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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 is RseP and RpoE-dependent.(A) ToxR immunoblot of cultures of O395 wild-type or ΔtoxR, ΔrseP or ΔrpoE grown for 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) Autoagglutination of O395 ΔtcpA, ΔtoxR, wild-type (WT), ΔrseP, ΔrpoE, toxR248, toxR248ΔrseP and toxR248ΔrpoE grown under inducing conditions (LB starting pH 6.5, 30°C) for 15 hours. Autoagglutination can be visualized as a pellet at the bottom of the tube. (C) TcpA and (D) ToxR immunoblots of the cultures in (B).
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pgen.1005145.g002: Proteolysis of ToxR is RseP and RpoE-dependent.(A) ToxR immunoblot of cultures of O395 wild-type or ΔtoxR, ΔrseP or ΔrpoE grown for 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) Autoagglutination of O395 ΔtcpA, ΔtoxR, wild-type (WT), ΔrseP, ΔrpoE, toxR248, toxR248ΔrseP and toxR248ΔrpoE grown under inducing conditions (LB starting pH 6.5, 30°C) for 15 hours. Autoagglutination can be visualized as a pellet at the bottom of the tube. (C) TcpA and (D) ToxR immunoblots of the cultures in (B).

Mentions: The levels of TcpP have been shown to be controlled by RIP through the site-2 protease RseP [18]. We determined whether the levels of ToxR in response to nutrient limitation at alkaline pH may also be influenced by proteolysis through RseP. As shown in Fig 2A, ToxR was largely undetectable in strain O395 after growth for 48 hours in LB with a starting pH of 7.0 or 9.3, whereas the levels of ToxR in the ΔrseP mutant under these conditions were similar to wild-type grown in LB buffered at pH 7.0 with 100 mM HEPES. This finding indicates that RseP is involved in the proteolysis of ToxR during late stationary phase at alkaline pH, possibly functioning as a site-2 protease.


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 is RseP and RpoE-dependent.(A) ToxR immunoblot of cultures of O395 wild-type or ΔtoxR, ΔrseP or ΔrpoE grown for 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) Autoagglutination of O395 ΔtcpA, ΔtoxR, wild-type (WT), ΔrseP, ΔrpoE, toxR248, toxR248ΔrseP and toxR248ΔrpoE grown under inducing conditions (LB starting pH 6.5, 30°C) for 15 hours. Autoagglutination can be visualized as a pellet at the bottom of the tube. (C) TcpA and (D) ToxR immunoblots of the cultures in (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4388833&req=5

pgen.1005145.g002: Proteolysis of ToxR is RseP and RpoE-dependent.(A) ToxR immunoblot of cultures of O395 wild-type or ΔtoxR, ΔrseP or ΔrpoE grown for 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) Autoagglutination of O395 ΔtcpA, ΔtoxR, wild-type (WT), ΔrseP, ΔrpoE, toxR248, toxR248ΔrseP and toxR248ΔrpoE grown under inducing conditions (LB starting pH 6.5, 30°C) for 15 hours. Autoagglutination can be visualized as a pellet at the bottom of the tube. (C) TcpA and (D) ToxR immunoblots of the cultures in (B).
Mentions: The levels of TcpP have been shown to be controlled by RIP through the site-2 protease RseP [18]. We determined whether the levels of ToxR in response to nutrient limitation at alkaline pH may also be influenced by proteolysis through RseP. As shown in Fig 2A, ToxR was largely undetectable in strain O395 after growth for 48 hours in LB with a starting pH of 7.0 or 9.3, whereas the levels of ToxR in the ΔrseP mutant under these conditions were similar to wild-type grown in LB buffered at pH 7.0 with 100 mM HEPES. This finding indicates that RseP is involved in the proteolysis of ToxR during late stationary phase at alkaline pH, possibly functioning as a site-2 protease.

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