The Vibrio cholerae Quorum-Sensing Protein VqmA Integrates Cell Density, Environmental, and Host-Derived Cues into the Control of Virulence

Quorum sensing (QS) is a process of chemical communication that bacteria use to orchestrate collective behaviors. QS communication relies on chemical signal molecules called autoinducers. QS regulates virulence in Vibrio cholerae, the causative agent of the disease cholera. Transit into the human small intestine, the site of cholera infection, exposes V. cholerae to the host environment. In this study, we show that the combination of two stimuli encountered in the small intestine, the absence of oxygen and the presence of host-produced bile salts, impinge on V. cholerae QS function and, in turn, pathogenicity. We suggest that possessing a QS system that is responsive to multiple environmental, host, and cell density cues enables V. cholerae to fine-tune its virulence capacity in the human intestine.Quorum sensing is a chemical communication process in which bacteria use the production, release, and detection of signal molecules called autoinducers to orchestrate collective behaviors. The human pathogen Vibrio cholerae requires quorum sensing to infect the small intestine. There, V. cholerae encounters the absence of oxygen and the presence of bile salts. We show that these two stimuli differentially affect quorum-sensing function and, in turn, V. cholerae pathogenicity. First, during anaerobic growth, V. cholerae does not produce the CAI-1 autoinducer, while it continues to produce the DPO autoinducer, suggesting that CAI-1 may encode information specific to the aerobic lifestyle of V. cholerae. Second, the quorum-sensing receptor-transcription factor called VqmA, which detects the DPO autoinducer, also detects the lack of oxygen and the presence of bile salts. Detection occurs via oxygen-, bile salt-, and redox-responsive disulfide bonds that alter VqmA DNA binding activity. We propose that VqmA serves as an information processing hub that integrates quorum-sensing information, redox status, the presence or absence of oxygen, and host cues. In response to the information acquired through this mechanism, V. cholerae appropriately modulates its virulence output

SrrAB negatively influences <i>ahpC</i> and <i>kat</i> during the early exponential growth and fermentative growth phases upon culture in TSB medium.

<p>Panels A-B; The Δ<i>srrAB</i> strain is resistant towards H₂O₂ challenge when cultured aerobically to exponential growth (Panel A) or fermentatively (Panel B). The WT (JMB1100) with pCM28 empty vector (p<i>EV</i>) and the Δ<i>srrAB</i> strain (JMB1467) with pCM28 (p<i>EV</i>) or p<i>srrAB</i> were cultured in TSB aerobically to exponential growth phase (2 doublings) (Panel A) or fermentatively (Panel B). The cells were subsequently challenged with 2.6 mM (Panel A) or 0.22 mM H₂O₂ (Panel B) and growth was monitored aerobically. Panels C; The mRNA transcript abundances corresponding to <i>ahpC</i> and <i>kat</i> are increased in the Δ<i>srrAB</i> strain cultured fermentatively. The abundances of the <i>ahpC</i>, <i>kat</i>, <i>dps</i>, and <i>cydB</i> mRNA transcripts were determined in the WT and Δ<i>srrAB</i> strains cultured fermentatively. Panel D; Catalase (Kat) activity is increased in a Δ<i>srrAB</i> strain cultured fermentatively. Kat activity was assessed in cell-free lysates from the WT and Δ<i>srrAB</i> strains after fermentative culture. Panel E; The abundance of the <i>ahpC</i> transcript is increased in the Δ<i>srrAB</i> strain cultured aerobically to exponential growth. Transcript abundances corresponding to <i>ahpC</i> and <i>dps</i> were quantified in the WT and Δ<i>srrAB</i> strains cultured aerobically to exponential growth phase. The data in Panels C and E were normalized to 16s rRNA transcript levels and are presented as fold-change relative to the WT. Data shown in panels C-E represent the average of biological triplicates with standard deviations shown. Representative growth profiles are presented in Panels A and B and experiments were performed on least three independent occasions. Where indicated, two-tail student t-tests were performed on data and * denotes p< 0.05 and NS denotes not significant.</p

SrrAB positively influences Dps expression and iron chelation or introduction of a Δ<i>perR</i> allele alleviates the deficient survival of a Δ<i>srrAB</i> mutant upon H₂O₂ challenge.

<p>Panel A; The abundance of the <i>dps</i> transcript is lower in the Δ<i>srrAB</i> strain. The abundances of the <i>dps</i> and <i>spa</i> transcripts were determined in the WT (JMB1100) and Δ<i>srrAB</i> (JMB1467) strains from the cDNA libraries described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170283#pone.0170283.g001" target="_blank">Fig 1E</a>. The data were normalized to 16s rRNA transcript levels and are presented as fold-change relative to the WT. Panel B; Pre-incubation of the Δ<i>srrAB</i> and Δ<i>dps</i> strains with a metal chelator alleviates their sensitivity towards H₂O₂ challenge. The WT, Δ<i>srrAB</i>, and Δ<i>dps</i> (JMB2092) strains were cultured aerobically and subsequently incubated in buffer with vehicle control or 1 mM 2,2 dipyrydyl. Cells were then challenged with H₂O₂ and survival was determining colony-forming units (CFU). Panel C; Introduction of a Δ<i>perR</i> allele mitigates the H₂O₂ sensitivity phenotype of the Δ<i>srrAB</i> strain. The WT, Δ<i>srrAB</i>, Δ<i>perR</i> (JMB2151), and Δ<i>perR</i> Δ<i>srrAB</i> (JMB2615) strains were diluted into TSB and challenged with 1.57 mM H₂O₂ at the point of inoculation (indicated by arrow). Panel D; SrrAB and PerR influence <i>dps</i> transcriptional activity independent of one another. The transcriptional activity of <i>dps</i> was assessed in the WT, Δ<i>srrAB</i>, Δ<i>perR</i> and Δ<i>perR</i> Δ<i>srrAB</i> strains containing <i>gfp</i> under the transcriptional control of the <i>dps</i> promoter (pCM11_<i>dps</i>). Representative data are displayed in Panels B and C and experiments were performed on least three independent occasions. Data in Panels A and D represent the average of biological triplicates with standard deviations shown. Two-tail student t-tests were performed on the data in Panel A and * represents statistically significant data with <i>P</i>< 0.05.</p

A Δ<i>srrAB</i> strain incurs increased damage to aconitase when cultured aerobically.

<p>Panel A; The activity of aconitase (AcnA) is decreased in a Δ<i>srrAB</i> mutant and this phenotype is independent of <i>acnA</i> transcription levels. AcnA activity was assessed in the <i>acnA</i>::<i>Tn</i> (JMB 3537; parent), <i>acnA</i>::<i>Tn</i> Δ<i>nfu</i> (JMB 3538), and <i>acnA</i>::<i>Tn</i> Δ<i>srrAB</i> (JMB 4367) strains carrying p<i>acnA</i>, which contains <i>acnA</i> under the transcriptional control of a xylose inducible promoter. Strains were cultured aerobically in the presence (induced) or absence (not induced) of 1% xylose. Panel B; The <i>sufC</i> mRNA transcript accumulates to similar levels in the WT and Δ<i>srrAB</i> strains. The WT (JMB1100) and Δ<i>srrAB</i> (JMB1467) strains were cultured aerobically and challenged with either 10 mM H₂O₂ or vehicle control and the abundance of the <i>sufC</i> mRNA transcript was quantified. Data were normalized to the 16s rRNA transcript levels and are presented as fold-change relative to the WT strain. Panel C; The activity of AcnA is similar in the WT and Δ<i>srrAB</i> strains when cultured anaerobically. AcnA activity was assessed in the <i>acnA</i>::<i>Tn</i> (JMB 3537; parent) and <i>acnA</i>::<i>Tn</i> Δ<i>srrAB</i> (JMB 4367) strains carrying p<i>acnA</i> that were cultured either aerobically or anaerobically. Panel D; A Δ<i>srrAB</i> mutant does not display increased dioxygen damage to AcnA. The <i>acnA</i>::<i>Tn</i> (JMB 3537; parent) and <i>acnA</i>::<i>Tn</i> Δ<i>srrAB</i> (JMB 4367) strains carrying p<i>acnA</i> were cultured anaerobically for 4.5 hours, treated with a protein synthesis inhibitor (100 μg mL^-1 rifampicin) and either exposed to dioxygen or incubated anaerobically for 35 minutes subsequent to determining the activity of AcnA. Data in Panels A-D represent the average of biological triplicates. Where indicated, two-tail student t-tests were performed on data and * denotes p< 0.05 and NS denotes not significant.</p

SrrAB positively influences the transcription of <i>scdA</i>.

<p>Panel A; A <i>S</i>. <i>aureus</i> USA300_LAC Δ<i>scdA</i> mutant is sensitive to H₂O₂ intoxication. The WT (JMB1100), Δ<i>scdA</i> (JMB1254), and Δ<i>kat</i> (JMB2078) strains were cultured aerobically, diluted into fresh medium, and challenged with 1.57 mM H₂O₂ at the point of inoculation. Panel B; The Δ<i>scdA</i> strain is not defective in catalase (Kat) activity. Kat activity was assessed in cell-free lysates from the WT, Δ<i>scdA</i>, Δ<i>perR</i> (JMB2151), and Δ<i>kat</i> strains cultured at a HVR of 10. Panel C; The Δ<i>scdA</i> strain does not have decreased superoxide dismutase (Sod) activity. Sod activity was assessed in cell-free lysates from the WT, Δ<i>scdA</i>, and Δ<i>sodA</i>::<i>Tn</i> (JMB6326) strains cultured at a HVR of 10. Panel D; The abundance of the <i>scdA</i> mRNA transcript is lower in the Δ<i>srrAB</i> strain during post-exponential growth. The mRNA abundance corresponding to <i>scdA</i> was determined using the same cDNA libraries as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170283#pone.0170283.g002" target="_blank">Fig 2A</a>. The data were normalized to 16s rRNA transcript levels and are presented as fold-change relative to the WT strain. Representative growth profiles are presented in Panel A and experiments were performed on least three independent occasions. The data in Panels B-D represent the average of biological triplicates with standard deviations shown.</p

SrrA binds to DNA fragments immediately preceding the annotated transcriptional start sites for <i>srrA</i> and <i>dps</i>.

<p>Panels A and B; Electromobility gel shift assays (EMSAs) demonstrating binding of SrrA to DNAs that correspond to the 150 base pair segments immediately preceding the annotated transcriptional start sites for the <i>srrA</i> (A) and <i>dps</i> (B). EMSAs were performed with SrrA (15–146 ng) and 8 fM of biotin labeled DNA. For each gel, the samples in lane 1 contain 146 ng SrrA with labeled sample DNA and a 125-fold excess of non-labeled (cold) competitor DNA. The samples in lanes 2–5 contain labeled DNA with varying amounts of SrrA protein (15–146 ng). The samples in lane 6 contain labeled DNA, but no SrrA. The samples in lanes 7–9 show that the interaction of SrrA with DNA is specific. The samples in lane 7 contain 146 ng SrrA with <i>rpsC</i> promoter DNA and a 125-fold excess of non-labeled (cold) competitor DNA. The samples in lanes 8 contain <i>rpsC</i> promoter DNA with 146 ng of SrrA. The samples in lane 9 contain <i>rpsC</i> promoter DNA, but no SrrA.</p

Working model for the role of SrrAB in modulating the transcription of genes utilized in H₂O₂ resistance and dioxygen respiration.

<p>SrrAB modulates gene transcription in response to cellular respiratory flux [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170283#pone.0170283.ref036" target="_blank">36</a>]. We propose that increased culture aeration leads to increased respiratory flux during post-exponential growth, which results in altered kinase activity of SrrB and variation in the cellular pool of SrrA~P. An altered SrrA~P pool results in increased expression of genes under the SrrAB regulon that are utilized for H₂O₂ resistance and dioxygen respiration. The resultant physiological changes allow for cellular homeostasis by protecting macromolecules against H₂O₂ toxicity that arise during dioxygen respiration.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

The <i>Staphylococcus aureus</i> SrrAB Regulatory System Modulates Hydrogen Peroxide Resistance Factors, Which Imparts Protection to Aconitase during Aerobic Growth

<div><p>The SrrAB two-component regulatory system (TCRS) positively influences the transcription of genes involved in aerobic respiration in response to changes in respiratory flux. Hydrogen peroxide (H₂O₂) can arise as a byproduct of spontaneous interactions between dioxygen and components of respiratory pathways. H₂O₂ damages cellular factors including protein associated iron-sulfur cluster prosthetic groups. We found that a <i>Staphylococcus aureus</i> strain lacking the SrrAB two-component regulatory system (TCRS) is sensitive to H₂O₂ intoxication. We tested the hypothesis that SrrAB manages the mutually inclusive expression of genes required for aerobic respiration and H₂O₂ resistance. Consistent with our hypothesis, a Δ<i>srrAB</i> strain had decreased transcription of genes encoding for H₂O₂ resistance factors (<i>kat</i>, <i>ahpC</i>, <i>dps</i>). SrrAB was not required for the inducing the transcription of these genes in cells challenged with H₂O₂. Purified SrrA bound to the promoter region for <i>dps</i> suggesting that SrrA directly influences <i>dps</i> transcription. The H₂O₂ sensitivity of the Δ<i>srrAB</i> strain was alleviated by iron chelation or deletion of the gene encoding for the peroxide regulon repressor (PerR). The positive influence of SrrAB upon H₂O₂ metabolism bestowed protection upon the solvent accessible iron-sulfur (FeS) cluster of aconitase from H₂O₂ poisoning. SrrAB also positively influenced transcription of <i>scdA</i> (<i>ytfE</i>), which encodes for a FeS cluster repair protein. Finally, we found that SrrAB positively influences H₂O₂ resistance only during periods of high dioxygen-dependent respiratory activity. SrrAB did not influence H₂O₂ resistance when cellular respiration was diminished as a result of decreased dioxygen availability, and negatively influenced it in the absence of respiration (fermentative growth). We propose a model whereby SrrAB-dependent regulatory patterns facilitate the adaptation of cells to changes in dioxygen concentrations, and thereby aids in the prevention of H₂O₂ intoxication during respiratory growth upon dixoygen.</p></div