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Vibrio cholerae CsrA Regulates ToxR Levels in Response to Amino Acids and Is Essential for Virulence.

Mey AR, Butz HA, Payne SM - MBio (2015)

Bottom Line: Conversely, specific amino acid substitutions in CsrA were associated with defects in ToxR production in response to NRES.Unlike previously described effects of CsrA on virulence gene regulation, the effects of CsrA on ToxR were not mediated through quorum sensing and HapR.By linking environmental sensing to the ToxR regulon, CsrA effectively acts as a switch that controls pathogenesis in response to specific signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA armey@austin.utexas.edu.

No MeSH data available.


Related in: MedlinePlus

An Arg6His substitution in CsrA is sufficient to abolish the response to NRES, even when the Var system is functional. The wild-type strain N16961, the NcsrA.R6H mutant, the vector control strain NcsrA.R6H/pCC1, and the complemented strain NcsrA.R6H/pFCsrA were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 12.5 mM NRES mix. Cells were harvested in mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (A) or immunoblotted using polyclonal anti-ToxR antisera (B).
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fig7: An Arg6His substitution in CsrA is sufficient to abolish the response to NRES, even when the Var system is functional. The wild-type strain N16961, the NcsrA.R6H mutant, the vector control strain NcsrA.R6H/pCC1, and the complemented strain NcsrA.R6H/pFCsrA were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 12.5 mM NRES mix. Cells were harvested in mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (A) or immunoblotted using polyclonal anti-ToxR antisera (B).

Mentions: To test directly the role of CsrA in the regulation of ToxR protein levels, attempts were made to delete the entire csrA gene in strain N16961; however, none of these attempts was successful, suggesting that csrA is essential in V. cholerae. Instead, we tested whether a point mutation in CsrA alone is sufficient to abolish the ToxR/OMP response to the NRES mix. The csrA R6H point mutation, which was one of the most frequently isolated mutations, was constructed in the wild-type genetic background. The NcsrA.R6H strain exhibited wild-type growth (data not shown) but produced only OmpT, and no OmpU, in the presence of the NRES mix (Fig. 7A), indicating that the csrA point mutation, and not the varA deletion, caused the failure to respond to the NRES mix in the original NvarAL-1 strain. As in the NvarAL-1 strain, the ToxR levels in the NcsrA.R6H point mutant did not increase in the presence of NRES (Fig. 7B). This shows that changing just a single amino acid in CsrA profoundly affects the ability of V. cholerae to respond to amino acids. To verify that the NcsrA.R6H point mutant did not contain any additional suppressor mutations that might be responsible for the observed phenotype, the point mutant was complemented. A csrA plasmid clone was created in a single-copy-number vector, pCC1. When pFCsrA was introduced into NcsrA.R6H, both the OMP profile and the ToxR level were restored to wild type (Fig. 7A and B). This shows that the R6H point mutation in CsrA is solely responsible for the defect in ToxR/OmpU production. Expression of csrA from the pFCsrA plasmid did not interfere with regulation of the OMPs in the wild-type strain (see Fig. S2 in the supplemental material), most likely due to tight regulation of free CsrA levels by the VarS/A-CsrB/C/D system.


Vibrio cholerae CsrA Regulates ToxR Levels in Response to Amino Acids and Is Essential for Virulence.

Mey AR, Butz HA, Payne SM - MBio (2015)

An Arg6His substitution in CsrA is sufficient to abolish the response to NRES, even when the Var system is functional. The wild-type strain N16961, the NcsrA.R6H mutant, the vector control strain NcsrA.R6H/pCC1, and the complemented strain NcsrA.R6H/pFCsrA were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 12.5 mM NRES mix. Cells were harvested in mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (A) or immunoblotted using polyclonal anti-ToxR antisera (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526715&req=5

fig7: An Arg6His substitution in CsrA is sufficient to abolish the response to NRES, even when the Var system is functional. The wild-type strain N16961, the NcsrA.R6H mutant, the vector control strain NcsrA.R6H/pCC1, and the complemented strain NcsrA.R6H/pFCsrA were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 12.5 mM NRES mix. Cells were harvested in mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (A) or immunoblotted using polyclonal anti-ToxR antisera (B).
Mentions: To test directly the role of CsrA in the regulation of ToxR protein levels, attempts were made to delete the entire csrA gene in strain N16961; however, none of these attempts was successful, suggesting that csrA is essential in V. cholerae. Instead, we tested whether a point mutation in CsrA alone is sufficient to abolish the ToxR/OMP response to the NRES mix. The csrA R6H point mutation, which was one of the most frequently isolated mutations, was constructed in the wild-type genetic background. The NcsrA.R6H strain exhibited wild-type growth (data not shown) but produced only OmpT, and no OmpU, in the presence of the NRES mix (Fig. 7A), indicating that the csrA point mutation, and not the varA deletion, caused the failure to respond to the NRES mix in the original NvarAL-1 strain. As in the NvarAL-1 strain, the ToxR levels in the NcsrA.R6H point mutant did not increase in the presence of NRES (Fig. 7B). This shows that changing just a single amino acid in CsrA profoundly affects the ability of V. cholerae to respond to amino acids. To verify that the NcsrA.R6H point mutant did not contain any additional suppressor mutations that might be responsible for the observed phenotype, the point mutant was complemented. A csrA plasmid clone was created in a single-copy-number vector, pCC1. When pFCsrA was introduced into NcsrA.R6H, both the OMP profile and the ToxR level were restored to wild type (Fig. 7A and B). This shows that the R6H point mutation in CsrA is solely responsible for the defect in ToxR/OmpU production. Expression of csrA from the pFCsrA plasmid did not interfere with regulation of the OMPs in the wild-type strain (see Fig. S2 in the supplemental material), most likely due to tight regulation of free CsrA levels by the VarS/A-CsrB/C/D system.

Bottom Line: Conversely, specific amino acid substitutions in CsrA were associated with defects in ToxR production in response to NRES.Unlike previously described effects of CsrA on virulence gene regulation, the effects of CsrA on ToxR were not mediated through quorum sensing and HapR.By linking environmental sensing to the ToxR regulon, CsrA effectively acts as a switch that controls pathogenesis in response to specific signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA armey@austin.utexas.edu.

No MeSH data available.


Related in: MedlinePlus