Fig. 4: HGT can increase the duration of, but does not stabilize, cooperative virulence.

a Experimental system to determine the role of HGT in the stabilization of cooperative virulence. In both the mobile pVir and non-mobile pVir scenarios, the cooperator contains pVir^Low and a functional TTSS-1, making it virulent. In the mobile scenario, pVir can be transferred to cheaters. In the non-mobile scenario, transfer of pVir is blocked because of an incompatible plasmid, P2, in the cheater strain. b–g Ampicillin pretreated mice were orally infected with ~10² CFU of cheater (14028 ΔhilD ΔssaV; Kan^R, Amp^R) immediately followed by ~10² CFU of pVir^Low donor (14028 ΔhilD ΔssaV pVir; Cm^R encoded on pVir, Amp^R). The donor contained a functional invG allele, making it virulent (ssaV is deleted in both strains). Mice (n = 8 until day 4; n = 5 until day 10 for both groups) were given either a cheater with no plasmid (i.e., same strain used in Fig. 1; mobile pVir; black) or a cheater with P2 (incompatible with pVir; non-mobile pVir; pink). Dotted lines indicate detection limits. Medians are indicated by lines. Two-tailed Mann–Whitney U tests (p > 0.05 (ns), *p < 0.05, **p < 0.01, ***p < 0.001) are used to compare the mobile pVir and non-mobile pVir scenarios on each day. Donor, recipient, and transconjugant populations are presented in Fig. S6. Source data are provided as a Source Data file. b The pVir-bearing population was determined by selective plating on Cm-supplemented MacConkey agar. c the pVir-bearing population is reported as a percentage of the total population. Dashed line indicates 100% plasmid spread. d Inflammation was measured by a Lipocalin-2 ELISA on fecal samples. e Total S.Tm populations determined by the sum of Cm- and Kan-supplemented MacConkey agar. f, g Cm-supplemented MacConkey agar plates containing colonies from feces collected on day 10 post infection were analyzed for SipC expression as a proxy for TTSS-1 expression using a colony western blot and represented as the percentage of colonies that produced SipC are reported out of the total S.Tm population (f) or out of the pVir-containing population for the mobile pVir scenario (g).