and a linear increase to 60% B was performed over 5 min. The gradient was held isocratic from 6.5 min to 7.0 min and then increased to 100% B at 8.0 min. The column was washed at 100% B for 1 min and then returned to the starting conditions to allow equilibration for 1.0 min prior to the next injection.

Marfey’s analysis
Marfey’s analysis was conducted to determine the stereochemistry of amino acids in the sample, employing a modified version of the procedure27. Each amino acid standard was prepared in water at approximately 4 mg/mL. Each prepared standard (50 µL) was then combined with 1 M NaHCO3 (20 µL), and 1% Marfey’s reagent (Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide) in acetone (100 µL), and the mixture was incubated at 40°C for 1 h. The reaction was quenched by adding 10 µL of 2 N HCl, and the resulting mixture was dried under a stream of nitrogen. The residue was reconstituted in approximately 200 µL of methanol. Additionally, approximately 0.1 mg of purified hominicin was hydrolyzed with 0.5 mL of 6 N HCl at 90°C for 24 h. After hydrolysis, the sample was evaporated under a stream of nitrogen. The residue was reconstituted in 25 µL of water, followed by the addition of 10 µL of 1 M NaHCO3 and 50 µL of 1% Marfey’s reagent in acetone. The mixture was agitated at 40°C for 1 h, then quenched with 5 µL of 2 N HCl. The derivatized sample was dried under a stream of nitrogen and reconstituted in approximately 125 µL of methanol.

Each derivatized standard was injected individually (1 µL) onto the UPLC-MS, and a mixed standard containing aliquots of all derivatized standards was also prepared and injected. Samples were analyzed on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with a heated electrospray ionization source coupled to an Acquity ultra high-performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA). Chromatographic separation was performed using a BEH C18 column (150 mm × 2.1 mm, 1.7 µm particle size) at a flow rate of 0.3 mL/min. The mobile phase consisted of 0.1% formic acid in water (solvent A) and methanol (solvent B). A gradient of 10–70% solvent B over 10 min was employed, followed by re-equilibration to the initial conditions (90% A, 10% B) over 1.5 min with a total runtime of 11.5 min.

Mass spectrometric analysis was performed in positive with spray voltage 3000 V, capillary temperature 256°C, probe heater temperature 350°C, sheath gas flow 48 (arbitrary units), auxiliary gas flow 11 (arbitrary units), and S-Lens RF level 50. Data acquisition was performed in full-scan MS mode over a mass range of m/z 150–1000. Stereochemical assignments were made by comparing retention times and m/z values for the amino acids detected from hominicin to those of D- and L-amino acid standards (Supplementary Fig. 7).

Butanol extraction of AH5011 hominicin
n-Butanol (100 mL) was added to 300 mL of cell-free, conditioned media and thoroughly mixed before setting aside for several min until the phases settled. After centrifugation at 2000 x g for 5 min, the upper butanol phase was collected and evaporated in a rotatory vacuum evaporator (Buchi Rotavapor R-3000) at 45°C. The dried extract was resuspended and concentrated in nuclease-free water or PBS, which was stored at −80°C until use.

Isolation of the AH5011 hominicin
The bacteriocin was concentrated by liquid-liquid partitioning of culture media with chloroform (1:1, v/v). The chloroform was removed by evaporation under nitrogen gas, and the material was subjected to reversed-phase flash chromatography using an automated CombiFlash RF system (Teledyne-Isco). A 34.7-minute method was implemented with a gradient of water (A) and methanol (B). The gradient was performed at a flow rate of 75 mL/min on a 130 g C18 RediSep column beginning at 50% B and linearly increasing to 100% B, followed by an isocratic hold at 100% B for approximately 8.5 min. Subsequently, preparative scale reversed phase high performance liquid chromatography (HPLC) was performed for further purification on a Varian HPLC system (Agilent, Santa Clara, CA) equipped with ProStar 210 pumps, a ProStar 710 fraction collector, a ProStar 335 photodiode array detector with and Galaxie Chromatography Workstation software (version 1.9.3.2). A 40-minute method was implemented with a gradient of water (A) and acetonitrile (B) on a Phenomenex Gemini–NX C18 column (5 μm; 250 × 21.20 mm). The gradient separation was performed at a flow rate of 21.2 mL/min and began at 30% B and linearly increased to 50% B.

NMR data collection
NMR experiments were performed on an Agilent 700 MHz NMR spectrometer equipped with a cryoprobe, operating at 700 MHz for 1H and 175 MHz for 13C. Spectra were collected using a water suppression enhanced through T1 effects (WET) pulse sequence. The sample was dissolved in methanol-d4, and the residual solvent signals (δ = 3.31 ppm and δ = 49.0 ppm) were used as reference peaks.

Determination of minimum inhibitory concentration (MIC)
The MICs of the purified hominicin were determined for a panel of skin microbes using a broth microdilution assay modified from the Clinical Laboratory Standards Institute (CLSI) guidelines. Serial dilutions of hominicin were prepared in 96-well microtiter plates containing bacterial cultures adjusted to 5 × 105 CFU/mL in either TSB media (staphylococci) or in cation-adjusted Mueller-Hinton Broth with laked horse blood (streptococci). As a control, the MICs of vancomycin were also obtained for the same panel of skin microbes. Plates were incubated overnight at 37°C at 250 rpm, and bacterial growth was assessed by measuring optical density at 600 nm using a BioTek Synergy H1 microplate reader. The MIC was defined as the lowest concentration of hominicin that completely inhibited visible bacterial growth.

SYTOX Green influx to detect membrane permeabilization
Mid-log S. aureus cells were washed and then suspended in a final concentration of 2 µM SYTOX Green (Invitrogen) in nuclease-free water. 100 µL of SYTOX-bacterial suspension was dispensed into a 96-well black culture plate (Corning). Plates were incubated for 15 min in the dark. Then, 100 µL of extract from AH5011 or Δp1Δp2, 70% v/v ethanol, or water were added to achieve a final volume of 200 µL per well. Fluorescence intensity was measured with a Tecan plate reader at an excitation/emission wavelength of 480 nm/522 nm and normalized to “no bacteria” controls.

DiSC3(5) efflux to detect membrane depolarization
To measure DiSC3(5) efflux, mid-log S. aureus cells were washed and suspended in a final concentration of 4 µM DiSC3(5) (Invitrogen) in buffer (5 mM HEPES, 20 mM glucose, pH 7.2). The labeled bacteria were dispensed into a 96-well black culture plate and allowed to reach equilibrium. Next, 100 µL of extract from AH5011 or Δp1Δp2, 1% Triton X-100, or HEPES/glucose buffer were added to achieve a final volume of 200 µL per well. Fluorescence measurements were taken every 5 min at an excitation/emission wavelength of 620 nm/670 nm and normalized to “no bacteria” controls.

Voltage-clamp planar lipid bilayer
Electrophysiology experiments were performed using a 4-channel micro-electrode cavity array (100 µm MECA, Ionera Technologies) on an Orbit Mini (Nanion Technologies) system. Recordings were collected using the Elements Data Reader software, with parameters set to 200 pA gain and 1.25 kHz sampling rate and subsequently analyzed using the Elements Data Analyzer (v. 1.4.6). For preparation of the horizontal lipid bilayer system, 150 µL of the bathing solution (1 M KCl, 10 mM HEPES, pH 7.2) was added to the measurement chamber. 1,2-diphytanoyl-sn-glycerol-3-phosphocholine (DphpC) from Avanti Polar Lipids was dissolved in n-octane to a concentration of 10 mg/mL (Sigma, electronics grade). Membranes were painted over the micro-cavities using the air bubble technique75 until the capacitance reached 15–30 pF, as recommended by the manufacturer. A transmembrane voltage of +50 mV was applied, unless otherwise stated. Approximately 40 ng of hominicin, 40 ng of synthetic unmodified core peptide, 0.125% v/v methanol, or 8 ng of alpha-toxin was added per aperture, and each membrane was monitored for fusion spikes to indicate insertion of peptides into the lipid bilayer. Current traces were monitored until single-channel conductance events were observed at the indicated voltage. For current-voltage analysis, the current was recorded for 10 seconds at 20 mV increments with a return to 0 mV between each step, starting from −100 mV to +100 mV. Single-channel conductance was determined from the current divided by the transmembrane voltage.

Hemolysis assay
Whole human blood samples were collected separately from three healthy adult donors in sodium heparin blood collection tubes (BD Vacutainer). Overnight cultures of bacterial strains were pelleted, washed with PBS, and normalized to 5 × 107 CFU/mL. 200 µL of bacterial suspension, conditioned media extract, or water was incubated with 200 µL of whole blood at 37°C for 4 h. Following incubation, samples were centrifuged at 5500 x g, and 100 µL of the supernatant was transferred to a 96-well plate. Erythrocyte lysis was determined by measuring the absorbance at 543 nm. Percent hemolysis was determined by normalizing absorbance values to those of water-treated samples (i.e., 100% total lysis).

Lactate dehydrogenase leakage assay with N/TERT-2G keratinocytes
Immortalized human N/TERT-2G keratinocytes were obtained from Dr. James Rheinwald laboratory and were grown in serum-free keratinocyte growth medium (Gibco) supplemented with 25 µg/mL bovine pituitary extract, 0.2 ng/mL epidermal growth factor, and 0.3 mM calcium chloride as previously described76. Low passage (<15), undifferentiated N/TERT-2G cells were seeded to 1 × 105 cells/mL into 24-well tissue culture plates (Corning) and cultured at 37°C with 5% CO2 until reaching ~90% confluency. Cell monolayers were washed with PBS and media was replaced. Overnight cultures of bacteria were pelleted, washed, and then added to cell monolayers at 1 × 106 CFU and allowed to incubate for 4 h. LDH release was measured using the CyQUANT LDH Cytotoxicity Kit (Invitrogen), following the manufacturer’s instructions.

Murine epicutaneous infection with S. aureus
Age- and gender-matched five male and five female 8-week-old C57BL6/J mice were used for epicutaneous exposure to S. aureus5,52,77. 24 h prior to bacterial inoculation, the dorsal skin of anesthetized C57BL6/J mice (2% isoflurane) was shaved and depilated with Nair (Church & Dwight Co., Inc.). S. aureus strain AH6350 was subcultured 1:50 in TSB and allowed to reach an OD600 of 1. Cells were pelleted, washed, and then resuspended in PBS to achieve an inoculum of 1 × 108 CFU in 100 µL volume. S. aureus was inoculated on a sterile 2-cm2 gauze pad and affixed to the back skin with a Tegaderm dressing and secured with adhesive bandages (BAND-AID, Johnson and Johnson) for 72 h. For mice receiving treatment, hominicin (100 µg suspended in methanol) or vehicle (2% v/v methanol) were combined with S. aureus immediately prior to application on the gauze pad. Inoculum concentration was verified by serial dilution, plating, and colony counting. Transepithelial water loss was measured using a Tewameter TM300 device (Courage & Khazaka Electronic GmbH) before infection and at 72 h post-infection. Two sites per lesion were analyzed to minimize error in measurements. The disease score as a measure of skin inflammation severity was evaluated by a blinded observer from digital photographs and quantified as a sum of three individual grades for erythema, edema (each graded: 0, 1, 2, 3) and scaling/erosion (scaling graded: 0, 1, 2, 3 while erosion graded as 4, 5, 6, 7)52. To enumerate bacterial burden, the full-thickness 2-cm2 atopic lesions were excised, suspended in 0.50 mL PBS with 1-mm zirconia-silica homogenization beads (Biospec), and subsequently homogenized for three 1-minute intervals. The tissue homogenates were serially diluted and plated on nonselective (TSA) and selective (mannitol salt agar [MSA]) media. Plates were incubated overnight before counting colonies.

Whole-genome assembly and annotation
Genomic DNA from S. hominis AH5011 and S. aureus AH6350 were isolated by phenol-chloroform extraction for whole-genome sequencing, genome assembly, and annotation at the SeqCenter facility (Pittsburgh, USA). SeqCenter-prepared DNA libraries were sequenced on the NextSeq 2000 Illumina and the ONT MinION sequencer. Quality control and adapter trimming were performed with blc2fastq (version 2.20.0.445) and porechop (version 0.2.3_seqan2.1.1) for Illumina and Oxford Nanopore sequencing, respectively. Unicycler (version 0.5.0) was used to assemble Illumina and ONT reads78. Bandage (version 0.8.1)79 and BUSCO (version 5.2.2)80 were used to assess assembly completeness. Genomes were annotated using Prokka (version 1.14.5)81. BAGEL4 was used to detect biosynthetic gene clusters19. Multi-locus sequence typing of S. aureus genomes was performed using PubMLST database82. Unless otherwise stated, default parameters were used for all software. The hom BGC and its homologs were identified using BLASTp. Genomes of various staphylococci containing the hom BGC were downloaded from NCBI and are listed in Supplementary Table 3. Comparative gene cluster analysis and visualization were performed using CAGECAT, a web-based platform that integrates Cblaster83 and Clinker84 modules. Multi-sequence alignment was performed using Clustal Omega85 and visualized using Geneious Prime (version 2024.0.7). Structural protein homology was analyzed and visualized using Foldseek (version 10) and PyMOL (version 3.0.3).

Quantification and statistical analysis
Statistical details of experiments, including the methods used and defined significance values, can be found in the figure legends. All statistical analyses were performed with GraphPad Prism software (version 10.4.0). To determine the statistical significance of differences between two groups, we used two-tailed Student’s t-tests. For multiple comparisons involving more than two groups, we chose a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. For bacterial growth curve experiments, we selected a repeated measures two-way ANOVA analysis.

Ethics statement
All vertebrate animal experiments were approved and conducted in accordance with the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus under protocol number 00941. Eight-week-old male and female C57BL6/J mice (strain #000664) were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in specific pathogen-free facilities at the University of Colorado Anschutz Medical Campus Animal Care Facility under controlled conditions (12-h light/dark cycle; temperature: 20-22°C; humidity: 30-70%). Mice were provided ad libitum access to food and water and allowed to acclimate for 1 week prior to experimentation. At experimental endpoints, mice were euthanized via CO2 inhalation followed by cervical dislocation. This method is consistent with the recommendations of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. All procedures involving human blood samples were conducted in accordance with guidelines approved by the Colorado Multiple Institutional Review Board (COMIRB) under protocol number 17-1926 and the Institutional Biosafety Committee under protocol number IBC-00001264. Informed consent was obtained from all subjects prior to participation.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Manuscript Review: The antimicrobial daptide from human skin commensal Staphylococcus hominis protects against skin\npathogens\nSummary\nThe manuscript investigates the role of a daptide bacteriocin produced by a skin commensal Staphylococcus hominis strain\nD11 in antagonizing other skin commensals and pathogens. The authors utilize a combination of microbiology, genetics,\nbioinformatics, biochemistry, and biophysics to elucidate the machinery and mode of action of the S. hominis daptide. The\nauthors have also identified a novel gene encoding for an immunity protein that confers resistance to\ndaptide-mediated killing. Lastly, the authors have demonstrated the therapeutic capability of\ndaptide in an epicutaneous infection with S.\naureus in a murine model. Thus, this study highlights the importance of exploring commensal bacteria to identify novel\nbioactive molecules as antimicrobial agents. The most noteworthy results are the discovery of a novel antimicrobial and the\ndemonstration that this peptide has potential for treatment of S. aureus infections. Overall, the manuscript is presented with\nrigorous experiments and is well-written. There are only a few\nsuggestions offered.\nSpecific comments/suggestions\nThe work is of high significance to the field and related fields and compares very favorably with the established literature.\nThe rigor of the study is apparent and support the conclusions and claims made in the manuscript.\nThe methods are sound and rigorously performed. Sufficient detail is provided in the methods for the work to be reproduced.\nL22, 67: Suggest adding a few words to highlight the biological relevance of the N2-N2-dimethyl-1,2-propanediamine C-\nterminus feature. This becomes more apparent later in the manuscript but is not clear when introduced early on.\nL68-70: Please provide references for these studies.\nL102,115: Please provide a rationale for why S. hominis AH4553 was the strain of choice/ control strain to demonstrate\ndaptide-mediated killing if in Fig. 1B, there were several S. hominis strains that were also sensitive to conditioned media\nfrom S. hominis AH5011 strain.\nL141-153. Is the reaction mechanism for the fully modified daptide described here supported by data provided in the\nmanuscript or is this a proposed mechanism? There were few references provided to support this.\nL172: Please provide a reference for the sentence that states “daptide-resistant S. aureus strain RN4220”. Also, why is this\nstrain resistant and what is the mechanism underlying disparate sensitivities to the daptide (Fig. 1B). Do these encode\nhomI?\nL172 and Discussion/Fig. S14. There was little discussion/speculation as to the mechanism underlying how HomI provides\nresistance, other than it may be membrane spanning. Is there any evidence for HomI and daptide direct interactions?\nLine 256, Table S3, Fig. S11A. Are these gene clusters also located on plasmids or are some of these chromosomally\nencoded?\nL289-290: Suggest clarifying that the S. aureus strain expressing homABCDIJM1M2PlanMD is the RN4220 strain\nexpressing hom BGC.\nL303: Please specify the WT strain for this experiment.\nLines 327-335. It’s surprising that the 2-log reduction in AH5011 upon hominicin treatment, results in such profound effects\non skin barrier function and disease scores. There was no inclusion of a control with hominicin treatment alone in the\nabsence of infection to determine if there is some benefit to overall healing but it’s unclear if this model would address the\nquestion. Perhaps an incisional wound model would be better here.\nL895: The data in Fig. 1 A, D, and F are represented as OD600 and CFUs where either set of data conveys the same result.\nSince the experiments in the subsequent figures (Figs. 1E,G, 4C, 6A,B, S2 A-D) are all represented as OD600, consider\nrepresenting Fig. 1A, D, and F as OD600 only for consistency. Alternatively, provide a rationale for why both OD600 and\nCFU were chosen to be displayed.\nFig. 1C: Please change the label from Spot Strain to Lawn Strain.\nFig. 1F: Please remove the misplaced text box with “D” within the graph.\nFig. 7 and methods: Please report the sex of the mice used in these studies and the rationale for this choice. The methods\nsection should include whether sex and/or gender were considered in the study design and whether sex and/or gender of\nparticipants was determined based on self-report or assigned (and methodology used).", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "Daptides are a small, relatively new family of RiPPs characterized by the presence of an unusual (S)-N2-N2-dimethyl-1,2\npropanediamine (Dmp)-modified C-terminus. Homincin is retrospectively considered as the inaugural compound of the\ndaptide RiPP family, as it had previously been isolated and structurally characterized from Staphylococcus hominis MBBL\nby Pyoung Il Kim and co-workers (Biochemical and Biophysical Research Communications 2010, 399, 133–138). However,\nnothing was known about its biosynthesis. Recently, a seminal paper on daptides established most of the structural and\nbiosynthetic aspects of a family of daptide compounds isolated from Microbacterium paraoxydans (Ren H. et al., Nat\nCommun 2023, 14, 1624).\nIn this work, the authors report the discovery of the plasmid-encoded biosynthetic gene cluster (BGC) of hominicin from a\nhuman skin isolate of Staphylococcus hominis. The authors demonstrate the link between (the known) hominicin and the\n(unknown) hom BGC by heterologous expression and propose a plausible and logical biosynthetic pathway to the daptide\ncompound. However, the role or the output of some key enzymes in the BGC are just assumed but not proved. Of note is the\nrole of HomI in the self-resistance of the producing strain, the first time this mechanism of immunity is reported in daptide-\nproducing strains.\nThe authors clearly showcase the narrow-spectrum antibacterial activity of hominicin against a set of skin commensal\nStaphylococcus strains (and other genera of commensal bacteria), which aligns perfectly aligns with the ecological niche\nroles of bacteriocins. They further elaborate on the mechanism of action and successfully establish the pore-forming activity\nof hominicin. Furthermore, they demonstrate that hominicin is able to prevent S. aureus infection in vivo (mouse skin).\nOverall, the results are comprehensive, well-substantiated and very well presented. This work represents an advance in the\nknowledge of the biosynthesis, biological activity and mechanism of action of daptides, especially considering that daptides\nremain an understudied and slowly growing family of RiPPs.\nBefore publication, some points need to be addressed:\nA. Major comments:\n1) My main concern relates to the N,N-dimethyl Ile (DmIle1) at the N-terminus of homincin. Considering that this post\ntranslational modification (PTM) is so far restricted to hominicin but it is absent in the other few daptide compounds\ncharacterized so far (Ren H. et al., Nat Commun 2023, 14, 1624), I believe that author should provide experimental\nevidences about this N,N-dimethylation.\nThey propose that HomM1 is likely responsible for this methylation, but nothing has been done to prove it. I think they should\nconduct some experiments on it, as they have done for other enzymes in the BGC. Also, this experiment could give valuable\ninsight into the importance or not of this N-terminal dimethylation on the bioactivity or even the mechanism of action of\nhomincin. Of note, the N-terminal methylation has been proved to be essential in the bioactivity of cypemycin and other\nlinaridin RiPPs (e.g.; Claesen J, Bibb M. Proc Natl Acad Sci USA 2010, 107:16297–16302).\n2) Regarding the stereochemistry of hominicin, I greatly miss experimental confirmation. Hominicin is a previously known\ncompound, but the stereochemistry remains a knowledge gap in its structure.\nOf course, the authors know that being a RiPP, most of the constituent amino acids will be L. However, they claim that\ngenetically encoded Ser-10 and Ser-17 are converted to Dha by dehydratase HomMD and then reduced to D-Ala residues\nby oxidoreductase HomJ. This explanation is perfectly consistent with the biosynthetic logic of lantipeptides, but I think it\nshould be empirically tested by Marfey´s analysis. Moreover, this experiment would make it possible to completely rule out the\npresence of cryptic epimerases acting on the precursor peptide.\nInstead of tedious partial hydrolysis and derivatizations, a full hydrolysis and determination of the L-Ala to D-Ala ratio would\nsuffice, as has been done in previous works (e.g.; Ortiz-López et al. Angewandte Chemie International Edition, 2020, 59(31),\n12654-12658). In addition, this experiment would further confirm the presence of Glu-13 (of which most NMR signals are not\nobserved) in the structure.\nAs for the stereochemistry of the Dmp residue at the C-terminus, I think it is quite reasonable to assume the absolute (S)\nconfiguration based on biosynthetic arguments, but the authors should make this argument more clearly explicit in the\nhominicin structure characterization part of the manuscript.\nB. Apart from the above, some minor questions on the bioactivity and mechanism of action of hominicin:\n3) The authors provide valuable insights into the mechanism of action of hominicin. However, I am not sure about the\nextrapolation of these results to daptides in general, as the authors state in the introduction and conclusions (last sentence of\nthe last paragraph of the manuscript). As they themselves discuss in the manuscript, there is some genetic diversity within\nthe daptide BGCs found in staphylococcal strains, and these differences include the presence or not of PTM enzymes such\nas LanMD dehydratases and/or HomJ oxidoireductases. It is possible that other novel daptides with additional structural\nfeatures will later be discovered that modify the molecules to the extent that the mechanism of action changes (but they will\nstill be daptides).\n4) Although the authors showcase the in vivo activity of hominicin (mouse skin infection model), I think the therapeutic\napplicability remains doubtful unless additional experiments such as cytotoxicity and hemolytic activity are done. Do the\nauthors have data on this?\n5) Hominicin does not seem to inhibit S. capitis, any plausible explanation?\n6) I have not been able to find any information on the S. aureus strain AH6350, which was used in the work to show\nbioactivity in vitro and in vivo (mouse skin infection model). The authors should add an appropriate reference(s) on this\npathogenic strain, particularly those related to antimicrobial resistance mechanisms and/or membrane-level characterization.\nC. Finally, there are other very minor comments:\ni) Line 139: This part of the sentence is a bit confusing; the reader could understand that there are some structural\ndifferences between hominicin and the compound identified in this work (which is actually the same). Please rewrite it.\nii) Line 168: I would not say that NMR was just an orthogonal approach to confirm the structure, but that it was applied to\nunequivocally confirm the structure.\niii) Line 179: I miss the data of the amount of compound isolated, and I could not find this data in the supporting information.\nPlease include this data.\niv) Line 207: The reading of abbreviation for the mutant HomACIM1PlanMD was a bit confusing to me. I would change it to\nHomACIM1PLanMD in line 207 and the successive times it appears (at least in the manuscript text).\nv) Line 216: Actually, the first ion of the MS cluster should be the previous m/z (1026.0642, calculated with Bruker Isotope\nPattern tool). Please reflect this also in Figure S9C\nvi) Line 339: I would write “understudied” instead of “unstudied”.\nvii) Line 394: I agree that the differences between Mpa daptides and hominicin regarding their bioactivity profiles are probaly\ndue to the presence of dehydroamino acids in the latter, but the authors should not forget that the other key difference is the\nN-terminal dimethylation. This kind of modification has been proved essential for the bioactivity of cypemycin and other\nlinaridins, as commented above (comment 1)\nviii) Line 553: The NMR spectra were recorded in methanol-d3 (CD3OH). Were the experiments carried out under a solvent-\npresaturation scheme? If so, please indicate it in the experimental section.\nix) Figure 7C: Images are not clear. They appear blurry or pixelated. I think they should be improved.\nx) Supporting Information, Figure S3: I´m a bit surprised by the impressive accuracy of the LC-MS1 data shown across\ndifferent samples. The retention time and the m/z are completely identical for both hominicin AH5011(wt) and\nhomABCDIJM1M2PlanMD mutant. Perhaps the authors have inadvertently duplicated the same figure.\nxi) Supporting Information, Figure S9C: (see also comment v above).\n--At entry 1 of the table, ion y9 is not correct. It should be m/z 868.xxx instead 852.4864. In the corresponding peptide\nsequence, there is another typo: it reads Glu-Ile-Dhb-Dha-Ala-Val-Ile-Ala-Thr, but it should be Glu-Ile-Dhb-Ala-Dha-Val-Ile-\nAla-Thr.\n--From entry 7 (ion b4) to entry 12 (ion b11) there are some erroneous annotations of m/z values\nand/or aa sequence. Please check it before publishing.\nxii) Supporting Information, Table S4: The coupling constant (J) value for γ´-CH3 signal of Val-18 is not feasible. Please\nrevise this probable typo.", "reviewer_4": "Review of Nguyen et al\nThis manuscript is an activity-based natural product isolation story focused on a molecule made by members of the skin\nmicrobiome. The molecule in question is a RiPP (ribosomal natural product) called a daptide. These RiPPs are\ndistinguished by the presence of methylated amine groups found at both the N- and C-termini. Previous work on daptides\nclearly laid out the biosynthesis of these molecules but showed only minimal characterization of bioactivity. The current\nmanuscript convincingly shows that daptides originating from Staph. are membrane active and depolarize the membrane.\nIn this current work the authors also discover an immunity factor for daptides, a YidC homolog. Finally, the authors also show\nthat the daptide, named hominicin, is effective at combatting Staph aureus skin infections in mice. If there is any substantial\ncriticism of the work it is lack of novelty of the hominicin compound, but in my opinion this weakness is overcome by the new\ninsights into the structure-activity relationship, the discovery of a highly unusual immunity factor, the clear experiments into\nthe mechanism of action, and the demonstration of efficacy in a mouse skin infection model. I am highly supportive of\npublication pending some clarifications/edits delineated below.\n1) Line 60, make sure to include the AD abbreviation when atopic dermatitis is first mentioned\n2) Line 139, this sentence should be altered a bit to make it clear that the disagreement is at positions 10 and 17 of the core\npeptide, not the precursor. This is only clear when looking at Figure 2. Moreover, it is still unclear to me after reading the\npaper if there is any difference between the homincin in ref 17 and what is reported here. Finally, and related to this, can the\nauthors include a comparison or sequence alignment of hominicin(s) (if they are different) and the daptide from ref 12?\n3) Line 147, related to the above point, how does the hominicin BGC compare to the daptide BGC in ref 12?\n4) Line 172, is the RN4220 strain resistant because it is a hominicin producer? Or does it just harbor the gene encoding\nHomI?\n5) Line 179, can the authors provide the peptide yield after purification to homogeneity?\n6) Line 183: the reference to Figure S5 is incorrect, this is a standard curve\n7) Line 203: too many significant figures on the report zone sizes\n8) Line 207-217: this section was hard for me to follow because it is difficult to keep track of what all of the genes do. This\nmay be improved when the Figure 4 is put near this text, but I might still suggest that the authors explain briefly what each\nvariants is omitting\n9) Line 222: ref 12 did show that the original daptides had hemolysis activity. I agree that this isn’t the same as forming\npores, but the hemolysis activity should probably be mentioned here\n10) Line 283: this section discusses the presence of homI in the Staph BGCs, but what about the set of ~500 daptides\npredicted in ref 12? Is the presence of homI more general or is it Staph-specific?\n11) Line 286: when I see the phrase “homI mutant” I think of a deletion mutant, but this is an overexpression mutant. I\nsuggest the authors make this clear and mention whether the homI expression plasmid is multicopy\n12) Line 326: express should be expresses\n13) Line 328: I find the statement that “infection with S. aureus was augmented by the topical addition…” confusing. The\nword augment could suggest that homincin was helping colonization of S. aureus\n14) Line 339: I think the authors mean understudied\n15) Figure 1: there is an extra letter D floating in panel F\n16) Figure 1B caption: Table S1 in the SI does not seem to include information about all of the strains used for susceptibility\ntesting. This information is important to include if others want to follow up on the work"}, "author_response": "Response: We appreciate the positive comments from the reviewer.\nL22, 67: Suggest adding a few words to highlight the biological relevance of the N2-N2-dimethyl-\n1,2-propanediamine C-terminus feature. This becomes more apparent later in the manuscript but\nis not clear when introduced early on.\nResponse: To the best of our knowledge, the seminal work by Ren et al. (DOI: 10.1007/s12223-\n013-0259-1) has thoroughly investigated the biochemical and functional aspect of the N -N -\n2 2\ndimethyl-1,2-propanediamine (Dmp)-modified C-terminus. No other works have examined the\nDmp feature. To date, the identified Dmp-containing peptides include MpaA1, MpaA2, and MpaA3\nfrom M. paraoxydans and hominicin; however, the genetic and biosynthetic origin of the latter, as\na RiPP, remains undetermined. We now provide this information in the text with works cited (Line\n68-76).\nL68-70: Please provide references for these studies.\nResponse: We have added references citing these studies (Line 68).\nL102,115: Please provide a rationale for why S. hominis AH4553 was the strain of choice/ control\nstrain to demonstrate daptide-mediated killing if in Fig. 1B, there were several S. hominis strains\nthat were also sensitive to conditioned media from S. hominis AH5011 strain.\nResponse: We selected S. hominis AH4553 as a sensitive control strain based on several factors:\nit is a well-established reference strain from the American Type Culture Collection (also referred\nto as ATCC 27844) that was isolated from human skin, is commercially available, and has a\npublicly available genome in the National Center for Biotechnology Information database through\nthe National Institute of Health.\nThis information is provided in the text and Supplementary Table 1. “We observed that the\nantimicrobial function inhibits the S. hominis reference strain ATCC 27844 (AH4553) from the\nAmerican Type Culture Collection in a concentration-dependent manner (Fig. 1 c, d, e). This strain\nwas adopted as a sensitive control strain for subsequent experiments.”\nL141-153. Is the reaction mechanism for the fully modified daptide described here supported by\ndata provided in the manuscript or is this a proposed mechanism? There were few references\nprovided to support this.\nResponse: The description of the post-translational modifications described in this section\nrepresent a proposed biosynthetic model based on the genetic data and is later supported by\ncloning experiments, MS, NMR, and Marfey’s analysis. Furthermore, the chemical modification of\nthe C-terminal threonine into Dmp has been extensively investigated in previous work by Ren et\nal. (DOI: 10.1007/s12223-013-0259-1). The dehydration of serines and threonines, as well as the\noxidoreduction of dehydroalanine intermediates, are well documented in RiPP biosynthesis.\nAdditional references have been added to the text of this section to support the biosynthetic\nmodel.\nL172: Please provide a reference for the sentence that states “daptide-resistant S. aureus strain\nRN4220”. Also, why is this strain resistant and what is the mechanism underlying disparate\nsensitivities to the daptide (Fig. 1B). Do these encode homI?\nResponse: After careful review, we believe it is not appropriate to describe S. aureus strain\nRN4220 as “daptide-resistant” because we did not perform an antimicrobial MIC test to establish\nits resistance and have relied on the conditioned media growth experiment as an indicator of\nsusceptibility. We have now revised the text to read, “We cloned the 9.6-kb BGC on the plasmid\npCM28 and heterologously expressed the construct in a restriction-deficient cloning host S.\naureus strain RN4220.”\nWe confirmed that RN4220 (GenBank Accession CP076105) does not encode either a\nbiosynthetic gene cluster for hominicin or harbor the homI gene. RN4220 does encode two\nputative yidC_1 and yidC_2 genes, which share structural similarity with HomI and may contribute\nto the observed resistance phenotype. With limited number of sequencing data, we cannot verify\nwith certainty that all resistant strains encode homI and/or homologs of HomI. With that being\nsaid, we also cannot rule out other potential mechanism(s). The disparate sensitivities to\nhominicin could be attributed to canonical transporter systems for bacteriocins (e.g., ATP-binding\ncassette transporters), which can confer cross-resistance to other bacteriocins. Another plausible\nexplanation is that the concentration of hominicin in the CM may be insufficient to elicit observable\nsensitivity and could vary between batches. We have included additional text addressing these\nalternative explanations in the Discussion section.\nL172 and Discussion/Fig. S14. There was little discussion/speculation as to the mechanism\nunderlying how HomI provides resistance, other than it may be membrane spanning. Is there any\nevidence for HomI and daptide direct interactions?\nResponse: To the best of our knowledge, there is little to no literature reporting mechanisms of\ndaptide resistance and/or HomI-specific resistance. To investigate potential interactions, we\nperformed protein-ligand modelling using Boltz-1x with HomI protein sequence and hominicin\nligand SMILES as inputs (see Fig. 6, Supplementary Fig. 15). Our structural modeling shows\nhominicin docks within HomI’s charged core with high predictability. While YidC facilitates protein\ninsertion into the membrane, HomI may sequester hominicin within its core or participate in the\ntransport of hominicin. Other studies also suggest that YidC is involved in immunity against\nbacteriocin and lantibiotics. We have provided these speculations and the related references in\nthe Discussion section.\nLine 256, Table S3, Fig. S11A. Are these gene clusters also located on plasmids or are some of\nthese chromosomally encoded?\nResponse: In our initial analysis of fourteen staphylococcal assemblies, we identified one closed\nplasmid sequence from a fully assembled genome of S. epidermidis strain ADQBK (Accession\nCP134841) that encodes a daptide gene cluster. The remaining thirteen other assemblies are\nshotgun sequences and cannot be verified with certainty whether the daptide BGCs they encode\nare chromosomally or plasmid-derived. We have adjusted the text accordingly to clarify that the\ngenome assemblies used for comparison were sourced from shotgun sequencing data.\nL289-290: Suggest clarifying that the S. aureus strain expressing homABCDIJM1M2PlanMD is\nthe RN4220 strain expressing hom BGC.\nResponse: We thank the reviewer for their suggestion. We have revised the text to read, “the S.\naureus strain RN4220 expressing hom BGC,” which improves clarity.\nL303: Please specify the WT strain for this experiment.\nResponse: We thank the reviewer for their comment. We have revised the text to specify the\nstrain that now reads, “the wild-type strain S. aureus Newman,” which indicates that the strain\nNewman was used as the parental strain in this experiment.\nLines 327-335. It’s surprising that the 2-log reduction in AH5011 upon hominicin treatment, results\nin such profound effects on skin barrier function and disease scores. There was no inclusion of a\ncontrol with hominicin treatment alone in the absence of infection to determine if there is some\nbenefit to overall healing but it’s unclear if this model would address the question. Perhaps an\nincisional wound model would be better here.\nResponse: We have now included in vivo data showing hominicin treatment alone in the absence\nof S. aureus infection (see Fig. 7 and Supplementary Fig. 16). We believe that the improvements\nin skin barrier function and disease scores are attributed to the reduction or absence of S. aureus\nburden rather than to hominicin promoting skin healing. S. aureus is known to secrete various\nvirulence factors (e.g., exoenzymes, pore-forming toxins, superantigens) that contribute to skin\nbarrier disruption and inflammation. We have revised the text to clarify this.\nL895: The data in Fig. 1 A, D, and F are represented as OD600 and CFUs where either set of\ndata conveys the same result. Since the experiments in the subsequent figures (Figs. 1E,G, 4C,\n6A,B, S2 A-D) are all represented as OD600, consider representing Fig. 1A, D, and F as OD600\nonly for consistency. Alternatively, provide a rationale for why both OD600 and CFU were chosen\nto be displayed.\nResponse: We appreciate the reviewer’s suggestion and agree that the growth curve\nrepresented as OD conveys the same results as CFUs. We believe that displaying the CFU\n600\ndata strengthens the accuracy and biological relevance of the OD measurements. While OD\n600 600\nprovides information on culture turbidity and allows quick real-time monitoring of growth, it does\nnot distinguish cell viability. CFU counts reflect actual viability, cytotoxicity, and antimicrobial\neffects, thus serve as a standard validation metric for growth curve experiments. Having validated\nour growth experiments using CFU counts, we rationalized that OD measurements can be used\n600\nas indicators of inhibition in subsequent experiments.\nFig. 1C: Please change the label from Spot Strain to Lawn Strain.\nResponse: We thank the reviewer for the comment and fixed the typo for Figure 1c.\nFig. 1F: Please remove the misplaced text box with “D” within the graph.\nResponse: We thank the reviewer for the comment and removed the misplaced label.\nFig. 7 and methods: Please report the sex of the mice used in these studies and the rationale for\nthis choice. The methods section should include whether sex and/or gender were considered in\nthe study design and whether sex and/or gender of participants was determined based on self-\nreport or assigned (and methodology used).\nResponse: We used five male and five female 8-week-old C57BL6/J mice per condition for the\nepicutaneous infection experiment. The sex of mice is mentioned in the Result, and the rationale\nhas been added in the Methods section, along with relevant literature citing the use of this model.\nFor the isolation of bacteria from skin swabs, we recruited human participants of both sexes and\nall ethnicities. Each bacterial isolate is linked to anonymized, self-reported information about the\nage and sex of sampled volunteer. This information is now explicitly stated in the Methods section.\nReviewer #2\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We appreciate the reviewer’s evaluation of this manuscript.\nReviewer #3\nDaptides are a small, relatively new family of RiPPs characterized by the presence of an unusual\n(S)-N2-N2-dimethyl-1,2 propanediamine (Dmp)-modified C-terminus. Hominicin is retrospectively\nconsidered as the inaugural compound of the daptide RiPP family, as it had previously been\nisolated and structurally characterized from Staphylococcus hominis MBBL by Pyoung Il Kim and\nco-workers (Biochemical and Biophysical Research Communications 2010, 399, 133–138).\nHowever, nothing was known about its biosynthesis. Recently, a seminal paper on daptides\nestablished most of the structural and biosynthetic aspects of a family of daptide compounds\nisolated from Microbacterium paraoxydans (Ren H. et al., Nat Commun 2023, 14, 1624).\nIn this work, the authors report the discovery of the plasmid-encoded biosynthetic gene cluster\n(BGC) of hominicin from a human skin isolate of Staphylococcus hominis. The authors\ndemonstrate the link between (the known) hominicin and the (unknown) hom BGC by\nheterologous expression and propose a plausible and logical biosynthetic pathway to the daptide\ncompound. However, the role or the output of some key enzymes in the BGC are just assumed\nbut not proved. Of note is the role of HomI in the self-resistance of the producing strain, the first\ntime this mechanism of immunity is reported in daptide-producing strains.\nThe authors clearly showcase the narrow-spectrum antibacterial activity of hominicin against a\nset of skin commensal Staphylococcus strains (and other genera of commensal bacteria), which\nperfectly aligns with the ecological niche roles of bacteriocins. They further elaborate on the\nmechanism of action and successfully establish the pore-forming activity of hominicin.\nFurthermore, they demonstrate that hominicin is able to prevent S. aureus infection in vivo (mouse\nskin).\nOverall, the results are comprehensive, well-substantiated and very well presented. This work\nrepresents an advance in the knowledge of the biosynthesis, biological activity and mechanism\nof action of daptides, especially considering that daptides remain an understudied and slowly\ngrowing family of RiPPs.\nBefore publication, some points need to be addressed:\nA. Major comments:\n1) My main concern relates to the N,N-dimethyl Ile (DmIle1) at the N-terminus of hominicin.\nConsidering that this post translational modification (PTM) is so far restricted to hominicin but it is\nabsent in the other few daptide compounds characterized so far (Ren H. et al., Nat Commun\n2023, 14, 1624), I believe that author should provide experimental evidence about this N,N-\ndimethylation. They propose that HomM1 is likely responsible for this methylation, but nothing\nhas been done to prove it. I think they should conduct some experiments on it, as they have done\nfor other enzymes in the BGC. Also, this experiment could give valuable insight into the\nimportance or not of this N-terminal dimethylation on the bioactivity or even the mechanism of\naction of hominicin. Of note, the N-terminal methylation has been proved to be essential in the\nbioactivity of cypemycin and other linaridin RiPPs (e.g.; Claesen J, Bibb M. Proc Natl Acad Sci\nUSA 2010, 107:16297–16302).\nResponse: In response to this point, we made an expression strain lacking the homM1 gene and\nobserved a pronounced loss of antimicrobial activity (Fig. 4). We performed mass spectrometric\nanalysis of the spent media from the mutant and detected the proposed molecule displayed in\nSupplementary Fig. 11b, which is supported by MS/MS analysis. As expected, this peptide lacks\nthe N-terminal methylations while retaining formation of D-Ala from L-Ser. Surprisingly, the\nobserved product also lacked the Dmp modification on the C-terminus. The biosynthetic rationale\nfor this is unclear as the installation of the C-terminal modifications should precede removal of the\nleader peptide needed for N-terminal methylation (Ren H. et al., Nat Commun 2023, 14, 1624).\nWe speculate that perhaps the removal of homM1 impacted expression of the daptide specific\ngenes or that the Dmp biosynthetic enzymes are not leader dependent. To evaluate this further,\nwe attempted to generate a knockout of the homM2 but was unsuccessful. We next produced a\nhomD knockout. As shown in Supplementary Fig. 11c, we observed formation of a hominicin\nanalogue with the expected N-terminal methylations and D-Alas, but a C-terminal ketone instead\nof the fully modified Dmp. This indicates that the N-terminal methylations can be installed even\nwithout formation of the Dmp motif.\n2) Regarding the stereochemistry of hominicin, I greatly miss experimental confirmation.\nHominicin is a previously known compound, but the stereochemistry remains a knowledge gap in\nits structure. Of course, the authors know that being a RiPP, most of the constituent amino acids\nwill be L. However, they claim that genetically encoded Ser-10 and Ser-17 are converted to Dha\nby dehydratase HomMD and then reduced to D-Ala residues by oxidoreductase HomJ. This\nexplanation is perfectly consistent with the biosynthetic logic of lantipeptides, but I think it should\nbe empirically tested by Marfey´s analysis. Moreover, this experiment would make it possible to\ncompletely rule out the presence of cryptic epimerases acting on the precursor peptide.\nInstead of tedious partial hydrolysis and derivatizations, a full hydrolysis and determination of the\nL-Ala to D-Ala ratio would suffice, as has been done in previous works (e.g.; Ortiz-López et al.\nAngewandte Chemie International Edition, 2020, 59(31), 12654-12658). In addition, this\nexperiment would further confirm the presence of Glu-13 (of which most NMR signals are not\nobserved) in the structure.\nAs for the stereochemistry of the Dmp residue at the C-terminus, I think it is quite reasonable to\nassume the absolute (S) configuration based on biosynthetic arguments, but the authors should\nmake this argument more clearly explicit in the hominicin structure characterization part of the\nmanuscript.\nResponse: In response to these important points, we have now performed a full hydrolysis of\nhominicin followed by Marfey’s derivatization and LC-MS analysis to determine the ratio of L-Ala\nto D-Ala residues. The results confirmed the presence of D-Ala in hominicin, consistent with the\nbiosynthetic model and supporting the stereochemical assignment. These new data have been\nincorporated into the Supporting Information (refer to Supplementary Fig. 7) and summarized in\nthe manuscript text (Line 173-181). Moreover, we have quantified the ratio of L:D Ala and found\nit to be 1.2:1. This is consistent with our hypothesis of three L-Alas and two D-Alas (1.5:1).\nRegarding the Dmp residue, we agree that the (S) configuration is most likely because published\nwork (DOI: 10.1007/s12223-013-0259-1) confirmed the (S) configuration for other daptides.\nWe have added a statement to this effect in the structure elucidation section of the manuscript,\nas follows:\n“The configuration of the stereocenter of the Dmp group on the C-terminus was not confirmed,\nalthough previous work has shown this functional group to have an (S) configuration in other\ndaptides.”\nB. Apart from the above, some minor questions on the bioactivity and mechanism of action\nof hominicin:\n3) The authors provide valuable insights into the mechanism of action of hominicin. However, I\nam not sure about the extrapolation of these results to daptides in general, as the authors state\nin the introduction and conclusions (last sentence of the last paragraph of the manuscript). As\nthey themselves discuss in the manuscript, there is some genetic diversity within the daptide\nBGCs found in staphylococcal strains, and these differences include the presence or not of PTM\nenzymes such as LanMD dehydratases and/or HomJ oxidoreductases. It is possible that other\nnovel daptides with additional structural features will later be discovered that modify the molecules\nto the extent that the mechanism of action changes (but they will still be daptides).\nResponse: We agree with the reviewer that the mechanism of action for hominicin–specifically\npore formation–may not apply to all daptides. Ren et al. (DOI: 10.1007/s12223-013-0259-1)\ndemonstrated that the Mpa daptides are not antimicrobial but exhibit hemolytic activity. The\nauthors suggested that the combined internal hydrophobicity and the charge modification of the\nterminus may indicate a shared membrane-targeting mode of action among daptides. To address\nthis, we revised the text (Line 85, 497) to clarify that the proposed mode of action pertains to\nhominicin.\n4) Although the authors showcase the in vivo activity of hominicin (mouse skin infection model), I\nthink the therapeutic applicability remains doubtful unless additional experiments such as\ncytotoxicity and hemolytic activity are done. Do the authors have data on this?\nResponse: We thank the reviewer for these suggestions and have now included hemolysis data\nusing whole human blood as well as cytotoxicity data using immortalized N/TERT keratinocytes\n(Supplementary Fig. 13). We did not observed differences in hemolysis of the AH5011 extract\nwhen compared to treatment with the ∆p1∆p2 extract. This finding is supported by data using live\nbacteria. While the S. aureus strains exhibited pronounced hemolysis and cytotoxicity, S. hominis\nAH5011 is comparable to the control treatment (PBS). Additionally, we’ve included the hominicin\ntreatment alone without S. aureus infection on mouse skin and observed minimal skin damage\n(Fig. 7 and Supplementary Fig. 16).\n5) Hominicin does not seem to inhibit S. capitis, any plausible explanation?\nResponse: We observed varying levels of susceptibility to AH5011’s conditioned media (CM) in\nS. capitis, and one plausible explanation is that the concentration of hominicin in the CM is\ninsufficient to elicit observable sensitivity in S. capitis. While this work primarily focuses on the\nmechanism of producer immunity–specifically the role of HomI–we cannot rule out other potential\nmechanism(s). It is also possible that S. capitis may encode homologs of HomI or canonical\ntransporter systems for bacteriocins (e.g., ATP-binding cassette transporters).\n6) I have not been able to find any information on the S. aureus strain AH6350, which was used\nin the work to show bioactivity in vitro and in vivo (mouse skin infection model). The authors should\nadd an appropriate reference(s) on this pathogenic strain, particularly those related to\nantimicrobial resistance mechanisms and/or membrane-level characterization.\nResponse: The S. aureus strain AH6350 has not been previously published. Characterization of\nthis strain was primarily done through this work, which included genomic information, antimicrobial\nsusceptibility test, and hemolysis and cytotoxicity data. We rationalized that AH6350 is a suitable\norganism for in vivo studies due to its ability to cause cutaneous damage on mouse skin. To\nsupport this, we now include hemolysis and cytotoxicity data for AH6350 (Supplementary Fig. 13),\ncomparing it to the well-known USA300 lineage strain AH1263 and the ∆agr mutant to highlight\nits pathogenic potential. While we cannot provide references to cite this specific clinical strain,\nwe have added additional work citing the epidemiology, antimicrobial resistance, virulence, and\ncharacterization of the sequence type 15 (ST15).\nC. Finally, there are other very minor comments:\nLine 139: This part of the sentence is a bit confusing; the reader could understand that there are\nsome structural differences between hominicin and the compound identified in this work (which is\nactually the same). Please rewrite it\nResponse: We believe the confusion arises from a textual error. The difference is at positions 10\nand 17 of the core peptide, not the “precursor peptide” as originally stated. We apologize for this\nerror and have revised the text accordingly.\n“We detected differences at the 10th and 17th residues between the core peptide of the AH5011\nbacteriocin and the final structure of hominicin (Supplementary Fig. 14d). Although the genetic\nand biosynthetic information of the hominicin-producing strain MBBL 2-9 are not publicly available,\nwe infer that the AH5011 bacteriocin is likely hominicin.”\nLine 168: I would not say that NMR was just an orthogonal approach to confirm the structure,\nbut that it was applied to unequivocally confirm the structure.\nResponse: We thank Reviewer 3 for the observation and agree with the comment. We have\naddressed the suggestion and rephrased the sentence accordingly. “1D and 2D NMR\nexperiments were carried out to confirm the proposed structure of AH5011 hominicin.”\nLine 179: I miss the data of the amount of compound isolated, and I could not find this data in\nthe supporting information. Please include this data.\nResponse: We have now included the amount of compound isolated (approximately 6.4 mg) in\nthe main text.\nLine 207: The reading of abbreviation for the mutant HomACIM1PlanMD was a bit confusing to\nme. I would change it to HomACIM1PLanMD in line 207 and the successive times it appears (at\nleast in the manuscript text).\nResponse: We thank the reviewer for their thoughtful suggestion and have revised the text\naccordingly.\nLine 216: Actually, the first ion of the MS cluster should be the previous m/z (1026.0642,\ncalculated with Bruker Isotope Pattern tool). Please reflect this also in Figure S9C.\nResponse: We believe this confusion may arise from the way the mass spectrum was labeled in\nFigure 9a. According to ChemDraw’s isotope distribution calculator, the most abundant isotope\nhas an m/z of 1026.5659, which aligns with our experimental data (m/z 1026.5684). The m/z\n1026.0642 corresponds to the second most abundant isotope. We have revised Figure S9a,\nwhich is now Supplementary Fig 10a, to clarify this.\nLine 339: I would write “understudied” instead of “unstudied”.\nResponse: We thank the reviewer for the comment and the typo has been fixed.\nLine 394: I agree that the differences between Mpa daptides and hominicin regarding their\nbioactivity profiles are probaly due to the presence of dehydroamino acids in the latter, but the\nauthors should not forget that the other key difference is the N-terminal dimethylation. This kind\nof modification has been proved essential for the bioactivity of cypemycin and other\nlinaridins, as commented above (comment 1).\nResponse: We have revised the text to mention that the Mpa daptides lack a dimethylated N-\nterminus.\nLine 553: The NMR spectra were recorded in methanol-d3 (CD3OH). Were the experiments\ncarried out under a solvent-presaturation scheme? If so, please indicate it in the experimental\nsection.\nResponse: Following the reviewer's suggestion, we have included the pulse sequence used for\nthe NMR data collection, and the corresponding section in the method has been rephrased as\nfollows: NMR experiments were performed on an Agilent 700 MHz NMR spectrometer equipped\nwith a cryoprobe, operating at 700 MHz for 1H and 175 MHz for 13C. Spectra were collected using\na water suppression enhanced through T1 effects (WET) pulse sequence. The sample was\ndissolved in methanol-d , and the residual solvent signals (δ = 3.31 ppm and δ = 49.0 ppm)\nwere used as reference peaks.\nFigure 7C: Images are not clear. They appear blurry or pixelated. I think they should be improved.\nResponse: The blurry images of mouse skin have been replaced with the highest resolution\navailable.\nSupporting Information, Figure S3: I´m a bit surprised by the impressive accuracy of the LC-MS1\ndata shown across different samples. The retention time and the m/z are completely identical for\nboth hominicin AH5011(wt) and homABCDIJM1M2PlanMD mutant. Perhaps the authors have\ninadvertently duplicated the same figure.\nResponse: We thank the reviewer for catching this error. Upon review, we found that panels\nSupplementary Fig. 3a and 3c were inadvertently duplicated during figure assembly. Panels 3b\nand 3d are correct as presented. We have now replaced Supplementary Fig. 3a with the correct\ndata and updated the Supporting Information accordingly.\nSupporting Information, Figure S9C:\n--At entry 1 of the table, ion y9 is not correct. It should be m/z 868.xxx instead 852.4864. In the\ncorresponding peptide sequence, there is another typo: it reads Glu-Ile-Dhb-Dha-Ala-Val-Ile-Ala-\nThr, but it should be Glu-Ile-Dhb-Ala-Dha-Val-Ile-Ala-Thr.\n--From entry 7 (ion b4) to entry 12 (ion b11) there are some erroneous annotations of m/z values\nand/or aa sequence. Please check it before publishing.\nResponse: We thank the reviewer for their careful examination of the table. Upon review, we\nconfirmed that ion y9 was not observed in the fragmentation spectrum and have removed this\nentry from the table. Additionally, we have corrected the erroneous entries noted from entry 7 -12.\nThe updated table now accurately reflects the correct m/z values and fragment assignments.\nThese revisions have been incorporated into the Supporting Information.\nSupporting Information, Table S4: The coupling constant (J) value for γ´-CH3 signal of Val-18 is\nnot feasible. Please revise this probable typo.\nResponse: We apologize for this typo and thank Reviewer 3 for pointing it out. The J value for\nγ´-CH3 signal of Val-18 was corrected. The new J value is 6.6.\nReviewer #4\nThis manuscript is an activity-based natural product isolation story focused on a molecule made\nby members of the skin microbiome. The molecule in question is a RiPP (ribosomal natural\nproduct) called a daptide. These RiPPs are distinguished by the presence of methylated amine\ngroups found at both the N- and C-termini. Previous work on daptides clearly laid out the\nbiosynthesis of these molecules but showed only minimal characterization of bioactivity. The\ncurrent manuscript convincingly shows that daptides originating from Staph. are membrane active\nand depolarize the membrane. In this current work the authors also discover an immunity factor\nfor daptides, a YidC homolog. Finally, the authors also show that the daptide, named hominicin,\nis effective at combatting Staph aureus skin infections in mice. If there is any substantial criticism\nof the work it is lack of novelty of the hominicin compound, but in my opinion this weakness is\novercome by the new insights into the structure-activity relationship, the discovery of a highly\nunusual immunity factor, the clear experiments into the mechanism of action, and the\ndemonstration of efficacy in a mouse skin infection model. I am highly supportive of publication\npending some clarifications/edits delineated below.\n1) Line 60, make sure to include the AD abbreviation when atopic dermatitis is first mentioned.\nResponse: We appreciate the reviewer’s comment and have included the abbreviation for atopic\ndermatitis (AD).\n2) Line 139, this sentence should be altered a bit to make it clear that the disagreement is at\npositions 10 and 17 of the core peptide, not the precursor. This is only clear when looking at Figure\n2. Moreover, it is still unclear to me after reading the paper if there is any difference between the\nhominicin in ref 17 and what is reported here. Finally, and related to this, can the authors include\na comparison or sequence alignment of hominicin(s) (if they are different) and the daptide from\nref 12?\nResponse: We thank the reviewer for catching this error and have revised the text to clarify that\nthe differences are located in the core peptide. “We detected differences at the 10th and 17th\nresidues between the core peptide of the AH5011 bacteriocin and the final structure of hominicin\n(Supplementary Fig. 14d). Although the genetic and biosynthetic information of the hominicin-\nproducing strain MBBL 2-9 are not publicly available, we infer that the AH5011 bacteriocin is likely\nhominicin.”\nBased on the MS and NMR analysis, we confirmed the AH5011 daptide is structurally identical to\nthe hominicin reported by Kim et al. (DOI: 10.1016/j.bbrc.2010.07.024). This information is stated\nin the Result and Discussion sections. Importantly, with the genetic data, we resolved the\nbiosynthetic gap between the precursor peptide, which to our knowledge has not been reported,\nand the final structure of hominicin.\nAdditionally, we have included a comparison of the precursor sequences between hominicin from\nS. hominis and MpaA1, MpaA2, and MpaA3 from M. paraoxydans in Supplementary Fig. 14.\n3) Line 147, related to the above point, how does the hominicin BGC compare to the daptide BGC\nin ref 12?\nResponse: We appreciate the suggestion by the reviewer and have included a visual comparison\nof the hominicin BGC from S. hominis AH5011 and the Mpa BGC from M. paraoxydans DSM\n15019 in Supplementary Fig. 14. The presence of homBC (decarboxylase), homD\n(aminotransferase), and homM (methyltransferase) is shared between the two gene clusters.\nHowever, key differences include the presence of predicted ATP-binding cassette transporters\n(MpaT) in DSM 15019, which are absent in AH5011, and the presence of lanthipeptide\ndehydratase (LanM) and oxidoreductase (HomJ) in AH5011, which are absent in DSM 15019.\n4) Line 172, is the RN4220 strain resistant because it is a hominicin producer? Or does it just\nharbor the gene encoding HomI?\nResponse: We have confirmed that S. aureus strain RN4220 (GenBank Accession CP076105)\ndoes not encode either a biosynthetic gene cluster for hominicin or harbor the homI gene.\nFurthermore, we did not observe antimicrobial activity in either the live bacteria or its conditioned\nmedia (Fig. 4b and 4c), indicating that that it is not a hominicin producer. While RN4220 does not\nharbor homI, it does encode two putative yidC_1 and yidC_2 genes (Supplementary Fig. 15),\nwhich share structural similarity with HomI and may contribute to the observed resistance\nphenotype.\n5) Line 179, can the authors provide the peptide yield after purification to homogeneity?\nResponse: We have now included the peptide yield in the manuscript. Specifically, we isolated\n~6.4 mg of purified peptide from approximately 802 mg of crude chloroform extract, corresponding\nto a yield of approximately 0.8%. This information has been added to the main text.\n6) Line 183: the reference to Figure S5 is incorrect, this is a standard curve.\nResponse: We thank the reviewer’s comment. We have made changes to the main text, figure,\nand legend to reflect that the graph is a standard curve.\n7) Line 203: too many significant figures on the report zone sizes.\nResponse: The reported diameters of the zones have been adjusted to reflect the appropriate\nsignificant numbers.\n8) Line 207-217: this section was hard for me to follow because it is difficult to keep track of what\nall of the genes do. This may be improved when the Figure 4 is put near this text, but I might still\nsuggest that the authors explain briefly what each variant is omitting.\nResponse: We thank the reviewer for their suggestion and have added a brief statement clarifying\nthe omitted components of each variant in this section.\n9) Line 222: ref 12 did show that the original daptides had hemolysis activity. I agree that this isn’t\nthe same as forming pores, but the hemolysis activity should probably be mentioned here.\nResponse: We thank the reviewer for their suggestion and have added a brief statement about\nthe hemolytic activity of Mpa daptides from M. paraoxydans to the main text.\n10) Line 283: this section discusses the presence of homI in the Staph BGCs, but what about the\nset of ~500 daptides predicted in ref 12? Is the presence of homI more general or is it Staph-\nspecific?\nResponse: Previous work by Ren et al. (DOI: 10.1007/s12223-013-0259-1) reported that the\nglobal co-occurrence percentage of HomI and YidC (PF02096, TIGR03592) with daptide gene\nclusters is 0.8%, which corresponded to 4 out of 483 gene clusters. All four entries originated from\ngenomes of Staphylococcus pseudintermedius. This information has been added to the main text\n(Line 326).\n11) Line 286: when I see the phrase “homI mutant” I think of a deletion mutant, but this is an\noverexpression mutant. I suggest the authors make this clear and mention whether the homI\nexpression plasmid is multicopy.\nResponse: We agree with the reviewer and have rephrased the sentence to read, “we cloned\nhomI on a multicopy plasmid and observed no growth defects between the wild-type and homI-\nexpressing mutant in TSB.”\n12) Line 326: express should be expresses.\nResponse: We thank the reviewer’s comment and fixed the typo.\n13) Line 328: I find the statement that “infection with S. aureus was augmented by the topical\naddition…” confusing. The word augment could suggest that hominicin was helping colonization\nof S. aureus.\nResponse: We agree with the comment from the reviewer and rephased the sentence to read,\n“infection with S. aureus was attenuated by the topical addition…,” to emphasize that the addition\nof hominicin reduced S. aureus infection on mouse skin.\n14) Line 339: I think the authors mean understudied.\nResponse: We thank the reviewer’s comment and fixed the typo.\n15) Figure 1: there is an extra letter D floating in panel F.\nResponse: We thank the reviewer for the comment and removed the misplaced label.\n16) Figure 1B caption: Table S1 in the SI does not seem to include information about all of the\nstrains used for susceptibility testing. This information is important to include if others want to\nfollow up on the work.\nResponse: We appreciate the reviewer for their comment. The strains tested for susceptibility in\nthe conditioned media of AH5011, as represented in Fig. 1b, are listed in Supplementary Table 6."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done an excellent job of responding to the critiques raised in the prior review and there are no additional\nconcerns.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "Dear corresponding author,\nI appreciate the additional experimental work and thank the detailed responses to all my questions", "reviewer_4": "The authors have satisfactorily addressed my concerns"}, "author_response": ""}]

Decision letter