The Transcriptional Regulator CzcR Modulates Antibiotic Resistance and Quorum Sensing in Pseudomonas aeruginosa

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

Primers used for qRT-PCR.

<p>Primers used for qRT-PCR.</p

CzcR stimulates the expression of QS genes.

<p>Autoinducer production and expression of QS genes in the wt and the Δ<i>czcRS</i> complemented strains. pMMB66EH: empty vector; pSWT: <i>czcS</i> gene; pRWT: <i>czcR</i> gene. A) Quantification of AHL in the supernatant of LB culture using the beta-galactosidase reporter assay. Error bars represent the standard deviations of three independent quantifications. B) Semi-quantitative RT-PCR analysis of the <i>las</i> and <i>rhl</i> QS transcripts for the wt and the Δ<i>czcRS</i> complemented strains. Negative controls lacked reverse transcriptase (RT-).</p

CzcR represses pyocyanin biosynthesis.

<p>A) Pyocyanin production on KingA agar plate in the wt and the Δ<i>czcRS</i> complemented strains. pMMB66EH: empty vector; pSWT: <i>czcS</i> gene; pRWT: <i>czcR</i> gene. B) Quantification of pyocyanin produced in KingA medium by the wt and the Δ<i>czcRS</i> complemented strains. The experiment was performed three times and standard deviations (errors bars) are indicated. C) Schematic representation for the synthesis of pyocyanin, according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038148#pone.0038148-Mavrodi1" target="_blank">[29]</a>. Genes involved in the different steps are boxed. D) Transcription of the wt and the Δ<i>czcRS</i> complemented strains analyzed by semi-quantitative RT-PCR. Negative controls lacked reverse transcriptase (RT-).</p

Cellular pharmacokinetics and intracellular activity of the bacterial fatty acid synthesis inhibitor, afabicin desphosphono against different resistance phenotypes of Staphylococcus aureus in models of cultured phagocytic cells.

Antibiotics with new modes of action that are active against intracellular forms of Staphylococcus aureus are sorely needed to fight recalcitrant infections caused by this bacterium. Afabicin desphosphono (Debio 1452, the active form of afabicin [Debio 1450]) is an inhibitor of FabI enoyl-Acyl carrier protein reductase and has specific and extremely potent activity against Staphylococci, including strains resistant to current antistaphylococcal agents. Using mouse J774 macrophages and human THP-1 monocytes, we showed that afabicin desphosphono: (i) accumulates rapidly in cells, reaching stable cellular-to-extracellular concentration ratios of about 30; (ii) is recovered entirely and free in the cell-soluble fraction (no evidence of stable association with proteins or other macromolecules). Afabicin desphosphono caused a maximum cfu decrease of about 2.5 log after incubation in broth for 30 h, including against strains resistant to vancomycin, daptomycin, and/or linezolid. Using a pharmacodynamic model of infected THP-1 monocytes (30 h of incubation post-phagocytosis), we showed that afabicin desphosphono is bacteriostatic (maximum cfu decrease: 0.56 to 0.73 log) towards all strains tested, a behaviour shared with the comparators (vancomycin, daptomycin, and linezolid) when tested against susceptible strains. We conclude that afabicin desphosphono has a similar potential as vancomycin, daptomycin or linezolid to control the intracellular growth and survival of phagocytized S. aureus and remains fully active against strains resistant to these comparators

CzcR is required for virulence factor production and biofilm formation.

<p>Rhamnolipids, elastase production and biofilm formation in the wt and the Δ<i>czcRS</i> complemented strains. pMMB66EH: empty vector; pSWT: <i>czcS</i> gene; pRWT: <i>czcR</i> gene A) Rhamnolipid production on SW blue plate assay. The blue halo is due to the presence of rhamnolipids produced by the colony. Diameters of these halos are indicated below the figure. B) Elastase production determined by the Elastine-Congo Red assay. The determination was performed three times independently and standard deviations (errors bars) are indicated. C) Biofilm formation in M9 minimal medium on polypropylene tubes after 8 h of static growth. Biofilms were stained with cristal violet and quantified at OD₅₉₀ after solubilization in acetic acid. Quantification was performed in three independent experiments and standard deviations (errors bars) are indicated.</p

CzcR is associated to the <i>oprD</i>, <i>phzA1</i> and <i>czcR</i> promoters.

<p>A) Western blot analysis of the wild-type and the CzcR-HA strain carrying the HA-tagged version integrated into the chromosome. Total proteins from the wild-type or the CzcR-HA strain grown in the presence or absence of 0.5 mM ZnCl₂ in LB medium were analyzed by western blot. Antibodies used for the analysis are indicated in the left of the panel. B) Chromosome immunoprecipitation performed on the CzcR-HA strain using anti-HA antibody. Experiment was performed on culture grown in the absence or in the presence of 0.5 mM ZnCl₂, as indicated. Results represent the average of 3 independent experiments and standard deviations are indicated.</p

CzcR is required for the full virulence of <i>P. aeruginosa</i>.

<p>Killing of <i>C. elegans</i> fed with wild-type <i>P. aeruginosa</i> or with the Δ<i>czcRS</i> complemented strains (pMMB66EH: empty vector; pSWT: <i>czcS</i> gene; pRWT: <i>czcR</i> gene). <i>E. coli</i> 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 <i>ΔczcRS</i> pMBB66EH, <i>ΔczcRS</i> pMMB66EH and <i>ΔczcRS</i> 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 <i>ΔczcRS</i> pMMB66EH and <i>ΔczcRS</i> 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.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p