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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

V. cholerae shows reduced culturability over time at alkaline pH.(A) CFU/ml of O395 wild-type strain grown at different time points in LB pH 7.0 with 100 mM HEPES (Buff), or LB starting pH 9.3 unbuffered (pH 9.3). The bars represent the mean of four independent experiments and the error bars indicate the standard deviation. Statistical comparisons were made using the student’s t-test and compare samples relative to 12h Buff. ***P < 0.0005. (B) CFU/ml of O395 wild-type (WT), ΔtoxR, or ΔtoxR pVM7 strains grown at different time points in LB starting pH 9.3 unbuffered. The bars represent the mean of four independent experiments and the error bars indicate the standard deviation. Statistical comparisons were made using the student’s t-test and compare samples relative to wild-type on LB pH 9.3 at that specific time point. *P < 0.05, ***P < 0.0005.
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pgen.1005145.g003: V. cholerae shows reduced culturability over time at alkaline pH.(A) CFU/ml of O395 wild-type strain grown at different time points in LB pH 7.0 with 100 mM HEPES (Buff), or LB starting pH 9.3 unbuffered (pH 9.3). The bars represent the mean of four independent experiments and the error bars indicate the standard deviation. Statistical comparisons were made using the student’s t-test and compare samples relative to 12h Buff. ***P < 0.0005. (B) CFU/ml of O395 wild-type (WT), ΔtoxR, or ΔtoxR pVM7 strains grown at different time points in LB starting pH 9.3 unbuffered. The bars represent the mean of four independent experiments and the error bars indicate the standard deviation. Statistical comparisons were made using the student’s t-test and compare samples relative to wild-type on LB pH 9.3 at that specific time point. *P < 0.05, ***P < 0.0005.

Mentions: The proteolysis of ToxR under nutrient limitation at alkaline pH might provide an advantage to V. cholerae by preventing the expression of genes with roles in nutrient rich environments, such as OmpU, and promoting those with roles in nutrient poor conditions, such as OmpT [36–38]. Under conditions that are not conducive to active growth, such as nutrient limitation, V. cholerae is capable of entering a dormant, nonculturable state referred to as VBNC or CVEC that facilitates its survival and persistence [30,31,49–51]. We investigated whether the loss of ToxR is associated with the entry of V. cholerae into a nonculturable state. To assess this, the culturability of V. cholerae was measured by plating cultures of V. cholerae O395 between 12 and 48 hours after inoculation on LB medium with a starting pH of 9.3 and also in LB medium buffered at pH 7.0 with 100 mM HEPES at 6 hour intervals, and determining the colony forming units (CFUs) at each time point (Fig 3A). As shown in Fig 3A, the number of CFU/ml of O395 grown in LB 100 mM HEPES do not change over time whereas the number of CFU/ml of cultures grown on LB with a starting pH of 9.3 start getting reduced in some cultures around 24 hours of growth with a final CFU count nearly 5 logs lower than the cultures grown in LB buffered at pH 7.0 (Fig 3A). Interestingly, the culturability of V. cholerae is reduced more or less in parallel with the proteolysis of ToxR (Fig 1B). These results indicate that growth of V. cholerae to late stationary phase at alkaline pH, defined as ToxR proteolysis inducing (TPI) conditions, decreases the culturability of V. cholerae.


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)

V. cholerae shows reduced culturability over time at alkaline pH.(A) CFU/ml of O395 wild-type strain grown at different time points in LB pH 7.0 with 100 mM HEPES (Buff), or LB starting pH 9.3 unbuffered (pH 9.3). The bars represent the mean of four independent experiments and the error bars indicate the standard deviation. Statistical comparisons were made using the student’s t-test and compare samples relative to 12h Buff. ***P < 0.0005. (B) CFU/ml of O395 wild-type (WT), ΔtoxR, or ΔtoxR pVM7 strains grown at different time points in LB starting pH 9.3 unbuffered. The bars represent the mean of four independent experiments and the error bars indicate the standard deviation. Statistical comparisons were made using the student’s t-test and compare samples relative to wild-type on LB pH 9.3 at that specific time point. *P < 0.05, ***P < 0.0005.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005145.g003: V. cholerae shows reduced culturability over time at alkaline pH.(A) CFU/ml of O395 wild-type strain grown at different time points in LB pH 7.0 with 100 mM HEPES (Buff), or LB starting pH 9.3 unbuffered (pH 9.3). The bars represent the mean of four independent experiments and the error bars indicate the standard deviation. Statistical comparisons were made using the student’s t-test and compare samples relative to 12h Buff. ***P < 0.0005. (B) CFU/ml of O395 wild-type (WT), ΔtoxR, or ΔtoxR pVM7 strains grown at different time points in LB starting pH 9.3 unbuffered. The bars represent the mean of four independent experiments and the error bars indicate the standard deviation. Statistical comparisons were made using the student’s t-test and compare samples relative to wild-type on LB pH 9.3 at that specific time point. *P < 0.05, ***P < 0.0005.
Mentions: The proteolysis of ToxR under nutrient limitation at alkaline pH might provide an advantage to V. cholerae by preventing the expression of genes with roles in nutrient rich environments, such as OmpU, and promoting those with roles in nutrient poor conditions, such as OmpT [36–38]. Under conditions that are not conducive to active growth, such as nutrient limitation, V. cholerae is capable of entering a dormant, nonculturable state referred to as VBNC or CVEC that facilitates its survival and persistence [30,31,49–51]. We investigated whether the loss of ToxR is associated with the entry of V. cholerae into a nonculturable state. To assess this, the culturability of V. cholerae was measured by plating cultures of V. cholerae O395 between 12 and 48 hours after inoculation on LB medium with a starting pH of 9.3 and also in LB medium buffered at pH 7.0 with 100 mM HEPES at 6 hour intervals, and determining the colony forming units (CFUs) at each time point (Fig 3A). As shown in Fig 3A, the number of CFU/ml of O395 grown in LB 100 mM HEPES do not change over time whereas the number of CFU/ml of cultures grown on LB with a starting pH of 9.3 start getting reduced in some cultures around 24 hours of growth with a final CFU count nearly 5 logs lower than the cultures grown in LB buffered at pH 7.0 (Fig 3A). Interestingly, the culturability of V. cholerae is reduced more or less in parallel with the proteolysis of ToxR (Fig 1B). These results indicate that growth of V. cholerae to late stationary phase at alkaline pH, defined as ToxR proteolysis inducing (TPI) conditions, decreases the culturability of V. cholerae.

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