RpoS Controls the Vibrio cholerae Mucosal Escape Response

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Abstract
Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the “mucosal escape response,” this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle. Vibrio cholerae, a pathogenic microbe, causes a severe diarrhoeal disease mainly in Third World countries. Although the pathogenicity of this organism has been intensively studied for more than a century, most research has focused on the initial stages of the infection, especially colonization of the intestine and virulence gene expression. However, the last stages of the infectious process have received very little attention. In the present manuscript, the authors use the rabbit ileal loop model of cholera to show how this organism, late in the infection, detaches from the epithelial surface and migrates into the luminal fluid, a process the authors termed the “mucosal escape response.” This study identifies, for the first time, how the alternative starvation sigma factor RpoS regulates this process. Features of this genetic program include the dramatic induction of genes involved in motility and chemotaxis functions. This study furthermore identifies RpoS as an important regulator of virulence gene expression and shows that the mucosal escape response may coincide with diminished virulence gene expression. This work is essential for understanding a key and under-appreciated step in the life cycle of this important human pathogen: its exit from the intestine and how this serves to prepare it for transmission into environmental reservoirs or to new human hosts.