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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

Reduction in culturability of V. cholerae depends on the loss of ToxR.CFU/ml of O395 wild-type (WT) and mutant strains in LB pH 7.0 with 100 mM HEPES (Buff), or LB starting pH 9.3 unbuffered (pH 9.3) for 48 hours. The bars represent the mean of at least 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 48h Buff. ***P < 0.0005.
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pgen.1005145.g004: Reduction in culturability of V. cholerae depends on the loss of ToxR.CFU/ml of O395 wild-type (WT) and mutant strains in LB pH 7.0 with 100 mM HEPES (Buff), or LB starting pH 9.3 unbuffered (pH 9.3) for 48 hours. The bars represent the mean of at least 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 48h Buff. ***P < 0.0005.

Mentions: The same conditions that trigger the proteolysis of ToxR induce the loss of culturability of V. cholerae (Fig 1A and 3A). Additionally, a ΔtoxR mutant becomes nonculturable at a significantly faster rate than the wild-type strain and its phenotype can be complemented by ectopic expression of ToxR (Fig 3B). To further investigate the relationship between ToxR and the culturability of V. cholerae, the CFUs under TPI conditions were determined for wild-type O395, ΔtoxR, ΔrseP and ΔrpoE mutants. As shown in Fig 4, the number of CFU/ml for wild-type, which is able to proteolyze ToxR, was around 104 and the ΔtoxR mutant ranged between 0 and 102. In contrast, the number of CFU/ml for ΔrseP and ΔrpoE mutants that are unable to proteolyze ToxR was approximately 109 under this condition, similar to the number of CFUs recovered when the wild-type strain was grown on LB buffered at pH 7.0 with 100 mM HEPES (Fig 4). Introduction of the ΔtoxR mutation into the ΔrseP and ΔrpoE backgrounds decreased the CFUs under TPI conditions similar to that of the ΔtoxR mutant alone.


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)

Reduction in culturability of V. cholerae depends on the loss of ToxR.CFU/ml of O395 wild-type (WT) and mutant strains in LB pH 7.0 with 100 mM HEPES (Buff), or LB starting pH 9.3 unbuffered (pH 9.3) for 48 hours. The bars represent the mean of at least 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 48h Buff. ***P < 0.0005.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005145.g004: Reduction in culturability of V. cholerae depends on the loss of ToxR.CFU/ml of O395 wild-type (WT) and mutant strains in LB pH 7.0 with 100 mM HEPES (Buff), or LB starting pH 9.3 unbuffered (pH 9.3) for 48 hours. The bars represent the mean of at least 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 48h Buff. ***P < 0.0005.
Mentions: The same conditions that trigger the proteolysis of ToxR induce the loss of culturability of V. cholerae (Fig 1A and 3A). Additionally, a ΔtoxR mutant becomes nonculturable at a significantly faster rate than the wild-type strain and its phenotype can be complemented by ectopic expression of ToxR (Fig 3B). To further investigate the relationship between ToxR and the culturability of V. cholerae, the CFUs under TPI conditions were determined for wild-type O395, ΔtoxR, ΔrseP and ΔrpoE mutants. As shown in Fig 4, the number of CFU/ml for wild-type, which is able to proteolyze ToxR, was around 104 and the ΔtoxR mutant ranged between 0 and 102. In contrast, the number of CFU/ml for ΔrseP and ΔrpoE mutants that are unable to proteolyze ToxR was approximately 109 under this condition, similar to the number of CFUs recovered when the wild-type strain was grown on LB buffered at pH 7.0 with 100 mM HEPES (Fig 4). Introduction of the ΔtoxR mutation into the ΔrseP and ΔrpoE backgrounds decreased the CFUs under TPI conditions similar to that of the ΔtoxR mutant alone.

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