<<The>> expression of virulence genes increases membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium

Salmonella enterica serovar Typhimurium (S. Tm) represents one of the most prevalent enteric pathogens infecting humans and animals, associated with diarrheal disease. To trigger disease in the host, S. Tm critically relies on expression of specialized virulence factors. The transcription regulator HilD plays a central role in S. Tm regulatory network driving expression of virulence. Remarkably, the HilD regulon is only expressed in a fraction of S. Tm population both in vitro and in vivo. HilD-expressing cells are able to invade host epithelial cells and trigger gut inflammation, which fosters growth of S. Tm in the gut lumen. Expression of virulence factors modulates the host immune response therefore creating a favorable niche is an example of cooperative trait. In case of S. Tm inflammation represents a public good that helps S. Tm to outcompete the microbiota and to maximize the transmission to the next host. However, expression of virulence factors imposes a significant fitness cost to S. Tm at the single-cell level. Cost of virulence promotes emergence of virulence attenuated mutants – namely cheaters, which can benefit from inflammation, without contributing to its production. Therefore, understanding the cost of virulence and how it relates to virulence regulation could allow the identification and modulation of ecological factors to drive the evolution of S. Tm toward attenuation. Until now, the only described cost of virulence expression in S. Tm is a 2-fold reduction of the growth rate in cells expressing HilD regulon. Since most invasion factors controlled by HilD are embedded within envelope, we hypothesized that expression of the HilD regulon could render S. Tm intrinsically more sensitive to envelope stress. Moreover, it is known that in response to stress affecting membrane homeostasis, S. Tm generally downregulate expression of HilD-regulated functions. Therefore, we speculated that expression of HilD-controlled genes could be an integral part of general envelope stress response specific to S. Tm. We addressed this hypothesis, by comparing the membrane permeability, death rate and HilD activity in presence of membrane targeting stress in populations of S. Tm strains in which the expression of regulators and functions downstream of HilD was genetically tuned. Our results reveal a trade-off between envelope integrity and virulence expression in S. Tm which could explain the downregulation of virulence expression in response to hostile environmental conditions. This effect of virulence expression on S. Tm membrane status and stress resistance is independent from previously described growth rate reduction and presents a significant burden during in vivo infection in mice. Herein, the described fitness cost of virulence expression is a novel factor contributing to the inherent instability of virulence in S. Tm and selection for virulence attenuated mutants. This genetic instability of virulence in S. Tm could be exploited to fight against this pathogen, which is becoming increasingly resistant to antibiotics. Since membrane embedded virulence factors are critical during infection process of many bacterial pathogens, our findings can inspire development of new anti-virulence strategies.