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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

Sequence conservation, structural features, and locations of suppressor mutations in V. cholerae CsrA. (A) An alignment of the V. cholerae CsrA protein with CsrA from additional Gram-negative species, E. coli, Salmonella enterica serovar Typhimurium, and Pseudomonas aeruginosa, showing a high degree of conservation among these species. The positions of the predicted β sheets (β1 to β5) and α helix (α1) in the CsrA protein are indicated above the alignment. The colored arrows point to the positions of the CsrA suppressor mutations relative to the secondary structural elements. The angled black arrow shows the translational start of the csrA open reading frame. The mutated residues in the suppressor strains are highlighted in the V. cholerae CsrA protein sequence. The thick arrows above the alignment show the positions of the transposon insertions within the published E. coli csrA::kan (open arrow) (29) and V. cholerae csrA::Tn5 (closed arrow) (25) mutant strains. (B) List of the CsrA point mutations identified in the NvarAL and the NtoxR, varAL suppressor strains (color coded to match the arrows in panel A), their phenotypes with respect to OMP and ToxR production, and their frequency of isolation. The ARM759L strains were not tested for their NRES response, since they are toxR mutants and do not make OmpU. The NvarAL suppressor mutants are in the V. cholerae N16961 ΔvarA::cam background, and the ARM759L suppressor mutants are in the N16961 ΔtoxR::kan ΔvarA::cam background.
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fig4: Sequence conservation, structural features, and locations of suppressor mutations in V. cholerae CsrA. (A) An alignment of the V. cholerae CsrA protein with CsrA from additional Gram-negative species, E. coli, Salmonella enterica serovar Typhimurium, and Pseudomonas aeruginosa, showing a high degree of conservation among these species. The positions of the predicted β sheets (β1 to β5) and α helix (α1) in the CsrA protein are indicated above the alignment. The colored arrows point to the positions of the CsrA suppressor mutations relative to the secondary structural elements. The angled black arrow shows the translational start of the csrA open reading frame. The mutated residues in the suppressor strains are highlighted in the V. cholerae CsrA protein sequence. The thick arrows above the alignment show the positions of the transposon insertions within the published E. coli csrA::kan (open arrow) (29) and V. cholerae csrA::Tn5 (closed arrow) (25) mutant strains. (B) List of the CsrA point mutations identified in the NvarAL and the NtoxR, varAL suppressor strains (color coded to match the arrows in panel A), their phenotypes with respect to OMP and ToxR production, and their frequency of isolation. The ARM759L strains were not tested for their NRES response, since they are toxR mutants and do not make OmpU. The NvarAL suppressor mutants are in the V. cholerae N16961 ΔvarA::cam background, and the ARM759L suppressor mutants are in the N16961 ΔtoxR::kan ΔvarA::cam background.

Mentions: Since loss of VarA is predicted to result in overaccumulation of active CsrA due to low levels of the CsrA-sequestering sRNAs (Fig. 1), we reasoned that suppressor mutations could arise in the csrA gene itself. The csrA gene from the NvarAL strain was sequenced and was found to contain a point mutation that replaces the arginine residue at amino acid position 6 with a histidine (R6H). Several independently derived NvarAL isolates were analyzed to determine whether they also carried suppressor mutations in csrA and whether there was variation in the type of csrA point mutation that could arise in the absence of functional VarA. All of the NvarAL isolates tested carried point mutations affecting csrA. In contrast, none of the small colony phenotype NvarAS strains had mutations in csrA. Most of the NvarAL strain mutations were in the csrA coding region, but several isolates carried mutations in the predicted Shine-Dalgarno (SD) sequence upstream of the csrA translational start (Fig. 4). All coding region mutations resulted in a single amino acid substitution in CsrA (Fig. 4). The OMP profiles of several NvarAL suppressor mutants were analyzed. Many of these mutants did not produce OmpU in response to the NRES mix (Fig. 5A). Rather, they produced OmpT at high levels in minimal medium both with and without NRES supplementation. Thus, these mutants were not stimulated to switch OMPs in the presence of NRES. The amino acid substitutions in this group of mutants generally clustered in the N-terminal half of CsrA (R6H, R6L, T11P, I14T, and T19P) (Fig. 4). Another class of point mutants produced both OmpT and OmpU in T medium alone, and this pattern did not change with the addition of NRES, suggesting defects in OMP regulation in response to environmental cues (Fig. 5A). Several mutants of this class had amino acid substitutions localizing to a region within the C-terminal half of the CsrA protein (A36S and P37L) (Fig. 4). Some of the suppressor strains had mutations in the putative SD sequence (Fig. 4). These mutants were phenotypically similar to the class of mutants exhibiting no OmpU production in response to NRES (Fig. 5A; also data not shown).


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

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

Sequence conservation, structural features, and locations of suppressor mutations in V. cholerae CsrA. (A) An alignment of the V. cholerae CsrA protein with CsrA from additional Gram-negative species, E. coli, Salmonella enterica serovar Typhimurium, and Pseudomonas aeruginosa, showing a high degree of conservation among these species. The positions of the predicted β sheets (β1 to β5) and α helix (α1) in the CsrA protein are indicated above the alignment. The colored arrows point to the positions of the CsrA suppressor mutations relative to the secondary structural elements. The angled black arrow shows the translational start of the csrA open reading frame. The mutated residues in the suppressor strains are highlighted in the V. cholerae CsrA protein sequence. The thick arrows above the alignment show the positions of the transposon insertions within the published E. coli csrA::kan (open arrow) (29) and V. cholerae csrA::Tn5 (closed arrow) (25) mutant strains. (B) List of the CsrA point mutations identified in the NvarAL and the NtoxR, varAL suppressor strains (color coded to match the arrows in panel A), their phenotypes with respect to OMP and ToxR production, and their frequency of isolation. The ARM759L strains were not tested for their NRES response, since they are toxR mutants and do not make OmpU. The NvarAL suppressor mutants are in the V. cholerae N16961 ΔvarA::cam background, and the ARM759L suppressor mutants are in the N16961 ΔtoxR::kan ΔvarA::cam background.
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fig4: Sequence conservation, structural features, and locations of suppressor mutations in V. cholerae CsrA. (A) An alignment of the V. cholerae CsrA protein with CsrA from additional Gram-negative species, E. coli, Salmonella enterica serovar Typhimurium, and Pseudomonas aeruginosa, showing a high degree of conservation among these species. The positions of the predicted β sheets (β1 to β5) and α helix (α1) in the CsrA protein are indicated above the alignment. The colored arrows point to the positions of the CsrA suppressor mutations relative to the secondary structural elements. The angled black arrow shows the translational start of the csrA open reading frame. The mutated residues in the suppressor strains are highlighted in the V. cholerae CsrA protein sequence. The thick arrows above the alignment show the positions of the transposon insertions within the published E. coli csrA::kan (open arrow) (29) and V. cholerae csrA::Tn5 (closed arrow) (25) mutant strains. (B) List of the CsrA point mutations identified in the NvarAL and the NtoxR, varAL suppressor strains (color coded to match the arrows in panel A), their phenotypes with respect to OMP and ToxR production, and their frequency of isolation. The ARM759L strains were not tested for their NRES response, since they are toxR mutants and do not make OmpU. The NvarAL suppressor mutants are in the V. cholerae N16961 ΔvarA::cam background, and the ARM759L suppressor mutants are in the N16961 ΔtoxR::kan ΔvarA::cam background.
Mentions: Since loss of VarA is predicted to result in overaccumulation of active CsrA due to low levels of the CsrA-sequestering sRNAs (Fig. 1), we reasoned that suppressor mutations could arise in the csrA gene itself. The csrA gene from the NvarAL strain was sequenced and was found to contain a point mutation that replaces the arginine residue at amino acid position 6 with a histidine (R6H). Several independently derived NvarAL isolates were analyzed to determine whether they also carried suppressor mutations in csrA and whether there was variation in the type of csrA point mutation that could arise in the absence of functional VarA. All of the NvarAL isolates tested carried point mutations affecting csrA. In contrast, none of the small colony phenotype NvarAS strains had mutations in csrA. Most of the NvarAL strain mutations were in the csrA coding region, but several isolates carried mutations in the predicted Shine-Dalgarno (SD) sequence upstream of the csrA translational start (Fig. 4). All coding region mutations resulted in a single amino acid substitution in CsrA (Fig. 4). The OMP profiles of several NvarAL suppressor mutants were analyzed. Many of these mutants did not produce OmpU in response to the NRES mix (Fig. 5A). Rather, they produced OmpT at high levels in minimal medium both with and without NRES supplementation. Thus, these mutants were not stimulated to switch OMPs in the presence of NRES. The amino acid substitutions in this group of mutants generally clustered in the N-terminal half of CsrA (R6H, R6L, T11P, I14T, and T19P) (Fig. 4). Another class of point mutants produced both OmpT and OmpU in T medium alone, and this pattern did not change with the addition of NRES, suggesting defects in OMP regulation in response to environmental cues (Fig. 5A). Several mutants of this class had amino acid substitutions localizing to a region within the C-terminal half of the CsrA protein (A36S and P37L) (Fig. 4). Some of the suppressor strains had mutations in the putative SD sequence (Fig. 4). These mutants were phenotypically similar to the class of mutants exhibiting no OmpU production in response to NRES (Fig. 5A; also data not shown).

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