Skin androgens regulate Staphylococcus aureus pathogenicity via quorum sensing

John, Maria Sindhura; Chinnappan, Mahendran; Sturges, Camille I.; Artami, Methinee; Bhattacharya, Mohini; Keogh, Rebecca A.; Kavanaugh, Jeffrey S.; Jain, Shivani; Gedamu, Hanna; Komarovsky, Jessica; Velasquez, Mauricio; Sharma, Tripti; McDonald, Jeffrey G.; Horswill, Alexander R.; Harris-Tryon, Tamia A.

doi:10.1038/s41564-026-02261-2

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Published: 27 February 2026

Skin androgens regulate Staphylococcus aureus pathogenicity via quorum sensing

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Abstract

Skin cells secrete testosterone, with greater amounts secreted at the skin surface of males compared with females. Males are also more susceptible to skin infections than females. Here we report that mice engineered with testosterone-deficient skin are resistant to methicillin-resistant Staphylococcus aureus infections. Testosterone promoted the expression of S. aureus cytotoxic virulence factors by activating the accessory gene regulator (agr) quorum-sensing pathway in a concentration-dependent manner and independent of quorum-sensing-activating auto-inducing peptides. Mutational analysis revealed that a functional histidine kinase AgrC in S. aureus was required for testosterone to exert its effect, with in silico evidence indicating a direct interaction between testosterone and AgrC. An isomer of testosterone, enantiomer-testosterone, that blocked bacterial quorum sensing, inhibited S. aureus-induced cytotoxicity of human cells. These findings advance our understanding of how the skin regulates bacterial virulence and reveals a potential therapeutic strategy for the management of infections.

Main

Males are more susceptible to skin and soft tissue infections than females, yet the mechanisms driving these differences are unclear^1,2,3. One established distinction between males and females is androgen production, including testosterone and dihydrotestosterone (DHT), generated by the testes and the ovaries⁴. The skin also generates and secretes nanomolar concentrations of hormones, including testosterone, with our previous liquid chromatography–tandem mass spectrometry (LC–MS/MS) studies showing site-specific differences in the abundance and repertoire of hormones at the skin surface^5,6. Skin-colonizing bacteria are exposed to these hormones, yet their impact on bacterial pathogenesis remains unclear.

The majority of skin infections are caused by the opportunistic pathogen Staphylococcus aureus⁷. While Staphylococcus epidermidis and Staphylococcus hominis are established resident skin commensals, S. aureus is typically restricted to the anterior nares of approximately a third of healthy individuals, with skin colonization and infection occurring infrequently^8,9. To colonize the skin, S. aureus requires virulence factors that are dampened by coordinated regulatory networks that tune their expression depending on sensed environmental conditions^10,11. The most characterized regulatory system in S. aureus is the accessory gene regulator (agr) quorum-sensing system¹¹ that controls the expression of toxins and adhesins required for skin colonization and dermal invasion^12,13. Staphylococcus auto-activates agr signalling through the production of auto-inducing peptide (AIP) signals¹¹. Strains that lack functional agr signalling or agr-regulated factors, such as the phenol soluble modulin (PSM) peptides, show impaired skin infection^12,13. Host and bacterially derived small molecules that inhibit agr have been described^14,15,16. However, the skin cues that promote S. aureus pathogenesis at the skin surface remain incompletely understood. Increased understanding is critical to address the rising antibiotic resistance and lethality of methicillin-resistant S. aureus (MRSA)^17,18.

Here we investigated whether testosterone promotes S. aureus infection and found that mice engineered to secrete reduced levels of testosterone and DHT were resistant to MRSA infection. Further, testosterone and DHT activate MRSA and methicillin-sensitive S. aureus strains through stimulation of the agr quorum-sensing system. Using isogenic agr mutants and bioluminescent reporters, we found that testosterone is sufficient to activate quorum sensing, independent of the bacterially derived AIP. In addition, activation requires both the agr membrane histidine kinase receptor AgrC and the transcription factor AgrA. In silico modelling identified a testosterone binding site on AgrC distinct from the AIP binding site¹⁹. We further identified a stereoisomer of testosterone, enantiomer-testosterone (ent-T), as an inhibitor of agr signalling and S. aureus-mediated cytotoxicity. Together, these findings establish androgens as essential host signals for S. aureus skin infection and identify ent-T as a therapeutic candidate for the treatment of skin infections.

Results

Androgen secretion at the skin surface is required for S. aureus skin infection

Higher serum androgen production is associated with S. aureus colonization and infection^3,20. However, there is limited understanding of androgen dynamics at the skin surface. Using LC–MS/MS, we quantified testosterone secretion from human skin through daily sebum sampling over 6 days in age-matched male and female participants without skin disease. Human skin consistently secreted between 5 nM and 10 nM testosterone with stable levels across days (Fig. 1a). In keeping with our previous study^5,6 of a larger cohort, the male participant secreted greater amounts of testosterone compared with the female subject (Fig. 1a,b). These findings demonstrate stable, sex-dependent secretion of nanomolar amounts of testosterone at the skin surface^5,6.

**Fig. 1: *Hsd3b6*^∆skin mice with reduced androgen production in skin are resistant to *S. aureus* skin infection.**

In addition to increased S. aureus in men versus women, S. aureus induces greater necrosis in male murine skin infections compared with female mice^2,3. To confirm these findings, we epicutaneously infected male and female age-matched C57BL/6 mice with a bioluminescent strain of MRSA SAP430 (MRSA::lux). The MRSA::lux strain generates luminescence in direct proportion to the number of colony forming units (CFUs) present at the skin surface²¹. We observed increased infection severity in male mice compared with female mice (Fig. 1c and Extended Data Fig. 1). Male mice showed greater MRSA-induced bioluminescence imaging (BLI) and worse disease scores compared with female mice (Fig. 1c and Extended Data Fig. 1a,b). They also showed greater skin barrier damage (Extended Data Fig. 1c). In keeping with our human studies (Fig. 1a)⁵, male mice secreted higher levels of the androgens testosterone and DHT at the skin surface compared with female mice (Extended Data Fig. 1d,e), again linking higher androgen secretion with increased infection severity.

To directly test the role of skin-derived androgens, we generated mice lacking epithelial expression of the steroidogenic enzyme 3β-hydroxysteroid dehydrogenase 6 (HSD3B6) (Fig. 1d)^22,23,24, a mouse orthologue of human HSD3B1 (Extended Data Fig. 2a–d)²⁴. Hsd3b6^∆skin (K14-Cre^+/−; Hsd3b6^fl/fl) mice had the same amounts of serum hormones and weights compared with controls (Extended Data Fig. 2e,h). Hsd3b6^∆skin mice also showed no differences in skin barrier function or histology compared with controls (Extended Data Fig. 2f,g). However, Hsd3b6^∆skin mice secreted lower levels of testosterone, progesterone and DHT at the skin surface compared with Hsd3b6^fl/fl mice (Fig. 1e,f and Extended Data Fig. 2i).

Next, we assessed the susceptibility of Hsd3b6^∆skin mice to skin infection. Across 4 days of epicutaneous infection with MRSA::lux, the Hsd3b6^∆skin mice showed reduced bioluminescence, lower diseases scores, less skin barrier damage and reduced epidermal thickness, suggesting that Hsd3b6^∆skin mice are more resistant to skin infection compared with controls (Fig. 1g–i and Extended Data Fig. 3a). Complementing these findings in male mice, bacterially induced bioluminescence of female Hsd3b6^∆skin mice infected with MRSA::lux were augmented by topical testosterone (Fig. 1j and Extended Data Fig. 3b,c). Topical testosterone also increased disease scoring, skin barrier disruption and epidermal thickness (Fig. 1k,l and Extended Data Fig. 3b). Reduction of skin-secreted hormones also minimized sex-dependent differences in infection between male and female mice (Fig. 1g,j, open circles and open squares). Thus, reduced secretion of testosterone, DHT and progesterone protected the skin from infection, and treatment with testosterone was sufficient to promote infection.

Testosterone and DHT activate agr quorum sensing and promote S . aureus pathogenesis

Next, to determine the direct impact of testosterone on the bacterial transcriptome, we sequenced RNA from a methicillin-sensitive strain of S. aureus (HG003 strain) treated with 10 nM of testosterone compared with vehicle treated control. Interestingly, testosterone had a narrow impact on the transcriptome of S. aureus, with increases in the expression of agrB, agrD, agrC, agrA, psmα, psmβ and RNAIII (Fig. 2a and Extended Data Fig. 4a–c), all of which were in the agr quorum-sensing pathway (Fig. 2b)^11,25. Transcripts for the haemolysins hlgB and hlgC, which lyse human red blood cells (RBCs), were also more abundant in S. aureus treated with 10 nM of testosterone compared with controls (Extended Data Fig. 4a). Conversely, genes whose expression is repressed upon agr activation, including coa, rot and spa, were less abundant in testosterone-treated S. aureus (Extended Data Fig. 4a). In contrast, pregnenolone, a hormone with a similar carbon count to testosterone, had no discernible impact on the HG003 transcriptome (Extended Data Fig. 4d). Notably, treatment with testosterone or DHT did not alter S. aureus HG003 growth in culture (Extended Data Fig. 5a). Taken together, these findings suggested that testosterone stimulated agr quorum sensing in the HG003 strain of S. aureus.

**Fig. 2: Testosterone and DHT stimulate *agr* quorum sensing and drive *S. aureus* pathogenesis.**

Activation of agr quorum sensing in S. aureus occurs through the stimulation of the P2, P3 and PSM promoters that regulate the production of AIP-I synthesis and the PSM peptides (Fig. 2b)¹¹. Therefore, to test the influence of sex steroids on agr signalling, we created P3 promoter fusions of a S. aureus strain (HG003agr-P3::lux) that generate bioluminescence in proportion to the activation of quorum sensing²⁶. Both testosterone and DHT activated the agr-P3 promoter, with similar kinetics to the established ligand AIP-I (Fig. 2c). However, oestradiol and progesterone had no impact on agr activation (Extended Data Fig. 5b). To confirm the effect of testosterone on S. aureus, we quantified the transcription of the read-outs of agr, psmα, agrA and RNAIII, and demonstrated that testosterone stimulates the expression of these transcripts (Fig. 2d and Extended Data Fig. 5c). Thus, testosterone and DHT activate the transcription of the agr regulon in S. aureus, but oestradiol, pregnenolone and progesterone do not. These findings suggested that the lower infectivity of MRSA in the Hsd3b6^∆skin mice compared with controls (Fig. 1g–l) was due to loss of testosterone and DHT at the skin surface, rather than reductions of progesterone (Extended Data Fig. 2i).

Every staphylococcal isolate contains a single copy of the agr system, and each species of S. aureus produces different types of AIP signal through variation in the agrBDCA operon¹¹. There are four types of AIP signal made by S. aureus and the HG003 strain falls into the agr type-I class. To test the generality of the effects of testosterone across agr types, we treated different agr types with testosterone, including USA100 (MRSA type II) and MW2 (MRSA type III)¹¹. All strains showed increased expression of psmα and RNAIII in response to 10 nM of testosterone (Fig. 2e,f and Extended Data Fig. 5d–i). Testosterone also activated the agr-P3 promoter in MRSA type-II and MRSA type-III luminescent strains (Extended Data Fig. 5e,h), indicating that testosterone stimulates virulence and quorum sensing across S. aureus strains. Again, treatment with testosterone and DHT had no impact on the growth curves of these strains in culture. More than 90% of S. aureus strains are type I–III²⁷, suggesting that testosterone stimulates virulence across S. aureus strains with active agr systems. Given the association between S. aureus and the skin disease atopic dermatitis (AD)^28,29, we also tested an array of strains obtained from diseased skin³⁰. Indeed, testosterone treatment increased the transcription of psmα, RNAIII and the cytoplasmic regulator agrA in bacterial strains obtained from AD skin (Fig. 2g and Extended Data Figs. 6a,b). Notably, the AD04.E17 strain of S. aureus showed a more robust response to testosterone than the other AD-associated strains tested, and the response was of a similar magnitude to AIP-II stimulation (Fig. 2d–f and Extended Data Fig. 6c). These findings suggest that testosterone stimulation correlates closely with AIP stimulation at the same dose and may reflect the baseline agr activity of the strain. In addition, testosterone stimulated the transcription of cytotoxic virulence factors stimulated by AIPs, including lukS-PV, hla and hld (Extended Data Fig. 6d–f), and increased bacterially induced cytotoxicity of human cells, including skin cell death, RBC haemolysis and neutrophil killing (Fig. 2h,i and Extended Data Fig. 6g). These effects were comparable to those of AIP-I (Fig. 2h,i).

Finally, we compared quorum-sensing activity in vivo using a bioluminescent quorum-sensing reporter of the hypervirulent community-associated USA300 MRSA (CA-MRSA-agr-P3::lux). Consistent with our previous in vivo data using the constitutive lux reporter (Fig. 1g–l), bacterial quorum signalling was lower in Hsd3b6^∆skin mice infected with the quorum-sensing reporter strain compared with Hsd3b6^fl/fl controls (Fig. 2j and Extended Data Fig. 6h). agr activation in vivo was also greater in male mice compared with female mice (Fig. 2j). Taken together, these data show that androgens stimulate the S. aureus quorum-sensing system in vitro and in vivo and have the capacity to increase bacterial cytotoxicity.

Testosterone stimulates S. aureus pathogenesis independently of the bacterial AIP

The established activator of agr type-I quorum sensing in S. aureus, AIP-I, is generated by the transcription of a propeptide AgrD, which is then processed by the endopeptidases AgrB and MroQ (Fig. 2b)²⁵. Thus, the biosynthesis mutant ∆agrBD strain of S. aureus (HG003), which cannot synthesize AIP-I, is unable to auto-stimulate the quorum-sensing system¹⁶. We hypothesized that testosterone would require AIP-I to activate quorum sensing. However, in the biosynthesis mutant strain of methicillin-sensitive S. aureus (HG003-∆agrBD), testosterone retained the ability to activate quorum-sensing phenotypes, including increased damage to skin cells, RBCs and neutrophils (Fig. 3a,b and Extended Data Fig. 7e). These effects were dose dependent, with greater concentrations of testosterone increasing the transcription of RNAIII, agrA, agrC and psmα (Extended Data Fig. 7a). In addition, we generated a luminescent reporter (∆agrBD::lux) and tested the impact of progesterone and oestradiol on S. aureus. As in the wild-type reporter HG003 agr-P3 strain (Extended Data Fig. 5b), neither progesterone nor oestradiol stimulated luminescence in ∆agrBD::lux (Extended Data Fig. 7c), and neither DHT nor testosterone influenced bacterial growth curves in the ∆agrBD strain (Extended Data Fig. 7b). However, DHT and testosterone were both able to stimulate bioluminescence in the absence of AIP-I (Extended Data Fig. 7c). As AIP and testosterone could act independently, we next tested the impact of both AIP and testosterone on agr signalling. Indeed, treatment of S. aureus with testosterone had the capacity to augment AIP-I signalling in a dose-dependent manner (Fig. 3d and Extended Data Fig. 7d), establishing that testosterone may synergize with AIP signals to regulate pathogenesis.

**Fig. 3: Testosterone stimulates *S. aureus* quorum sensing independently of the AIPs.**

Although AIP-I auto-stimulates quorum sensing in type I S. aureus strains, AIP-II and AIP-III, generated from type-II and type-III S. aureus strains respectively, inhibit quorum sensing in type-I strains (Extended Data Fig. 7f). Indeed, probiotics of strains that inhibit quorum sensing by generating inhibitory peptides are in development as S. aureus therapeutics^11,31. To understand how skin-secreted testosterone might impact these inhibitory signals from other bacteria, we tested how testosterone competes with inhibitory signals derived from strains that generate non-cognate AIPs. Interestingly, when we exposed a type-I S. aureus strain to equal low nanomolar concentrations of AIP-II and testosterone, quorum sensing was inhibited, demonstrating that testosterone was unable to overcome inhibitory signals at the same concentration (Fig. 3e and Extended Data Fig. 7g). However, at higher concentrations, testosterone stimulated quorum sensing and overcame the inhibitory AIP-II signal (Fig. 3e and Extended Data Fig. 7g). Similar dynamics were observed with AIP-III (Fig. 3f and Extended Data Fig. 7h). These findings suggest that skin testosterone participates in the established crosstalk between competing microorganisms at the skin surface and can overcome inhibitory signals from other bacteria in a dose-dependent manner.

Lastly, we used the mutant ∆agrBD::lux strain to examine how loss of the AIP-I signal would impact bacterial infections in vivo. Interestingly, the mutant ∆agrBD::lux strain was able to infect the Hsd3b6^fl/fl mice with similar kinetics to the MRSA::lux strain (black dots in Figs. 1g and 3g). In contrast, the Hsd3b6^∆skin mice, with reduced androgen production on skin, showed reduced luminescence (Fig. 3g,h). The addition of testosterone or AIP-I on day 0 was able to partially rescue the intensity of infection in the Hsd3b6^∆skin mice at day 1 post-inoculation (Fig. 3g). These findings show that skin-derived androgens are sufficient to facilitate S. aureus skin infection through activation of agr quorum sensing, even in the absence of AIPs.

Testosterone stimulation of S. aureus requires bacterial expression of the histidine kinase AgrC

AIPs stimulate the agr system through binding and activation of the AgrC histidine kinase^11,19. Once activated, AgrC phosphorylates the response regulator AgrA that auto-induces transcription of the agr machinery (Fig. 2b)^11,32. Given the specific impact of testosterone on the agr (Fig. 2a and Extended Data Fig. 4) and its ability to cooperate or compete with established ligands of AgrC (Fig. 3)¹⁹, we tested if testosterone would require a complete AgrCA two-component system to regulate S. aureus virulence. We generated a constitutive bioluminescent reporter deficient in AgrC (∆agrC::lux)¹⁶. In contrast to the biosynthetic ∆agrBD::lux mutant, the ∆agrC::lux mutant did not respond to testosterone in vitro (Fig. 4a). S. aureus required both AgrC and AgrA to respond to testosterone (Fig. 4b,c and Extended Data Fig. 7j–n). In ΔagrC mutants, testosterone failed to increase psmα or RNAIII expression and did not enhance haemolytic activity, neutrophil killing or skin cell cytotoxicity. Complementation of the ΔagrC mutant with AgrC in trans restored responsiveness to testosterone and AIP. In vivo, the loss of AgrC also decreased skin infections compared with infection with intact AgrC (Fig. 4d). Similar to HG003 and ∆agrBD, testosterone treatment did not affect ∆agrC bacterial growth (Extended Data Fig. 7i). As expected, neither exogenous testosterone nor AIP-I were able to restore infection phenotypes in the agrCA-deficient strains (Fig. 4b,c,e and Extended Data Fig. 7j–m). These results show that S. aureus skin infection is influenced by both the skin secretion of androgens and a functioning AgrCA two-component system.

**Fig. 4: Testosterone stimulation of quorum sensing requires the histidine kinase AgrC.**

Finally, to gain further insight on interactions between AgrC and testosterone, we predicted the structure of the AgrC type-I dimer using AlphaFold 3 (AF3)^33,34 and docked testosterone and AIP-I on the AgrC sensory domain^35,36,37 (Fig. 4f). In silico analysis predicts testosterone binding to a hydrophobic cleft distinct from the established AIP binding site. A binding site distinct from the catalytic site and from the site of AIP-I binding is consistent with our data demonstrating cooperative interactions between AIP-I and testosterone in type-I strains (Fig. 3d–f).

The enantiomer of testosterone inhibits MRSA pathogenesis

To further investigate the specificity of testosterone’s interaction with the agr system, we tested the impact of a stereoisomer of testosterone, ent-T, on S. aureus pathogenesis. Enantiomer steroids have the same physiochemistry and impact on biological membranes, but can have distinct effects on receptors and signalling^38,39. Similarly to other synthetic enantiomers, ent-T has a mirror image orientation at all six chiral centres of testosterone that leads to altered rotation of polarized light (Fig. 5a)^39,40. In contrast to the other hormones tested with neutral effects on quorum sensing (Fig. 2c and Extended Data Fig. 5b), ent-T acted as an inhibitor of agr (Fig. 5 and Extended Data Fig. 8). ent-T decreased S. aureus-dependent RBC haemolysis and neutrophil killing (Fig. 5b,c). Further, ent-T decreased the expression of quorum-sensing-dependent virulence factors in MRSA type-II, MRSA type-III and AD strains of S. aureus (Fig. 5d–f, and Extended Data Fig. 8a). At higher concentrations, ent-T could also outcompete testosterone and turn off quorum sensing in vitro (Extended Data Fig. 8b). In keeping with their shared hydrophobicity properties, in silico analysis identified a high-affinity binding solution for ent-T bordered by residues T141, T197 and S201 of AgrC. Testosterone is predicted to bind to the same location; however, the orientation of its hydroxyl group is predicted to form hydrogen bonds with T190 and S194, which are not predicted for the chiral orientation of the ent-T hydroxyl groups (Extended Data Fig. 8c). Lastly, we tested how ent-T would impact S. aureus agr quorum sensing in vivo using the CA-MRSA-agr-P3::lux. Topical application of ent-T to mice skin infected with the CA-MRSA-agr-P3::lux reporter lowered quorum sensing in vivo in both male and female mice (Fig. 5g and Extended Data Fig. 8d–f). These data demonstrate that ent-T inhibits S. aureus cytotoxicity of human skin cells, RBCs and neutrophils by antagonizing agr quorum-sensing signalling⁴¹.

**Fig. 5: *ent-*T inhibits *S. aureus* quorum sensing and cell damage.**

Discussion

Here we have identified that the androgens testosterone and DHT regulate the pathogenesis of S. aureus, including lethal antibiotic-resistant MRSA strains (Fig. 1). Testosterone regulates S. aureus specifically through stimulation of a bacterial communication system called quorum sensing. Moreover, androgens can signal this system independently of the well-characterized, bacterially produced agonist of quorum sensing, AIP (Figs. 2 and 3). Further, in the absence of skin androgens, bacterial infection is diminished and sex-dependent differences in infection are markedly reduced (Figs. 1g,j). Our data also show that testosterone signalling of quorum sensing requires the expression of the S. aureus histidine kinase receptor AgrC and the response regulator AgrA (Fig. 4 and Extended Data Fig. 7a–d). In silico analysis supports testosterone binding directly to the transmembrane protein AgrC at a hydrophobic pocket formed within the transmembrane portion of α helices 4 and 8, areas that have been shown to shift during autophosphorylation of the AgrC protein¹⁹. While an earlier version of our study was publicly available as a preprint, a related study⁴² showed the impact of testosterone on S. aureus. Our findings, using mice with epithelial-specific reductions of testosterone on the skin surface, bacterial transcriptomics and bioluminescent quorum-sensing models, add depth and context for all previous observations of sex-specific differences in S. aureus skin infection. These data provide definitive support for the direct sensing of testosterone by S. aureus and the function of this interaction in how S. aureus colonizes skin and causes skin damage. Although testosterone and DHT increase the virulence of S. aureus, other hormones such as progesterone, oestradiol and pregnenolone have no effect. Testosterone and DHT both have carbonyl and hydroxyl groups that extend beyond the steroid rings (Fig. 1d). The hormones that lack both moieties and have larger side chains are unable to stimulate quorum sensing, potentially explaining the differences in activity between hormone classes. These findings define testosterone and DHT as cues sensed by bacteria at the skin surface that regulate the expression of key virulence factors required for skin invasion and infection^12,13.

In contrast to progesterone and oestradiol that had a neutral effect on S. aureus, ent-T, a stereoisomer of testosterone, blocked the production of psmα and bacterially induced RBC and neutrophil killing. The topical application of ent-T also inhibited agr quorum sensing in vivo. Therefore, S. aureus responds to testosterone in an enantioselective manner, with the natural testosterone generating activating signals and its chiral counterpart, ent-T, leading to inhibition of the same phenotypes. Notably, all sex steroids are found naturally in a single form and ent-steroids are chemically synthesized. The enantioselectivity of steroids has been reported for several other sex steroids in their interactions with G-protein-coupled receptors (GPCRs) and GABA (γ-aminobutyric acid) A receptors (GABA_ARs) in the brain. At GABA_ARs and GPCRs, natural sex steroids and their enantiomers bind to the same site of the protein, but demonstrate distinct effects on receptor signalling, which have been leveraged as therapeutics^39,40,43,44. Our findings provide an example of this phenomenon in bacteria and the potential of ent-steroids for therapeutics.

The skin is known to generate antimicrobial proteins, antimicrobial lipids and nitric oxide that inhibit S. aureus pathogenesis^16,45,46. Our work describes how the skin potentiates the infectious phenotypes of a pathogen at the skin surface through the secretion of androgens. Indeed, although the biosynthetic mutant had delayed kinetics in vitro (Fig. 3a), it retained the ability to sustain in vivo skin infections in mice with normal hormone production (Fig. 3g). Thus, the host-derived hormone signal is sufficient to activate S. aureus in vivo. In the in vivo biosynthetic mutant experiments, both exogenous testosterone and AIP-I only partially rescue luminescence (Fig. 3g). The rescue was probably not sustained due to the established instability of both AIP-I and testosterone in biological systems. AIPs contain a thiolactone ring and peptide backbone that are sensitive to oxidation and protease degradation, respectively^47,48. Similarly, testosterone is actively metabolized in peripheral tissues such as the skin by enzymes including 17β-HSD and aromatase^49,50. As a result, exogenous testosterone and AIP may not persist at biologically active levels in vivo. In contrast, endogenous production of AIP and testosterone, by S. aureus and the skin, respectively, allows continuous and localized accumulation of the signal, maintaining sustained agr activation over time (Figs. 1j and 3g). Taken together with our data showing that testosterone can directly stimulate agr (Fig. 2a) and augment AIP-I signalling (Fig. 3d and Extended Data Fig. 7d), the in vivo finding suggests that testosterone may be an initiating signal for S. aureus skin infection that is then sustained by AIPs generated by bacterial growth. These findings complement other examples of host hormones and cholesterol-derived molecules that supply activating interkingdom signals to microorganisms with which they have co-evolved, such as the ability of specific Gram-negative bacteria to respond to bile acids and adrenaline^51,52. Our findings identify the ability of mammalian hormones to curate the virulence phenotypes of an opportunistic Gram-positive pathogen at the skin surface.

Given the capacity of skin androgens to cue the microbiota, our findings define the need for greater characterization of skin-secreted small molecules. These findings would also support the hypothesis that interventions that lower androgen amounts at the skin surface would improve the course of S. aureus infections, as we observed in the androgen-deficient mice (Fig. 1). We have previously quantified the secretion of skin sex steroids and illuminated topographic differences across the skin surface⁵. Previous work also shows that skin testosterone amounts shift during skin inflammation^53,54 and that IL-4 receptor signalling directly regulates androgen production through STAT6 binding to the HSD3B1 promoter²⁴. Specific strains of S. aureus may also influence androgen amounts through sex steroid metabolism^55,56 and stimulation of IL-4 receptor signalling³⁰. Therefore, therapeutic approaches that aim to regulate S. aureus virulence through inhibition of quorum sensing may need to take into account the concentration-dependent stimulation of quorum sensing by skin androgens^31,57. Further, these findings suggest that the observed pathogenicity of S. aureus at the skin surface is an aggregate read-out of environmental signals generated from the host and signals generated by the microbiota⁵⁸.

Methods

Reagent and resource sharing

Reagents created for this study, including the Hsd3b6^Δskin mice and bioluminescent strains of S. aureus, are available upon request from the corresponding author and may require a completed materials transfer agreement if there is potential for commercial application. Requests for resources should be directed to and will be fulfilled by the corresponding author.

Experimental model and participant details

Participant recruitment

This study was approved by the University of Texas (UT) Southwestern Institutional Review Board (STU 2019-0145), and written informed consent was obtained from study participants. Recruitment occurred from January 2024 to June 2024, and the human samples were collected between June 2024 and August 2024. Inclusion criteria included participants aged 18‒40 years, a body mass index between 20 kg m⁻² and 35 kg m⁻², and willingness and ability to comply with the requirements of the protocol. Exclusion criteria included participants outside the ages of 18‒30 years, the use of antibiotics in the last 6 months, the use of topical medications, chronic skin disorders or other chronic medical conditions, the use of lipid or hormone-altering medications (except for oral contraceptives), the use of immunomodulator medications, women with irregular menstrual periods, or participants with a history of surgery to endocrine organs. Basic demographic and medical information was collected from each participant. Two healthy control participants, a 29-year-old male and a 26-year-old female, were selected and informed consent was obtained. Study participants were compensated in accordance with the approved study protocol.

Human skin sampling

Skin excretions were collected via Sebutape after completion of skin preparation to decrease external variables⁵. At the time of sampling, the forehead was cleaned with alcohol and four Sebutapes from Clinical and Derm LLC were applied for 15 minutes. Participants were sampled daily for 6 days between the hours of 12 pm and 3 pm. Following collection, tapes were stored in glass vials at −20 °C until processing. Hormones were extracted into 3 ml of LC–MS-grade methanol, and quantified via LC–MS/MS using a SCIEX QTRAP 6500+ (ref. ⁵). Outliers within the four biological replicate samples were identified using a dual-sided Grubbs’ test, also called the extreme studentized deviate method, and were removed. The remaining biological replicate values for each day were averaged. These averaged values were translated from picogram per tape to nanomoles per litre (nM) using the total volume of the tape as provided in US patent US4532937A. Using GraphPad Prism v.10.4.1 (GraphPad Software), a linear regression model was created and plotted for each participant, along with the 95% confidence interval of each regression. An ordinary two-way analysis of variance (ANOVA) was performed to determine statistical significance of time and sex.

Human peripheral blood

Human peripheral blood samples were obtained from fully consented and institutional-review-board-approved donors through BioIVT, Innovative Research and IQ Biosciences.

Mice

Conventionally raised C57BL/6 male and female mice aged 6–9 weeks old were purchased from the Jackson Laboratory. C57BL/6 wild-type, K14-Cre^+/− and Hsd3b6^fl/fl (Extended Data Fig. 2a) mice were bred and maintained in the specific pathogen-free barrier facility at the UT Southwestern Medical Center at Dallas⁵⁹. The generation of Hsd3b6^Δskin (K14-Cre^+/−; Hsd3b6^fl/fl) is described below. Mice were co-housed with three to five mice per cage in all experiments. All mice were housed under a 12-hour light:12-hour dark cycle, at an ambient temperature of 22 ± 2 °C and relative humidity of 40–60%. Mice were fed ad libitum with free access to drinking water according to protocols approved by the Institutional Animal Care and Use Committees of the UT Southwestern Medical Center. All experiments involving live animals were approved by the Institutional Animal Care and Use Committees of the UT Southwestern Medical Center (protocol number 2015-101064).

Hsd3b6^fl/fl (C57BL/6), with loxP sites surrounding the first coding exon of Hsd3b6, were generated using CRISPR–Cas9 genome editing with guide RNAs targeting regions of the Hsd3b6 locus (Extended Data Fig. 2a). Guide RNAs were injected into fertilized C57BL/6J embryos by the Children’s Research Institute Mouse Genome Engineering facility at UT Southwestern. Healthy blastocytes were implanted in pseudo-pregnant mice. The resulting litter was screened by genomic sequencing to detect insertion of loxP sites and mice were bred to homozygosity and backcrossed with wild-type C57BL/6 mice. To generate K14-Cre^+/−; Hsd3b6^fl/fl (Hsd3b6^Δskin) mice, Hsd3b6^fl/fl mice were crossed with K14-Cre^+/− mice to generate K14-Cre^+/−; Hsd3b6^fl/+ mice; K14-Cre; Hsd3b6^fl/+ mice were crossed to Hsd3b6^fl/fl mice to obtain experimental mice K14-Cre^+/−; Hsd3b6^fl/fl (Hsd3b6^Δskin) and corresponding controls, Hsd3b6^fl/fl. Hsd3b6^fl/fl and Hsd3b6^Δskin status was determined using PCR primers (Extended Data Table 2) and resolving on a 3% agarose gel.

Method details

Quantification of hormones in mouse samples

Age- (7–8 weeks) and sex-matched Hsd3b6^fl/fl and Hsd3b6^Δskin mice were analysed. Blood samples were obtained from the retro-orbital vein of anaesthetized mice followed by serum isolation with the micro sample tube Serum Gel (catalogue number 41.1378.005; Sarstedt). Skin hormones were quantified from skin secretions. After anaesthesia with isoflurane, hair was removed using depilatory cream and shaving. After 24 hours, Sebutape (Clinical and Derm LLC) was applied to the dorsal surface for 15 minutes (Extended Data Fig. 2e). Steroid extraction was performed as previously described⁵. Sebutape was removed and placed in 3 ml of chromatography–mass spectrometry grade methanol (catalogue number A456-500; Thermo Fisher Scientific) in an 8 ml polytetrafluorethylene/rubber-lined vial (catalogue number 03-343-3E; Thermo Fisher Scientific). The sample was then dried by vacuum centrifuge at 40 °C and stored at −20 °C until analysis. For analysis, samples were reconstituted with 100 μl of appropriate hormone kit assay buffer. Steroid hormone quantification of progesterone, testosterone and DHT was measured by mouse-specific immunoassay (catalogue numbers MBS7606191, MBS266250 and MBS760829; My BioSource) following the manufacturer’s instructions.

Immunofluorescence microscopy

Mouse skin samples were fixed in formalin and embedded in paraffin by the UT Southwestern Histology Core. Samples were deparaffined with xylene followed by rehydration with decreasing concentrations of ethanol. Heat-induced antigen retrieval was attained in 10 mM sodium citrate buffer. Sections were washed briefly and blocked for an hour in blocking/permeabilization buffer (PBS, 5% goat serum and 0.5% Triton X-100). Sections were incubated in blocking/permeabilization buffer overnight with the following antibodies: anti-HSD3B6 (2.5 µg ml⁻¹; orb592071; Biorbyt), anti-cytokeratin-14 (1 µg ml⁻¹; sc-53253; Santa Cruz Biotechnology). After a brief wash in PBST (PBS + 0.2% Tween-20), sections were incubated with corresponding secondary antibodies: donkey anti-rabbit Alexa Fluor 647 (2 µg ml⁻¹; 711-605-152; Jackson Immunoresearch) and donkey anti-mouse Alexa Fluor 594 (2 µg ml⁻¹; A-21203; Thermo Fisher Scientific). The slides were then washed briefly in PBST and mounted with DAPI containing mounting medium (0100-20; SouthernBiotech). Images were processed using a Zeiss 780 confocal microscope.

Bacterial strains and plasmids

S. aureus strains (Extended Data Table 1) were streaked on tryptic soy agar plates and grown overnight at 37 °C. Single colonies were selected and cultured in tryptic soy broth (TSB) at 150 rpm at 37 °C in a shaking incubator overnight, followed by a 1:100 subculture at 37 °C in a shaking incubator to obtain bacteria from the mid-log phase (optical density at 600 nm (OD_600 nm) = 0.6). For reporter strains, all in vitro cultures were performed using TSB in the presence of 10 µg ml⁻¹ of chloramphenicol. Bacteria were pelleted, washed with PBS and resuspended in either TSB for in vitro experiments or PBS for in vivo experiments. HG003, ΔagrC, ΔagrBD and ΔagrA mutant strains were obtained from Dr Ferric Fang, University of Washington School of Medicine¹⁶. S. aureus strains from AD skin were obtained from Dr Julie Segre and Dr Heidi Kong³⁰. All other strains from the collections of the laboratories of A.R.H. and T.A.H.-T.

Construction of lux-expressing strains

The integrated luxCDABEG cassette was transduced into S. aureus strains HG003, ΔagrBD and ΔagrC obtained from the laboratory of Dr. Ferric Fang, University of Washington School of Medicine^16,40 using phage 11 generating strains AH6222 (lux+), AH6224 (lux+) and AH6223 (lux+), respectively.

Construction of complemented ΔagrC strain

The HG003 ΔagrC strain was complemented by introducing the pAgrC1AgrA plasmid⁶⁰ by phage transduction⁶¹ using phage 11.

In vitro luminescence assays

S. aureus strains, HG003, ΔagrBD and ΔagrC with constitutive Lux (φ11::LL29luxCDABEG) and quorum-sensing-dependent lux (pAmiAgrP3lux) plasmids (HG003 (AH6225) agr type I, USA100 (AH430) MRSA type II and MW2 (AH1747) MRSA type III)^14,18 were grown in TSB supplemented with antibiotic selection and subcultured 1:200 into fresh TSB containing steroid hormone. The assay was completed in opaque-sided, 96-well, clear-bottom, tissue-culture-treated plates with a final well volume of 200 μl. Bioluminescent signals (photons per 0.1 second acquisition time) were measured using a BioTek H1 Synergy plate reader. Experiments were completed in triplicate, with the agr-type-specific AIPs AIP-I (catalogue number 4515-v; Peptide Institute), AIP-II (catalogue number 4516-v; Peptide Institute) and AIP-III (catalogue number 4517-v; Peptide Institute) as positive control.

In vitro growth assay

S. aureus strains HG003 (agr type I), ΔagrBD, ΔagrC, USA100 (AH430; MRSA type II) and MW2 (AH1747; MRSA type III) were cultured overnight in TSB at 37 °C with shaking. Overnight cultures were diluted 1:100 in fresh TSB and grown to mid-log phase. Bacterial suspensions were inoculated into 96-well clear-bottom plates containing 10 nM testosterone, 10 nM DHT or vehicle control. OD₆₀₀ was measured over a 24-hour incubation period to assess bacterial growth using a BioTek H1 Synergy plate reader.

Haemolysis assay

Overnight cultures of HG003, ΔagrBD and ΔagrC strains were inoculated 1:200 into 10 ml of TSB containing testosterone, AIP-I or vehicle alone at concentrations of 10 nM. Cells were grown to mid-log phase. The supernatant of 1 ml of culture was filter sterilized using Millex sterile syringe filters with a pore size of 0.22 µm (catalogue number SLGV033RS). Filtered supernatant diluted 1:1 with PBS was added to 25 µl of human blood (catalogue numbers HUMANWBK2-0110649 and HUMANWBK2-0110717; BioIVT; catalogue number IWB1K2E-10ML; Innovative Research) to a 96-well V-bottom plate and incubated with agitation at 37 °C for 1 hour. After spinning at 1,000 rpm for 10 minutes, the supernatant was transferred to a flat-bottom 96-well plate. Absorbance was read at 541 nm (A₅₄₁) for haemoglobin using a BioTek H1 Synergy plate reader. The percentage of haemolysis was calculated using the following formula: (A₅₄₁ of RBC-treated sample − A₅₄₁ of buffer)/(A₅₄₁ of H₂O − A₅₄₁ of buffer); where buffer (PBS) = baseline, H₂O = 100% haemolysis⁷⁰.

Skin cell cytotoxicity assay

Cultures of HG003, ΔagrBD and ΔagrC strains were grown overnight with TSB containing testosterone, AIP-I or vehicle alone at concentrations of 10 nM. Bacteria were pelleted, followed by filter sterilization of the supernatant using Millex sterile syringe filters with a pore size of 0.22 µm (catalogue number SLGV033RS). Human keratinocytes (HaCaT cells; catalogue number T0020001; AddexBio) were used for cytotoxicity assays. These cells are spontaneously transformed keratinocytes derived from histologically normal skin of a male donor (62 years old). Cells were cultured in AddexBio-optimized DMEM supplemented with 10% FBS under standard conditions. HaCaT cells were treated with sterile-filtered bacterial supernatants at 5% by volume for 24 hours. After PBS washing, the resulting supernatants were used to measure lactate dehydrogenase (LDH) release from damaged cells using the LDH Cytotoxicity Detection Kit (catalogue number 2570393; Invitrogen).

Neutrophil killing assay

HG003, ΔagrBD and ΔagrC strains of bacteria were treated with testosterone, AIP-I or vehicle at concentrations of 10 nM and allowed to grow to mid-log phase (OD₆₀₀ = 0.6). Purified human neutrophils (catalogue number IQB-Hu1-Nu10; IQ Biosciences; catalogue numbers HUMANNEUT-0127862 and HUMANNEUT-0127879; BioIVT) were seeded at 1 × 10⁵ cells per well into a 96-well plate in 90 μl of RPMI. Bacterial supernatants (10 μl) were added (final concentration of 10%). After 3 hours incubation at 37 °C, 5% CO₂, the plates were centrifuged at 250g for 10 minutes, and the resulting supernatants were used to measure LDH leakage from damaged cells as the marker of neutrophil lysis with an LDH Cytotoxicity Detection Kit (catalogue number 2570393; Invitrogen). The percentage of neutrophil lysis was calculated using neutrophils incubated with 10% RPMI as 0% lysis control, and neutrophils incubated with 0.2% Triton X-100 were defined as 100% lysis⁷¹.

Quantitative real-time PCR

HG003, USA100 (AH3684), MW2 (AH843), ΔagrBD, ΔagrA, ΔagrC and AD strains were treated with testosterone and/or the respective AIPs at concentrations of 10 nM and allowed to grow to mid-log phase. Cells were pelleted and lysed using lysis matrix B tubes containing 0.1 mm silica spheres (catalogue number 174701; MP Lysing Matrix Tubes) and lysostaphin (catalogue number L7386; Sigma) at room temperature, and RNA was purified using the RNeasy Mini Kit (catalogue number 74104; Qiagen). RNA was quantified by absorbance at 260 nm, and its purity was evaluated by the ratios of absorbance at 260 nm:280 nm. RNA was used as a template to generate cDNA using the High-Capacity Reverse Transcription Kit (catalogue number 01071619; Applied Biosystems). Quantitative real-time PCR was performed by amplifying cDNA using Power SYBR Green Master Mix (catalogue 2749999; Applied Biosystems) and QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Relative expression values were calculated using the comparative Ct (ΔΔCt) method, and transcript abundances were normalized to gyrA transcript abundance for S. aureus and Rplp0 for mouse tissue samples. Primer sequences are shown in Extended Data Table 2.

RNA sequencing

Briefly, cultures of HG003 were grown in TSB with 10 nM or 100 nM of testosterone, 10 nM pregnenolone or DMSO alone in triplicate to an optical density of 0.4 at OD₆₀₀. Cells were collected and treated with RNA Protect Bacteria Reagent (catalogue number 76526; Qiagen). Cells were lysed using lysostaphin (catalogue number L7386; Sigma) and RNA purified using the RNeasy Mini Kit (catalogue number 74104; Qiagen). Sample quality was affirmed via Bioanalyzer (Agilent). Ribosomal RNA was depleted using RiboCop from the Bacterial META Removal Kit (Lexogen). cDNA libraries were generated at the University of Michigan Microbiome core using the CORALL RNA-seq Library Prep Kit (Lexogen). Samples were barcoded, pooled and sequenced in 125 × 125 paired-end reads on an Illumina HiSeq 2000 sequencer. Raw sequencing reads in FASTQ format were aligned and annotated to the S. aureus NCTC8325 reference genome with annotated small RNA⁶² using QiagenCLC Genomics Workbench default settings (v.21.0.5): mismatch cost, 2; insertion and deletion cost, 3; length and similarity fraction, 0.8. Normalization and differential expression calculations of uniquely mapped bacterial transcripts were performed using CLC. All transcripts with a false-discovery-rate-adjusted P < 0.05 were considered significant.

S. aureus epicutaneous skin infections

Before infection studies, mice were acclimatized to the animal biosafety level 2 animal housing facility. Age- (7–8 weeks), strain- and sex-matched C57BL/6 male and female mice, Hsd3b6^fl/fl and Hsd3b6^Δskin were used in the study. A previously described mouse model of epicutaneous S. aureus exposure was followed^21,46. Briefly, the dorsal skin of anaesthetized mice (2% isoflurane) was shaved and depilated (Nair cream). After 24 hours, bioluminescent S. aureus strains were grown to mid-log phase, pelleted and resuspended in PBS to achieve inoculum containing 1 × 10⁶ CFU. A 100 μl volume of PBS containing 1 × 10⁶ CFU with or without 10 nmoles of testosterone, AIP-I or the same volume of vehicle was placed on a sterile gauze pad and attached to the shaved skin with transparent bio-occlusive dressing (catalogue number 1622W; Tegaderm 3M; Henry Schein Medicals), and secured with adhesive bandages (catalogue number 1275033; Band-Aid; Johnson & Johnson, American White Cross) for 4 days. Photons emitted from luminescent bacteria were collected during an auto-exposure using the IVIS Lumina 3 imager machine and living image software (Xenogen) over the course of 1 minute. Bioluminescent image data are presented on a pseudo-colour scale (blue representing least intense and red representing the most intense signal) overlaid onto a greyscale photographic image. Using the image analysis tools in living image software, circular analysis windows (of uniform area) were overlaid onto dorsal regions of the infection area, and the corresponding bioluminescence values (total flux) were measured and plotted versus time after infection. Mice were randomly assigned to treatment groups, and at experimental endpoints, mice were euthanized using carbon dioxide inhalation.

Disease scoring

The severity of skin inflammation was assessed by a blinded observer from digital photographs and the total disease score was quantified²¹. The sum of the individual grades for erythema, oedema, erosion and scaling were each graded as 0 (none), 1 (mild), 2 (moderate) or 3 (severe).

Transepidermal water loss measurement

Transepidermal water loss, a measure of barrier function and integrity, of mice dorsal skin was measured using Vapometer (Delfin Technologies) according to manufacturer instructions⁶³.

Histology

Skin biopsy specimens were collected, fixed in 10% formalin and paraffin embedded. Skin cross-sections of 4 μm were mounted onto glass slides and stained with haematoxylin and eosin by the UT Southwestern Histology Core, according to guidelines for clinical samples. Epidermal thickness was measured by taking ten epidermal thickness measurements per mouse, averaged from images (Echo Revolve Microscopy) using ImageJ software.

Measurement of quorum sensing in vivo

S. aureus strains expressing quorum-sensing lux (pAmiAgrP3lux) plasmids were grown in TSB medium containing chloramphenicol overnight at 37 °C in a shaking incubator set to 150 rpm. Overnight cultures were diluted 1:100 TSB with chloramphenicol to mid-log phase and then pelleted and washed 2× in PBS and resuspended in sterile saline. PBS inoculum suspensions (100 μl) containing 1 × 10⁶ CFU were placed on a sterile gauze pad (1 cm × 1 cm) and attached to the shaved skin with transparent bio-occlusive dressing, with or without testosterone, ent-T, AIP-I or vehicle (3M; Tegaderm) and secured with 2 layers of adhesive bandages (Band-Aid; Johnson & Johnson). Beginning immediately after infection, mice were imaged hourly under isoflurane inhalation anaesthesia (2%). Photons emitted from luminescent bacteria were collected during auto-exposure using the IVIS Lumina 3 imager machine and living image software (Xenogen). Corresponding bioluminescence values (total flux) were measured and plotted versus time after infection⁴⁰.

In silico docking of testosterone to the AgrC receptor

The dimeric structure of AgrC was predicted using AlphaFold 2 (refs. ^33,34) as implemented in Google Colab. The sensory domain of a single subunit (residues 1–207) was selected for ligand docking. AIP-I was docked in silico using SwissDock^35,37 with nuclear-magnetic-resonance-derived coordinates for AIP-I. The docking pose for AIP-I that best aligned with reported structure–activity relationships^11,41 was selected as the AgrC–AIP-I complex for subsequent steroid docking. Stereospecific compound templates were retrieved from PubChem (testosterone; compound identifier 6013). Initial docking produced an AgrC model featuring a depression on the sensory domain surface, consistent with a putative membrane-exposed ligand-binding pocket. To refine the complex, co-folding of AgrC in the presence of both AIP-I and testosterone was performed using AF3⁶⁴, implemented in PXDesign^65,66 on the Protenix Server (https://protenix-server.com). AF3 successfully docked both ligands but introduced stereochemical distortions consistent with known AF3 limitations⁶⁴. These effects were also reproduced using Boltz-2 (ref. ⁶⁷). To correct for these artefacts, the co-folded AgrC–AIP-I complex was used as the target for in silico docking of testosterone with an updated version of AutoDock Vina^68,69. All structures were visualized and analysed using PyMOL v.2.5.2 (Schrödinger).

Quantification and statistical analysis

Statistical details of experiments can be found in the figure legends, including how significance was defined and the statistical methods used. Data represent mean ± s.e.m. Formal randomization techniques were not used; however, mice were allocated to experiments randomly. Mice that were determined to be in the anagen hair cycle at the initiation of the experiment were excluded. All statistical analyses were performed with GraphPad Prism software (v.10.0.1), except the bioluminescent imaging data that was analysed as described above. To assess the statistical significance of the difference between two treatments for in vitro models, we used unpaired two-tailed Student’s t-tests. To assess the statistical significance of the difference between two treatments for mouse models, we used the Mann–Whitney U-test and Kolmogorov–Smirnov test. To assess the statistical significance of differences between more than two treatments in vitro, we used one-way ANOVA with post-test corrections. To assess the statistical significance of differences between more than two treatment groups in mouse models, we used the Kruskal–Wallis test with post-test corrections. For experiments in vitro and in vivo where metrics were calculated over time, we used two-way ANOVA with post-test corrections. For the RNA-sequencing experiments, expression data were analysed with CLC. All transcripts with a false-discovery-rate-adjusted P < 0.05 were considered significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Sequencing data have been submitted to the National Center for Biotechnology Information Sequence Read Archive under BioProject accession number PRJNA1071176. Source data are provided with this paper.

Code availability

No unique code was generated for this study.

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Acknowledgements

We thank A. Croft for discussions and aid in experimental design. This work was supported by National Institutes of Health grant NIAMS-K08AR076459 (T.A.H.-T.), NIAID-AI162964 (A.R.H.), NIAID-AI153185 (A.R.H.), VA Merit Award BX002711 (A.R.H.), Burroughs Wellcome Fund 1022777 (T.A.H.-T.) and NIAID-AI187273 (A.R.H. and T.A.H.-T.).

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Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, TX, USA
Maria Sindhura John, Mahendran Chinnappan, Camille I. Sturges, Methinee Artami, Shivani Jain, Hanna Gedamu, Jessica Komarovsky & Tamia A. Harris-Tryon
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO, USA
Mohini Bhattacharya, Rebecca A. Keogh, Jeffrey S. Kavanaugh & Alexander R. Horswill
Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, USA
Mauricio Velasquez & Jeffrey G. McDonald
Childrens Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA
Tripti Sharma
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA
Jeffrey G. McDonald
Department of Veterans Affairs, Eastern Colorado Healthcare System, Aurora, CO, USA
Alexander R. Horswill
Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USA
Tamia A. Harris-Tryon

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Maria Sindhura John
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Mahendran Chinnappan
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Camille I. Sturges
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Methinee Artami
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Mohini Bhattacharya
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Rebecca A. Keogh
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Jessica Komarovsky
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Contributions

Conceptualization: T.A.H.-T., M.S.J., J.S.K., A.R.H. and J.G.M. Methodology: T.A.H.-T., M.S.J., M.B., J.S.K., T.S. and J.G.M. Investigation: M.S.J., M.C., T.S., M.A., C.I.S., M.B., R.A.K., J.S.K., M.V., H.G., J.K., S.J. and T.A.H.-T. Visualization: M.S.J., T.A.H.-T., C.I.S., T.S., R.A.K. and J.S.K. Funding acquisition: T.A.H.-T. and A.R.H. Project administration: T.A.H.-T. Supervision: T.A.H.-T., A.R.H. and J.G.M. Writing—original draft: T.A.H.-T. and M.S.J. Writing—review and editing: T.A.H.-T., M.S.J., M.C., C.I.S., M.B., T.S., J.S.K. and A.R.H.

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Correspondence to Tamia A. Harris-Tryon.

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T.A.H.-T., M.S.J., A.R.H. and J.K. have filed a patent (US patent application number 63/580,813). Mirofend L.L.C. (founder, T.A.H.-T.). The other authors declare no competing interests.

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Extended data

Extended Data Table 1 Staphylococcus aureus strains used in this work

Full size table

Extended Data Table 2 Primers for Quantitative RT-PCR and Gene Sequencing

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Extended Data Fig. 1 Greater bacterial growth and skin damage in male mice compared to female mice.

a,b, Male and female C57BL/6 wild-type (WT) mice were epicutaneously infected for 4 days with 1×10⁶ colony forming units (CFUs) of a bioluminescent strain of MRSA SAP430 (MRSA::lux). Bioluminescence quantified over time. In vivo bioluminescence images (a) and diseases scores (b) quantified on Day 4. c, Transepidermal water loss (TEWL), a measure of skin barrier damage, quantified by Vapometer on Day 0 and Day 4. d,e, Testosterone (d) and dihydrotestosterone (DHT) (e) quantified from the skin secretions of male and female mice by hormone immunoassay. Means ± SEM are plotted (n = 5/ group). (b) *p < 0.05 by two-sided Kolmogorov-Smirnov Test. (c) **p < 0.001 by two-way ANOVA (two-sided) with Sidak’s correction for multiple comparisons. (d,e) *p < 0.05 **p < 0.01 by Mann-Whitney U-test (two-sided).

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Extended Data Fig. 2 Generation and validation of Hsd3b6^∆skin mice by CRISPR/Cas9 genomic tar-geting and baseline analysis of the Hsd3b6^∆skin mice compared to control.

a, Schematic diagram of CRISPR/Cas9-mediated gene insertion of loxP sites flanking the first coding exon of the Hsd3b6 locus. Hsd3b6^fl/fl mice crossed with K14-Cre^+/- mice to generate Hsd3b6^∆skin mice (K14-Cre^+/-; Hsd3b6^fl/fl). b, Immunofluorescence staining of HSD3B6 expression in Hsd3b6^fl/fl and Hsd3b6^∆skin mice skin. Scale bar 50 µM. c, Genomic confirmation of loxP mediated deletion of Hsd3b6 in the K14Cre^+/- mice in the skin, with PCR of testicular samplings demonstrating tissue specificity. d, qRT-PCR for Hsd3b6 expression Hsd3b6^fl/fl (n = 4) and Hsd3b6^∆skin (n = 4) in the female skin and Hsd3b6^fl/fl (n = 9) and Hsd3b6^∆skin (n = 7) in the male skin, Hsd3b6^fl/fl (n = 3) and Hsd3b6^∆skin (n = 3) in testis, and Hsd3b6^fl/fl (n = 3) and Hsd3b6^∆skin (n = 3) in the adrenal gland normalized to Rplp0 expression. e, Serum progesterone in male Hsd3b6^fl/fl (n = 5) and Hsd3b6^∆skin (n = 5) and DHT in male Hsd3b6^fl/fl (n = 6) and Hsd3b6^∆skin (n = 5) quantified by hormone immunoassay. f, Transepidermal water loss (TEWL) of male Hsd3b6^fl/fl (n = 9) and Hsd3b6^∆skin (n = 7) and female Hsd3b6^fl/fl (n = 5) and Hsd3b6^∆skin (n = 10) mice measured by Vapometer device as a metric of barrier integrity. g, Hematoxylin and eosin staining of Hsd3b6^fl/fl and Hsd3b6^∆skin skin. Scale bar 220 µM. h, Weight measurements of male Hsd3b6^fl/fl (n = 9) and Hsd3b6^∆skin (n = 7) and female Hsd3b6^fl/fl (n = 6) and Hsd3b6^∆skin (n = 10) mice. i, Progesterone quantified from the skin secretions of male Hsd3b6^fl/fl (n = 11) and Hsd3b6^∆skin (n = 8) and female Hsd3b6^fl/fl (n = 7) and Hsd3b6^∆skin (n = 11) age-matched mice at 7 weeks by hormone immunoassay. Means ± SEM (error bars) are plotted. (d) *p < 0.05, ***p < 0.001, ns, not significant by one-way ANOVA. (e-f,h) ns by Mann-Whitney U-test (two-sided). (i) **p < 0.01, ****p < 0.0001 by Kruskal-Wallis test with correction for false discovery (two-sided).

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Extended Data Fig. 3 Hsd3b6^∆skin mice are resistant to skin infection with S. aureus and topical testosterone increases S. aureus infection.

Images related to Fig. 1g and j. a, Male Hsd3b6^fl/fl (n = 10) and Hsd3b6^Δskin (n = 5) mice epicutaneously infected for 4 days with 1×10⁶ CFUs of MRSA SAP430 (MRSA::lux). (i) Images related to Fig. 1g. (ii) Representative histology (H&E stain) and mean epidermal thickness (μm) ± SEM. Scale bars: 220 μm. *p < 0.05, by Mann-Whitney U-test (two-sided). Means ± SEM are plotted. (iii) Representative images showing skin lesions on day 4 post-infection. b, Female Hsd3b6^∆skin mice were epicutaneously challenged with 1×10⁶ CFUs with bioluminescent MRSA SAP430 (MRSA::lux) for 4 days with or without testosterone (vehicle, n = 3; testosterone n = 4). (i) Images related to Fig. 1j. (ii) Representative histology (H&E stain) and mean epidermal thickness (μm) ± SEM. Scale bars: 220 μm. ns, p ≥ 0.057 by Mann-Whitney U-test (two-sided). Means ± SEM are plotted. (iii) Representative images showing skin lesions on day 4 post-infection. c, Repeat of Female Hsd3b6^∆skin mice were epicutaneously challenged with 1×10⁶ CFUs with bioluminescent MRSA SAP430 (MRSA::lux) for 3 days with or without testosterone (vehicle, n = 5; testosterone, n = 5). Means ± SEM (error bars) are plotted. **p < 0.001 by two-way ANOVA. (i) Representative bioluminescence imaging (BLI) of mice at days 1, 2, and 3 post-infections. (ii) Quantification of infection progression over days.

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Extended Data Fig. 4 Testosterone stimulates quorum sensing readouts in S. aureus, but pregnenolone does not activate quorum sensing.

a-d, Transcriptomics (RNA-seq) of HG003 strain of S. aureus treated through the early-log phase (OD₆₀₀ 0.4) with 10 nM or 100 nM testosterone, 10 nM pregnenolone, or vehicle alone. Differential gene expression was analysed using DESeq2 (two-sided Wald test) with Benjamini-Hochberg correction for multiple comparisons (FDR). a, Volcano plot demonstrating genes fold change in expression in testosterone transcriptome compared to vehicle treated S. aureus, (n = 3) as shown in Fig. 2a. Here, additional significant genes with lower fold-change are labelled. b, Volcano plot demonstrating change in expression with 100 nM of testosterone transcriptome compared to vehicle treated S. aureus, (n = 3). c, Volcano plot demonstrating change in expression with 10 nM of testosterone transcriptome compared to pregnenolone treated S. aureus, (n = 3). d, Volcano plot comparing the transcriptome of S. aureus treated with pregnenolone compared to vehicle control (n = 3).

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Extended Data Fig. 5 Testosterone and DHT activate quorum sensing in S. aureus without affecting growth, and other hormone classes have no effect on quorum sensing.

a, Growth curve of HG003 (agr Type I) treated with 10 nM testosterone or DHT. ns by two-way ANOVA compared to Vehicle. b, Bioluminescence of agr-P3 reporter (HG003agrP3::lux) treated with testosterone, DHT, progesterone, or estradiol. #p < 0.0001 or ns by two-way ANOVA com-pared to estradiol. c, qRT-PCR of S. aureus HG003 strain (agr Type I) treated with 10 nM testos-terone or AIP-I (n = 3) till early-growth phase (OD600 0.6). Expression of target genes RNAIII and agrA normalized to gyrA expression. **p < 0.001, *p < 0.05by two-tailed unpaired t-test. d, Growth curve of USA100 (agr Type II) treated with 10 nM testosterone or DHT. ns, not significant by two-way ANOVA compared to vehicle. e, In vitro bioluminescence of USA100 (USA100 agrP3::lux) treated with 10 nM of testosterone or DHT (n = 3). #p < 0.0001, by two-way ANOVA compared to vehicle control. f, qRT-PCR of USA100 treated with 10 nM testosterone or respective AIP (n = 3) through mid-log phase (OD600 0.6). Expression of target genes RNAIII normalized to gyrA expres-sion. Means ± SEM (error bars) are plotted. *p < 0.05by two-tailed unpaired t-test. g, Growth curve of MW2 (agr Type III) treated with 10 nM testosterone or DHT. ns,not significant by two-way ANOVA compared to Vehicle. h, In vitro bioluminescence of MW2 (MW2 agrP3::lux) treated with 10 nM of testosterone or DHT (n = 3). #p < 0.0001, by two-way ANOVA compared to vehicle control. i, qRT-PCR of MW2 treated with 10 nM testosterone or respective AIP (n = 3) through mid-log phase (OD600 0.6). Expression of target genes RNAIII normalized to gyrA expression. Means ± SEM (error bars) are plotted. *p > 0.05, ns, not significant by two-tailed unpaired t-test.

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Extended Data Fig. 6 Testosterone stimulates the expression of virulence factors across strains of S. aureus and in vivo quorum sensing bioluminescence of female Hsd3b6^∆skin compared to controls.

a,b, qRT-PCR of strains of S. aureus obtained from patients with atopic dermatitis⁴⁶ and treated with 10 nM of testosterone to the mid-exponential growth (n = 3). Expression of target gene RNAIII (a), and agrA (b), normalized to housekeeping gene gyrA expression. Means ± SEM (error bars) are plotted. **p < 0.001, *p < 0.05; p > 0.05 ns, not significant by two-tailed unpaired t-test. c, qRT-PCR of AD04.E7 treated with 10 nM testosterone or AIP-II (n = 3) till late-exponential growth (OD₆₀₀ 1.0). Expression of target genes, psmα, and agrA normalized to gyrA expression. Means ± SEM (error bars) are plotted. *p < 0.05, p > 0.05 ns, not significant by one-way ANOVA. d-f, qRT-PCR of S. aureus agr Type I strain HG003 treated with 10 nM testosterone or AIP-I (n = 3) till late-exponential growth (OD₆₀₀ 1.0). Expression of target genes, lukS-PV, hld, and hla normalized to gyrA expression. Means ± SEM (error bars) are plotted.*p < 0.05; **p < 0.01 by two-tailed unpaired t-test. g, Percentage of bacterially-induced neutrophil killing from S. aureus treated with and without 10 nM testosterone, DHT or AIP-I. n = 4 human donors, 2 male and 2 female. Means ± SEM (error bars) are plotted. **p < 0.01, by one-way ANOVA. h, In vivo analysis of agr-P3 reporter. Female Hsd3b6^fl/fl and Hsd3b6^∆skin mice were epicutaneously infected with 1×10⁶ CFUs of the CA-MRSA-agr-P3lux and bioluminescence quantified over time in Fig. 2j.

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Extended Data Fig. 7 Testosterone stimulates S. aureus agr quorum sensing in a dose-dependent man-ner and requires AgrC and AgrA.

a, Biosynthetic mutant ΔagrBD treated with increasing concentrations of testosterone. RNAIII, agrA, agrC, and psmα quantified by qRT-PCR (n = 3).Means ± SEM (error bars) are plotted. *p < 0.05; ***p < 0.001, ns, not significant by one-way ANOVA with Tukey’s post-hoc test (two-sided), adjusted for multiple comparisons. b, Growth curves of ΔagrBD treated with 10 nM testosterone, DHT or vehicle (n = 3), no significant differences from vehicle by two-way ANOVA. Means ± SEM (error bars) are plotted. c, ΔagrBD::lux treated with 10 nM testosterone, DHT, progesterone, or estradiol (n = 3). Bioluminescence was measured. Means ± SEM (error bars) are plotted. d, RNAIII expression in ΔagrBD treated with 10 nM AIP-I and 10 or 100 nM testosterone (n = 3) was quantified. e, Neutrophil killing induced by ΔagrBD treated with 10 nM testosterone, AIP-I, or vehicle was measured (n = 3 from a single donor). Means ± SEM are plotted. (c-e) *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant by one-way ANOVA (two-sided). (c) ^#p < 0.0001 and ns by two-way ANOVA (two-sided). versus estradiol. f, Schematic showing inhibition of AgrC signaling by non-cognate AIPs. g,h, ΔagrBD strains were treated with AIP-II (g) or AIP-III (h) alone or with increasing concentrations of testosterone (10nM–10 μM).RNAIII, agrA, and agrC were quantified (n = 3). Means ± SEM (error bars) are plotted. *p < 0.05, **p < 0.01; ***p < 0.001, ****p < 0.0001, ns by one-way ANOVA. i, Growth curve of ΔagrC treated with 10 nM testosterone showed no significant difference from vehicle (n = 3) by two-way ANOVA. Means ± SEM (error bars) are plotted. j,k, Neutrophil killing (n = 3 replicates of RBCs from a single donor) (j) and skin cell cytotoxicity (n = 3) (k) analysed in ΔagrC treated with 10 nM testosterone, DHT, AIP-I, or combinations. ns by one-way ANOVA. Means ± SEM (error bars) are plotted. l,m, ΔagrC (l) and ΔagrA (m) mutants were treated with 10 nM testosterone, AIP-I, or vehicle, (n = 3) and psmα and RNAIII were quantified. Means ± SEM (error bars) are plotted. ns, not significant by one-way ANOVA. n, ΔagrC mutant strain complemented (Comp) with AgrC on a plasmid treated with 10 nM testosterone, AIP-I, or vehicle (n = 3) and agrA and RNAIII were quantified. Means ± SEM (error bars) are plotted. *p < 0.05 by one-way ANOVA. Panel f created in BioRender; Harris, T. https://biorender.com/dzfdpzh (2025).

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Extended Data Fig. 8 Enantiomer-testosterone (ent-T) inhibits the expression of RNA III in atopic dermatitis strains of S. aureus and also inhibits S. aureus quorum sensing in male and female mice in vivo.

a, qRT-PCR of strains of S. aureus obtained from patients with atopic dermatitis⁴⁶ and treated with 10 nM or ent-T, a stereoisomer of testosterone (n = 3). Expression of target gene RNAIII normalized to housekeeping gene gyrA expression. Means ± SEM (error bars) are plotted.*p < 0.05 by two-tailed paired t-test. b, Bioluminescence of agr-P3 reporter (HG003agrP3::lux) treated with testosterone, ent-T or testosterone with escalating amounts of ent-T at the same time as testosterone. ^#p < 0.0001 or ns by two-way ANOVA compared to testosterone alone. c, In silico analysis with testosterone (left) and ent-T (right) bound to a hydrophobic pocket of AgrC bound by the polar residues T141, T197, and S201. Testosterone (left) is predicted to form stable hydrogen bonds with S194 and T190. Ent-T (right)is predicted to bind to the same hydrophobic pocket, but without the equivalent stability of hydrogen-bond interactions. d, Bioluminescence images of Fig. 5g, male wild-type mice epicutaneously infected with 1×10⁶ CFUs CA-MRSA-agr-P3 S. aureus reporter treated with ent-T or vehicle. e, Bioluminescence images of female wild-type mice epicutaneously infected with 1×10⁶ CFUs CA-MRSA-agr-P3 S. aureus reporter treated with testosterone, ent-T, or vehicle, and f, Bioluminescence quantified over time ****p < 0.0001 by two-way ANOVA with comparisons shown. Means ± SEM (error bars) are plotted.

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John, M.S., Chinnappan, M., Sturges, C.I. et al. Skin androgens regulate Staphylococcus aureus pathogenicity via quorum sensing. Nat Microbiol (2026). https://doi.org/10.1038/s41564-026-02261-2

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Received: 20 March 2025
Accepted: 05 January 2026
Published: 27 February 2026
Version of record: 27 February 2026
DOI: https://doi.org/10.1038/s41564-026-02261-2

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Abstract

Main

Results

Androgen secretion at the skin surface is required for S. aureus skin infection

Testosterone and DHT activate agr quorum sensing and promote S . aureus pathogenesis

Testosterone stimulates S. aureus pathogenesis independently of the bacterial AIP

Testosterone stimulation of S. aureus requires bacterial expression of the histidine kinase AgrC

The enantiomer of testosterone inhibits MRSA pathogenesis

Discussion

Methods

Reagent and resource sharing

Experimental model and participant details

Participant recruitment

Human skin sampling

Human peripheral blood

Mice

Method details

Quantification of hormones in mouse samples

Immunofluorescence microscopy

Bacterial strains and plasmids

Construction of lux-expressing strains

Construction of complemented ΔagrC strain

In vitro luminescence assays

In vitro growth assay

Haemolysis assay

Skin cell cytotoxicity assay

Neutrophil killing assay

Quantitative real-time PCR

RNA sequencing

S. aureus epicutaneous skin infections

Disease scoring

Transepidermal water loss measurement

Histology

Measurement of quorum sensing in vivo

In silico docking of testosterone to the AgrC receptor

Quantification and statistical analysis

Reporting summary

Data availability

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