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The transcriptional regulator CzcR modulates antibiotic resistance and quorum sensing in Pseudomonas aeruginosa.

Dieppois G, Ducret V, Caille O, Perron K - PLoS ONE (2012)

Bottom Line: This unexpected co-regulation led us to address the role of CzcR in other cellular processes unrelated to the metal response.In agreement with this, the virulence of the czcRS deletion mutant is affected in a C. elegans animal killing assay.All together our data identify CzcR as a novel regulator involved in the control of several key genes for P. aeruginosa virulence processes.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Unit, Department of Botany and Plant Biology, Sciences III, University of Geneva, Switzerland.

ABSTRACT
The opportunistic pathogen Pseudomonas aeruginosa responds to zinc, cadmium and cobalt by way of the CzcRS two-component system. In presence of these metals the regulatory protein CzcR induces the expression of the CzcCBA efflux pump, expelling and thereby inducing resistance to Zn, Cd and Co. Importantly, CzcR co-regulates carbapenem antibiotic resistance by repressing the expression of the OprD porin, the route of entry for these antibiotics. This unexpected co-regulation led us to address the role of CzcR in other cellular processes unrelated to the metal response. We found that CzcR affected the expression of numerous genes directly involved in the virulence of P. aeruginosa even in the absence of the inducible metals. Notably the full expression of quorum sensing 3-oxo-C12-HSL and C4-HSL autoinducer molecules is impaired in the absence of CzcR. In agreement with this, the virulence of the czcRS deletion mutant is affected in a C. elegans animal killing assay. Additionally, chromosome immunoprecipitation experiments allowed us to localize CzcR on the promoter of several regulated genes, suggesting a direct control of target genes such as oprD, phzA1 and lasI. All together our data identify CzcR as a novel regulator involved in the control of several key genes for P. aeruginosa virulence processes.

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CzcR is required for the full virulence of P. aeruginosa.Killing of C. elegans fed with wild-type P. aeruginosa or with the ΔczcRS complemented strains (pMMB66EH: empty vector; pSWT: czcS gene; pRWT: czcR gene). E. coli OP50 was used as the non-pathogenic strain. The percentage of living worms, scored up to 6 days at 24 h-intervals, was plotted using GraphPad Prism 5 software. Data represent the average of three independent experiments. The comparison between the survival curves of WT pMMB66EH and ΔczcRS pMBB66EH, ΔczcRS pMMB66EH and ΔczcRS pRWT was performed by means of two different tests: the Log-rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test. Both tests showed that the curves were significantly different with a P value below 0.0001. The ΔczcRS pMMB66EH and ΔczcRS pSWT survival curves were also compared. This comparison gave a P value of 0.07 with the Log-rank (Mantel-Cox) test and 0.03 with the Gehan-Breslow-Wilcoxon test, indicating that these two curves are not significantly different.
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pone-0038148-g004: CzcR is required for the full virulence of P. aeruginosa.Killing of C. elegans fed with wild-type P. aeruginosa or with the ΔczcRS complemented strains (pMMB66EH: empty vector; pSWT: czcS gene; pRWT: czcR gene). E. coli OP50 was used as the non-pathogenic strain. The percentage of living worms, scored up to 6 days at 24 h-intervals, was plotted using GraphPad Prism 5 software. Data represent the average of three independent experiments. The comparison between the survival curves of WT pMMB66EH and ΔczcRS pMBB66EH, ΔczcRS pMMB66EH and ΔczcRS pRWT was performed by means of two different tests: the Log-rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test. Both tests showed that the curves were significantly different with a P value below 0.0001. The ΔczcRS pMMB66EH and ΔczcRS pSWT survival curves were also compared. This comparison gave a P value of 0.07 with the Log-rank (Mantel-Cox) test and 0.03 with the Gehan-Breslow-Wilcoxon test, indicating that these two curves are not significantly different.

Mentions: Since CzcR is involved in the expression of QS-regulated virulence factors, we wondered whether this regulator is important for the pathogenicity of P. aeruginosa. To this aim, we used the nematode Caenorhabditis elegans as a model of infection [40]. P. aeruginosa killing of the worm C. elegans is a multifactorial process involving both toxin-mediated mechanisms and a slower infectious process requiring bacterial proliferation within the gut [41]. The P. aeruginosa PAO1 strain used in this study is not cytotoxic and only slow killing was detected. In our assay, an infection with the wild type strain killed 50% of the population within circa 3 days of contact with bacteria and all worms were dead after 4 days (Fig. 4). In contrast, the virulence of the ΔczcRS mutant was significantly reduced since 50% of the worms were still alive after 5 days of contact. The virulence of the ΔczcRS mutant strain complemented with czcR on the pRWT plasmid was partially restored since 50% of the worms were dead after 4 days and less than 20% of the worm population was alive after 5 days, while no complementation was observed with the czcS gene (Fig. 4). The effect is statistically relevant as determined using the Log-rank and Gehan-Breslow-Wilcoxon tests (P value<0.0001). Deletion of this TCS did not completely abolish virulence. The delay we observed might suggest that the ΔczcR only partially affects the global virulence of P. aeruginosa. The observed result cannot account for a decrease of the bacterial number since no growth defect in plate and in liquid medium was observed in the ΔczcRS complemented strains (data not shown). A control with the non-virulent reference strain E. coli OP50 (non-pathogenic for C. elegans), showed that worm viability was stable after 5 days of contact with these bacteria (Fig. 4). Altogether these results strongly suggest that CzcR, by acting positively on quorum sensing gene expression, plays an important role in the pathogenicity of P. aeruginosa.


The transcriptional regulator CzcR modulates antibiotic resistance and quorum sensing in Pseudomonas aeruginosa.

Dieppois G, Ducret V, Caille O, Perron K - PLoS ONE (2012)

CzcR is required for the full virulence of P. aeruginosa.Killing of C. elegans fed with wild-type P. aeruginosa or with the ΔczcRS complemented strains (pMMB66EH: empty vector; pSWT: czcS gene; pRWT: czcR gene). E. coli OP50 was used as the non-pathogenic strain. The percentage of living worms, scored up to 6 days at 24 h-intervals, was plotted using GraphPad Prism 5 software. Data represent the average of three independent experiments. The comparison between the survival curves of WT pMMB66EH and ΔczcRS pMBB66EH, ΔczcRS pMMB66EH and ΔczcRS pRWT was performed by means of two different tests: the Log-rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test. Both tests showed that the curves were significantly different with a P value below 0.0001. The ΔczcRS pMMB66EH and ΔczcRS pSWT survival curves were also compared. This comparison gave a P value of 0.07 with the Log-rank (Mantel-Cox) test and 0.03 with the Gehan-Breslow-Wilcoxon test, indicating that these two curves are not significantly different.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3362554&req=5

pone-0038148-g004: CzcR is required for the full virulence of P. aeruginosa.Killing of C. elegans fed with wild-type P. aeruginosa or with the ΔczcRS complemented strains (pMMB66EH: empty vector; pSWT: czcS gene; pRWT: czcR gene). E. coli OP50 was used as the non-pathogenic strain. The percentage of living worms, scored up to 6 days at 24 h-intervals, was plotted using GraphPad Prism 5 software. Data represent the average of three independent experiments. The comparison between the survival curves of WT pMMB66EH and ΔczcRS pMBB66EH, ΔczcRS pMMB66EH and ΔczcRS pRWT was performed by means of two different tests: the Log-rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test. Both tests showed that the curves were significantly different with a P value below 0.0001. The ΔczcRS pMMB66EH and ΔczcRS pSWT survival curves were also compared. This comparison gave a P value of 0.07 with the Log-rank (Mantel-Cox) test and 0.03 with the Gehan-Breslow-Wilcoxon test, indicating that these two curves are not significantly different.
Mentions: Since CzcR is involved in the expression of QS-regulated virulence factors, we wondered whether this regulator is important for the pathogenicity of P. aeruginosa. To this aim, we used the nematode Caenorhabditis elegans as a model of infection [40]. P. aeruginosa killing of the worm C. elegans is a multifactorial process involving both toxin-mediated mechanisms and a slower infectious process requiring bacterial proliferation within the gut [41]. The P. aeruginosa PAO1 strain used in this study is not cytotoxic and only slow killing was detected. In our assay, an infection with the wild type strain killed 50% of the population within circa 3 days of contact with bacteria and all worms were dead after 4 days (Fig. 4). In contrast, the virulence of the ΔczcRS mutant was significantly reduced since 50% of the worms were still alive after 5 days of contact. The virulence of the ΔczcRS mutant strain complemented with czcR on the pRWT plasmid was partially restored since 50% of the worms were dead after 4 days and less than 20% of the worm population was alive after 5 days, while no complementation was observed with the czcS gene (Fig. 4). The effect is statistically relevant as determined using the Log-rank and Gehan-Breslow-Wilcoxon tests (P value<0.0001). Deletion of this TCS did not completely abolish virulence. The delay we observed might suggest that the ΔczcR only partially affects the global virulence of P. aeruginosa. The observed result cannot account for a decrease of the bacterial number since no growth defect in plate and in liquid medium was observed in the ΔczcRS complemented strains (data not shown). A control with the non-virulent reference strain E. coli OP50 (non-pathogenic for C. elegans), showed that worm viability was stable after 5 days of contact with these bacteria (Fig. 4). Altogether these results strongly suggest that CzcR, by acting positively on quorum sensing gene expression, plays an important role in the pathogenicity of P. aeruginosa.

Bottom Line: This unexpected co-regulation led us to address the role of CzcR in other cellular processes unrelated to the metal response.In agreement with this, the virulence of the czcRS deletion mutant is affected in a C. elegans animal killing assay.All together our data identify CzcR as a novel regulator involved in the control of several key genes for P. aeruginosa virulence processes.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Unit, Department of Botany and Plant Biology, Sciences III, University of Geneva, Switzerland.

ABSTRACT
The opportunistic pathogen Pseudomonas aeruginosa responds to zinc, cadmium and cobalt by way of the CzcRS two-component system. In presence of these metals the regulatory protein CzcR induces the expression of the CzcCBA efflux pump, expelling and thereby inducing resistance to Zn, Cd and Co. Importantly, CzcR co-regulates carbapenem antibiotic resistance by repressing the expression of the OprD porin, the route of entry for these antibiotics. This unexpected co-regulation led us to address the role of CzcR in other cellular processes unrelated to the metal response. We found that CzcR affected the expression of numerous genes directly involved in the virulence of P. aeruginosa even in the absence of the inducible metals. Notably the full expression of quorum sensing 3-oxo-C12-HSL and C4-HSL autoinducer molecules is impaired in the absence of CzcR. In agreement with this, the virulence of the czcRS deletion mutant is affected in a C. elegans animal killing assay. Additionally, chromosome immunoprecipitation experiments allowed us to localize CzcR on the promoter of several regulated genes, suggesting a direct control of target genes such as oprD, phzA1 and lasI. All together our data identify CzcR as a novel regulator involved in the control of several key genes for P. aeruginosa virulence processes.

Show MeSH
Related in: MedlinePlus