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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

Effect of nutrient limitation in the stability of ToxR, culturability and morphology of Vibrio parahaemolyticus.(A) ToxR immunoblot of V. parahaemolyticus RIMD2210633 wild-type or ΔtoxR grown for either 12 or 48 hours in LB starting pH 7.0 unbuffered (LB), or LB buffered to pH 7.0 with 100 mM HEPES (Buff) (B) Culturability of V. parahaemolyticus RIMD2210633 grown for 48 hours in LB starting pH 7.0 unbuffered (LB), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). 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 48h Buff. ***P < 0.0005. (C) Fluorescent (F) and differential interference contrast (DIC) images of V. parahaemolyticus RIMD2210633 grown for 48 hours under conditions as in (B). 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.g007: Effect of nutrient limitation in the stability of ToxR, culturability and morphology of Vibrio parahaemolyticus.(A) ToxR immunoblot of V. parahaemolyticus RIMD2210633 wild-type or ΔtoxR grown for either 12 or 48 hours in LB starting pH 7.0 unbuffered (LB), or LB buffered to pH 7.0 with 100 mM HEPES (Buff) (B) Culturability of V. parahaemolyticus RIMD2210633 grown for 48 hours in LB starting pH 7.0 unbuffered (LB), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). 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 48h Buff. ***P < 0.0005. (C) Fluorescent (F) and differential interference contrast (DIC) images of V. parahaemolyticus RIMD2210633 grown for 48 hours under conditions as in (B). 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: V. parahaemolyticus is an intestinal pathogen that causes bloody diarrhea and is generally transmitted through the consumption of raw or uncooked fish. Recently, Whitaker et al showed that ToxR is required for intestinal colonization of V. parahaemolyticus in the adult murine model [56]. V. parahaemolyticus is known to enter VBNC under starvation conditions [57]. We determined whether the entry of V. parahaemolyticus into dormancy was also associated with loss of ToxR. Unexpectedly, V. parahaemolyticus cannot grow on LB with a starting pH of 9.3, however, like V. cholerae, it also alkalinizes the pH of the media during growth. Nonetheless, we found that ToxR undergoes proteolysis in V. parahaemolyticus RIMD2210633 when the bacterium is cultured on LB pH 7.0 unbuffered for 48 hours (Fig 7A). Furthermore, the bacterium loses culturability and adopts a viable coccoid form under unbuffered conditions and nutrient limitation (Fig 7B and 7C). Our data suggests that entry into dormancy might be mediated by ToxR proteolysis among other species of the family Vibrionaceae encoding ToxR.


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)

Effect of nutrient limitation in the stability of ToxR, culturability and morphology of Vibrio parahaemolyticus.(A) ToxR immunoblot of V. parahaemolyticus RIMD2210633 wild-type or ΔtoxR grown for either 12 or 48 hours in LB starting pH 7.0 unbuffered (LB), or LB buffered to pH 7.0 with 100 mM HEPES (Buff) (B) Culturability of V. parahaemolyticus RIMD2210633 grown for 48 hours in LB starting pH 7.0 unbuffered (LB), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). 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 48h Buff. ***P < 0.0005. (C) Fluorescent (F) and differential interference contrast (DIC) images of V. parahaemolyticus RIMD2210633 grown for 48 hours under conditions as in (B). 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.g007: Effect of nutrient limitation in the stability of ToxR, culturability and morphology of Vibrio parahaemolyticus.(A) ToxR immunoblot of V. parahaemolyticus RIMD2210633 wild-type or ΔtoxR grown for either 12 or 48 hours in LB starting pH 7.0 unbuffered (LB), or LB buffered to pH 7.0 with 100 mM HEPES (Buff) (B) Culturability of V. parahaemolyticus RIMD2210633 grown for 48 hours in LB starting pH 7.0 unbuffered (LB), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). 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 48h Buff. ***P < 0.0005. (C) Fluorescent (F) and differential interference contrast (DIC) images of V. parahaemolyticus RIMD2210633 grown for 48 hours under conditions as in (B). 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: V. parahaemolyticus is an intestinal pathogen that causes bloody diarrhea and is generally transmitted through the consumption of raw or uncooked fish. Recently, Whitaker et al showed that ToxR is required for intestinal colonization of V. parahaemolyticus in the adult murine model [56]. V. parahaemolyticus is known to enter VBNC under starvation conditions [57]. We determined whether the entry of V. parahaemolyticus into dormancy was also associated with loss of ToxR. Unexpectedly, V. parahaemolyticus cannot grow on LB with a starting pH of 9.3, however, like V. cholerae, it also alkalinizes the pH of the media during growth. Nonetheless, we found that ToxR undergoes proteolysis in V. parahaemolyticus RIMD2210633 when the bacterium is cultured on LB pH 7.0 unbuffered for 48 hours (Fig 7A). Furthermore, the bacterium loses culturability and adopts a viable coccoid form under unbuffered conditions and nutrient limitation (Fig 7B and 7C). Our data suggests that entry into dormancy might be mediated by ToxR proteolysis among other species of the family Vibrionaceae encoding ToxR.

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