The effects of copper (Cu) on trace metal and antibiotic resistance of Pseudomonas aeruginosa have been investigated. Cu treatments induced resistance not only to this metal but also, surprisingly, to zinc (Zn). Quantitative reverse transcription-PCR (qRT-PCR) revealed that after Cu treatment the transcription of the czcRS two-component system (TCS) operon was enhanced as well as that of the czcCBA operon encoding an efflux pump specific for zinc, cadmium, and cobalt. Cu treatments at the same time caused a decrease in the production of OprD porin, resulting in resistance to the carbapenem antibiotic imipenem. The CzcR regulator was known to repress oprD . However, Cu was still able to decrease the production of OprD and induce imipenem resistance in a czcRS knockout mutant. This strongly suggested that another Cu-dependent regulatory system was acting negatively on oprD expression. TCS regulator genes copR-copS have been shown to be involved in Cu tolerance in P. aeruginosa . qRT-PCR showed that overproduction of the CopR or of the CzcR regulator resulted in increased transcription of the czcC gene as well as in a decrease in oprD gene transcription, either in the wild-type strain or in the czcRS knockout mutant. Overproduction experiments suggest that a metal-dependent mechanism operates at the posttranscriptional level to control the production of the CzcCBA efflux pump. This study shows that CopR is a new negative regulator of OprD porin and that it links Zn, Cu, and imipenem resistances by interacting with the CzcRS TCS.

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.

Zinc (Zn) is a trace element essential for life but can be toxic if present in excess. While cells have import systems to guarantee a vital Zn intracellular concentration, they also rely on export systems to avoid lethal Zn overload. In particular, the opportunistic pathogen Pseudomonas aeruginosa possesses four Zn export systems: CadA, CzcCBA, CzcD, and YiiP. In this work, we compare the importance for bacterial survival of each export system at high Zn concentrations. We show that the P-type ATPase CadA, and the efflux pump CzcCBA are the main efflux systems affecting the bacterium tolerance to Zn. In addition, cadA and czcCBA genes expression kinetics revealed a hierarchical organization and interdependence. In the presence of high Zn concentrations, cadA expression is very rapidly induced (15 min). Our present data show that the fast responsiveness of cadA to Zn excess is due to its transcriptional activator, CadR, which is constitutively present on its promoter and promptly activating cadA gene expression upon Zn binding. Moreover, we showed that CadA is essential for a timely induction of the CzcCBA efflux system. Finally, we observed an induction of cadA and czcCBA efflux systems upon phagocytosis of P. aeruginosa by macrophages, in which a toxic metal boost is discharged into the phagolysosome to intoxicate microbes. Importantly, we demonstrated that the regulatory link between induction of the CzcCBA system and the repression of the OprD porin responsible for carbapenem antibiotic resistance, is maintained in the macrophage environment.

Microbial infection of severe burn wounds is currently a major medical challenge. Of the infections by bacteria able to colonize such injuries, those by Pseudomonas aeruginosa are among the most severe, causing major delays in burn patient recovery or leading to fatal issues. In this study, we investigated the growth properties of several burn wound pathogens in biological fluids secreted from human burn wounds. We found that P. aeruginosa strains were able to proliferate but not those of the other pathogens tested. In addition, burn wound exudates (BWEs) stimulate the expression of virulence factors in P. aeruginosa . The chemical composition analysis of BWEs enabled us to determine the major components of these fluids. These data are essential for the development of an artificial medium mimicking the burn wound environment and for in vitro analysis of the initial step in the development of burn wound infections.

We extend the data-driven method of sparse identification of nonlinear dynamics (SINDy) developed by Brunton et al. , Proc. Natl Acad. Sci. USA 113 (2016) to the case of delay differential equations (DDEs). This is achieved in a bilevel optimization procedure by first applying SINDy for fixed delay and then subsequently optimizing the error of the reconstructed SINDy model over delay times. We test the SINDy-delay method on a noisy short dataset from a toy DDE and show excellent agreement. We then apply the method to experimental data of gene expressions in the bacterium Pseudomonas aeruginosa subject to the influence of zinc. The derived SINDy model suggests that the increase in zinc concentration mainly affects the time delay and not the strengths of the interactions between the different agents controlling the zinc export mechanism.