Abstract
Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the diarrheal disease, cholera. Two of its primary virulence regulators, TcpP and ToxR, are localized in the inner membrane. TcpP is encoded on the Vibrio Pathogenicity Island (VPI), a horizontally acquired mobile genetic element, and functions primarily in virulence gene regulation. TcpP has been shown to undergo regulated intramembrane proteolysis (RIP) in response to environmental conditions that are unfavorable for virulence gene expression. ToxR is encoded in the ancestral genome and is present in non-pathogenic strains of V. cholerae, indicating it has roles outside of the human host. In this study, we show that ToxR undergoes RIP in V. cholerae in response to nutrient limitation at alkaline pH, a condition that occurs during the stationary phase of growth. This process involves the site-2 protease RseP (YaeL), and is dependent upon the RpoE-mediated periplasmic stress response, as deletion mutants for the genes encoding these two proteins cannot proteolyze ToxR under nutrient limitation at alkaline pH. We determined that the loss of ToxR, genetically or by proteolysis, is associated with entry of V. cholerae into a dormant state in which the bacterium is normally found in the aquatic environment called viable but nonculturable (VBNC). Strains that can proteolyze ToxR, or do not encode it, lose culturability, experience a change in morphology associated with cells in VBNC, yet remain viable under nutrient limitation at alkaline pH. On the other hand, mutant strains that cannot proteolyze ToxR remain culturable and maintain the morphology of cells in an active state of growth. Overall, our findings provide a link between the proteolysis of a virulence regulator and the entry of a pathogen into an environmentally persistent state.
Highlights
The ability of microorganisms to alter their gene expression profiles when transitioning between environments is fundamental for their survival
We show that ToxR undergoes proteolysis under nutrient limitation at alkaline pH and this loss is associated with the entry of V. cholerae into a dormant state, similar to that found in its natural environment between epidemics
To our knowledge, we provide the first evidence of a link between the proteolysis of a virulence regulator and the entry of a bacterial pathogen into an environmentally persistent state
Summary
The ability of microorganisms to alter their gene expression profiles when transitioning between environments is fundamental for their survival. Upon entry of V. cholerae into the human host, the expression of its two major virulence factors is induced: the toxin co-regulated pilus (TCP), an essential intestinal colonization factor [5], and cholera toxin (CT), responsible for the diarrhea associated with the disease [5,6] The expression of these factors is coordinately regulated at the transcriptional level by a virulence cascade involving a number of regulatory proteins [7]. Central to this cascade is the cooperation between two pairs of membrane-localized transcriptional regulators, TcpPH, encoded on the Vibrio Pathogenicity Island (VPI) [8], and ToxRS, encoded in the ancestral genome and present in non-pathogenic isolates of V. cholerae These findings indicate that ToxR has roles outside of the human host. ToxT, encoded on the VPI, directly activates the expression of TCP and CT, as well as other genes [7,13,14]
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