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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