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Proteolysis of virulence regulator ToxR is associated with entry of Vibrio cholerae into a dormant state.

Almagro-Moreno S, Kim TK, Skorupski K, Taylor RK - PLoS Genet. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Viability and morphology of V. cholerae mutants after 48 hours at alkaline pH.Fluorescent (F) and differential interference contrast (DIC) images of O395 ΔtoxR, ΔrseP, ΔrsePΔtoxR, ΔrpoE or ΔrpoEΔtoxR, toxR248, toxR248ΔrseP, toxR248ΔrpoE, and toxR-phoA grown for 48 hours in LB starting pH 9.3 (unbuffered). The cells were observed after treatment with the LIVE/DEAD BacLight Bacterial Viability and Counting Kit. Viable and culturable cells appear green and elongated; viable but dormant cells appear green and round; dead cells appear red and round.
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pgen.1005145.g006: Viability and morphology of V. cholerae mutants after 48 hours at alkaline pH.Fluorescent (F) and differential interference contrast (DIC) images of O395 ΔtoxR, ΔrseP, ΔrsePΔtoxR, ΔrpoE or ΔrpoEΔtoxR, toxR248, toxR248ΔrseP, toxR248ΔrpoE, and toxR-phoA grown for 48 hours in LB starting pH 9.3 (unbuffered). The cells were observed after treatment with the LIVE/DEAD BacLight Bacterial Viability and Counting Kit. Viable and culturable cells appear green and elongated; viable but dormant cells appear green and round; dead cells appear red and round.

Mentions: The viability and morphology of the mutant strains tested in Fig 4 is consistent with their culturability (Fig 6). The strains that cannot proteolyze ToxR (ΔrseP, ΔrpoE, toxR248ΔrseP, and toxR-phoA) show a similar morphology and viability to cells in O/N cultures (Figs 5 and 6), whereas the mutants that do not encode ToxR (ΔtoxR, ΔrsePΔtoxR, and ΔrpoEΔtoxR) or proteolyze it (toxR248 and toxR248ΔrpoE) are viable and round (Fig 6). These findings indicate that in V. cholerae the loss of ToxR is associated with entry into a dormant state.


Proteolysis of virulence regulator ToxR is associated with entry of Vibrio cholerae into a dormant state.

Almagro-Moreno S, Kim TK, Skorupski K, Taylor RK - PLoS Genet. (2015)

Viability and morphology of V. cholerae mutants after 48 hours at alkaline pH.Fluorescent (F) and differential interference contrast (DIC) images of O395 ΔtoxR, ΔrseP, ΔrsePΔtoxR, ΔrpoE or ΔrpoEΔtoxR, toxR248, toxR248ΔrseP, toxR248ΔrpoE, and toxR-phoA grown for 48 hours in LB starting pH 9.3 (unbuffered). The cells were observed after treatment with the LIVE/DEAD BacLight Bacterial Viability and Counting Kit. Viable and culturable cells appear green and elongated; viable but dormant cells appear green and round; dead cells appear red and round.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388833&req=5

pgen.1005145.g006: Viability and morphology of V. cholerae mutants after 48 hours at alkaline pH.Fluorescent (F) and differential interference contrast (DIC) images of O395 ΔtoxR, ΔrseP, ΔrsePΔtoxR, ΔrpoE or ΔrpoEΔtoxR, toxR248, toxR248ΔrseP, toxR248ΔrpoE, and toxR-phoA grown for 48 hours in LB starting pH 9.3 (unbuffered). The cells were observed after treatment with the LIVE/DEAD BacLight Bacterial Viability and Counting Kit. Viable and culturable cells appear green and elongated; viable but dormant cells appear green and round; dead cells appear red and round.
Mentions: The viability and morphology of the mutant strains tested in Fig 4 is consistent with their culturability (Fig 6). The strains that cannot proteolyze ToxR (ΔrseP, ΔrpoE, toxR248ΔrseP, and toxR-phoA) show a similar morphology and viability to cells in O/N cultures (Figs 5 and 6), whereas the mutants that do not encode ToxR (ΔtoxR, ΔrsePΔtoxR, and ΔrpoEΔtoxR) or proteolyze it (toxR248 and toxR248ΔrpoE) are viable and round (Fig 6). These findings indicate that in V. cholerae the loss of ToxR is associated with entry into a dormant state.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus