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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

Viability and morphology of V. cholerae after 48 hours at alkaline pH.Fluorescent (F) and differential interference contrast (DIC) images of O395 wild-type grown in LB starting pH 7.0 unbuffered overnight and heat killed (O/N HK), LB starting pH 7.0 unbuffered overnight (O/N) as controls, 48 hours in LB buffered to pH 7.0 with 100 mM HEPES (48h Buff), and 48 hours in LB pH 9.3 unbuffered (48h pH 9.3).
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pgen.1005145.g005: Viability and morphology of V. cholerae after 48 hours at alkaline pH.Fluorescent (F) and differential interference contrast (DIC) images of O395 wild-type grown in LB starting pH 7.0 unbuffered overnight and heat killed (O/N HK), LB starting pH 7.0 unbuffered overnight (O/N) as controls, 48 hours in LB buffered to pH 7.0 with 100 mM HEPES (48h Buff), and 48 hours in LB pH 9.3 unbuffered (48h pH 9.3).

Mentions: To determine whether the decreased culturability of the wild-type and some of the mutants under TPI conditions is due to their entry into a dormant state and not cell death, the viability of the cells in the cultures in Fig 4 were examined using the LIVE/DEAD BacLight Bacterial Viability and Counting Kit. This kit allows for the discernment between viable cells (green) and dead cells (red) using fluorescent microscopy. The entry of V. cholerae into a dormant state is associated with loss of culturability (Fig 4), maintenance of viability (green cells), and a change in its morphology from elongated rods to round, coccoid-shaped cells [53,54]. Dead cells (O/N HK) can be seen as red under fluorescence (F), whereas cells from an overnight culture (O/N) are green and elongated (F and DIC) (Fig 5). We found that when grown in LB pH 7.0 buffered with 100 mM HEPES (48h Buff) the wild-type shows a morphology and viability similar to O/N (Fig 5), indicative of a culturable state. Under TPI conditions (48h pH 9.3), the cells remain viable and alive (green) and their morphology changes to a coccoid form, indicative of entry into a dormant state (Fig 5).


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)

Viability and morphology of V. cholerae after 48 hours at alkaline pH.Fluorescent (F) and differential interference contrast (DIC) images of O395 wild-type grown in LB starting pH 7.0 unbuffered overnight and heat killed (O/N HK), LB starting pH 7.0 unbuffered overnight (O/N) as controls, 48 hours in LB buffered to pH 7.0 with 100 mM HEPES (48h Buff), and 48 hours in LB pH 9.3 unbuffered (48h pH 9.3).
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005145.g005: Viability and morphology of V. cholerae after 48 hours at alkaline pH.Fluorescent (F) and differential interference contrast (DIC) images of O395 wild-type grown in LB starting pH 7.0 unbuffered overnight and heat killed (O/N HK), LB starting pH 7.0 unbuffered overnight (O/N) as controls, 48 hours in LB buffered to pH 7.0 with 100 mM HEPES (48h Buff), and 48 hours in LB pH 9.3 unbuffered (48h pH 9.3).
Mentions: To determine whether the decreased culturability of the wild-type and some of the mutants under TPI conditions is due to their entry into a dormant state and not cell death, the viability of the cells in the cultures in Fig 4 were examined using the LIVE/DEAD BacLight Bacterial Viability and Counting Kit. This kit allows for the discernment between viable cells (green) and dead cells (red) using fluorescent microscopy. The entry of V. cholerae into a dormant state is associated with loss of culturability (Fig 4), maintenance of viability (green cells), and a change in its morphology from elongated rods to round, coccoid-shaped cells [53,54]. Dead cells (O/N HK) can be seen as red under fluorescence (F), whereas cells from an overnight culture (O/N) are green and elongated (F and DIC) (Fig 5). We found that when grown in LB pH 7.0 buffered with 100 mM HEPES (48h Buff) the wild-type shows a morphology and viability similar to O/N (Fig 5), indicative of a culturable state. Under TPI conditions (48h pH 9.3), the cells remain viable and alive (green) and their morphology changes to a coccoid form, indicative of entry into a dormant state (Fig 5).

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