Fig. 1: Virulence can emerge through HGT in a population of cheaters in vivo, but is unstable.

a Experimental system to measure maintenance of cooperative virulence by HGT. Donors contain pVir encoding hilD, but cannot produce a functional TTSS-1 (invG mutant), making them avirulent. Recipients contain all genes for a functional TTSS-1 but do not have a functional copy of hilD (cheaters), preventing TTSS-1 expression and virulence. Transfer of pVir from the donor to the recipient forms a transconjugant that contains both functional TTSS-1-encoding genes and a copy of hilD from pVir, allowing TTSS-1-mediated virulence. Transconjugants can transfer pVir to additional recipients. b–e pVir is transferred to cheater recipients and allows cooperative virulence to emerge. Ampicillin pretreated mice were sequentially infected orally with donors (14028S ΔinvG ΔhilD ΔssaV; Cm^R, Amp^R) harboring pVir^Low (blue; n = 8), pVir^High (orange; n = 11), or P2 lacking hilD (control; white; n = 11), and recipients (14028 S ΔhilD ΔssaV; Kan^R, Amp^R). Each replicate is shown and bars indicate the median. Source data are provided as a Source Data file. b–d Statistics compare pVir^Low (blue asterisks) and pVir^High (orange asterisks) to the control on each day; Kruskal–Wallis test with Dunn’s multiple test correction (p > 0.05 not significant and not indicated, *p < 0.05, **p < 0.01, ***p < 0.001. Dotted line represents the detection limit. b Plasmid transfer was measured by selective and/or replica plating. The proportion of transconjugants is calculated by dividing the transconjugant population by the sum of recipients and transconjugants. c Inflammation was measured by a Lipocalin-2 ELISA on fecal samples. d Total population determined by summing all subpopulations. Donor, recipient, and transconjugant populations are presented in Fig. S3. e Transconjugants carrying pVir^Low (blue) and pVir^High (orange) were analyzed by colony western-blot and compared using a two-tailed Mann–Whitney U test (p > 0.05 not significant and not indicated, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). The percentage of colonies that expressed SipC are reported out of the total transconjugant population. Bars indicate the median. The black dotted line indicates the conservative detection limit, which is dependent on the number of colonies on the plate (values can therefore appear below the detection limit).