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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

Proteolysis of ToxR during late stationary phase at alkaline pH.(A) ToxR immunoblot of O395 wild-type or ΔtoxR grown for either 12 or 48 hours in LB starting pH 7.0 unbuffered (LB), LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). (B) ToxR immunoblots of O395 wild-type grown at different time points in LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). (C) ToxR immunoblots of O395 wild-type grown overnight in LB starting pH 7.0 unbuffered at 37°C, pelleted, and resuspended in phosphate buffered saline (PBS) at pH 7.0, pH 8.3, or pH 9.3 for 12 hours.
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pgen.1005145.g001: Proteolysis of ToxR during late stationary phase at alkaline pH.(A) ToxR immunoblot of O395 wild-type or ΔtoxR grown for either 12 or 48 hours in LB starting pH 7.0 unbuffered (LB), LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). (B) ToxR immunoblots of O395 wild-type grown at different time points in LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). (C) ToxR immunoblots of O395 wild-type grown overnight in LB starting pH 7.0 unbuffered at 37°C, pelleted, and resuspended in phosphate buffered saline (PBS) at pH 7.0, pH 8.3, or pH 9.3 for 12 hours.

Mentions: The expression of ompU is activated by ToxR in nutrient abundant environments, such as in rich medium [36] or in the host [41], whereas the expression of ompT is derepressed in nutrient limited conditions, such as in minimal medium [38] or during the late stationary phase of growth [37]. Since the levels of ToxR increase in the presence of nutrients to facilitate ompU expression [38], we hypothesized that the levels of ToxR would decrease during the growth of V. cholerae in late stationary phase to facilitate ompT expression. To test this, wild-type classical biotype strain O395 was grown in LB medium (starting pH 7.0, unbuffered) for 12 and 48 hours. As shown in Fig 1A, the levels of ToxR after 48 hours were significantly reduced compared to the 12 hour culture. The final pH of the 48 hour culture was determined to be 9.2–9.3, indicating that the medium became alkaline during growth. To determine whether alkaline pH contributed to the loss of ToxR after 48 hours, V. cholerae was grown for 12 and 48 hours in LB medium with a starting pH of 9.3 (unbuffered) and also in LB medium buffered at pH 7.0 with 100 mM HEPES. As shown in Fig 1A, ToxR was undetectable in cultures grown for 48 hours in LB at pH 9.3, whereas its levels remained high at this pH when grown for only 12 hours. This indicates that, in LB, ToxR appears to start being proteolyzed once the cultures reach both alkaline pH and nutrient limitation. When the medium was buffered at pH 7.0, ToxR levels remained high even after 48 hours (Fig 1A). Thus, in late stationary phase, ToxR levels significantly decrease after 48 hours due to an increase in the pH. As a control we examined the stability of a ToxR-unrelated protein, GbpA, and found that these conditions did not affect the stability of GbpA (S1A Fig) [42]. We then determined the dynamics of ToxR proteolysis by measuring ToxR protein levels between 12 hours and 48 hours post-inoculation in LB pH 9.3 at 6 hour intervals (Fig 1B). We found that ToxR proteolysis begins around 24 hours of growth at LB pH 9.3 (Fig 1B). ToxR becomes undetectable between 42 and 48 hours of culture (Fig 1B).


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)

Proteolysis of ToxR during late stationary phase at alkaline pH.(A) ToxR immunoblot of O395 wild-type or ΔtoxR grown for either 12 or 48 hours in LB starting pH 7.0 unbuffered (LB), LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). (B) ToxR immunoblots of O395 wild-type grown at different time points in LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). (C) ToxR immunoblots of O395 wild-type grown overnight in LB starting pH 7.0 unbuffered at 37°C, pelleted, and resuspended in phosphate buffered saline (PBS) at pH 7.0, pH 8.3, or pH 9.3 for 12 hours.
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pgen.1005145.g001: Proteolysis of ToxR during late stationary phase at alkaline pH.(A) ToxR immunoblot of O395 wild-type or ΔtoxR grown for either 12 or 48 hours in LB starting pH 7.0 unbuffered (LB), LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). (B) ToxR immunoblots of O395 wild-type grown at different time points in LB starting pH 9.3 unbuffered (pH 9.3), or LB buffered to pH 7.0 with 100 mM HEPES (Buff). (C) ToxR immunoblots of O395 wild-type grown overnight in LB starting pH 7.0 unbuffered at 37°C, pelleted, and resuspended in phosphate buffered saline (PBS) at pH 7.0, pH 8.3, or pH 9.3 for 12 hours.
Mentions: The expression of ompU is activated by ToxR in nutrient abundant environments, such as in rich medium [36] or in the host [41], whereas the expression of ompT is derepressed in nutrient limited conditions, such as in minimal medium [38] or during the late stationary phase of growth [37]. Since the levels of ToxR increase in the presence of nutrients to facilitate ompU expression [38], we hypothesized that the levels of ToxR would decrease during the growth of V. cholerae in late stationary phase to facilitate ompT expression. To test this, wild-type classical biotype strain O395 was grown in LB medium (starting pH 7.0, unbuffered) for 12 and 48 hours. As shown in Fig 1A, the levels of ToxR after 48 hours were significantly reduced compared to the 12 hour culture. The final pH of the 48 hour culture was determined to be 9.2–9.3, indicating that the medium became alkaline during growth. To determine whether alkaline pH contributed to the loss of ToxR after 48 hours, V. cholerae was grown for 12 and 48 hours in LB medium with a starting pH of 9.3 (unbuffered) and also in LB medium buffered at pH 7.0 with 100 mM HEPES. As shown in Fig 1A, ToxR was undetectable in cultures grown for 48 hours in LB at pH 9.3, whereas its levels remained high at this pH when grown for only 12 hours. This indicates that, in LB, ToxR appears to start being proteolyzed once the cultures reach both alkaline pH and nutrient limitation. When the medium was buffered at pH 7.0, ToxR levels remained high even after 48 hours (Fig 1A). Thus, in late stationary phase, ToxR levels significantly decrease after 48 hours due to an increase in the pH. As a control we examined the stability of a ToxR-unrelated protein, GbpA, and found that these conditions did not affect the stability of GbpA (S1A Fig) [42]. We then determined the dynamics of ToxR proteolysis by measuring ToxR protein levels between 12 hours and 48 hours post-inoculation in LB pH 9.3 at 6 hour intervals (Fig 1B). We found that ToxR proteolysis begins around 24 hours of growth at LB pH 9.3 (Fig 1B). ToxR becomes undetectable between 42 and 48 hours of culture (Fig 1B).

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