The CzcCBA Efflux System Requires the CadA P-Type ATPase for Timely Expression Upon Zinc Excess in Pseudomonas aeruginosa

Abstract

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 (<1 min), while czcCBA expression occurs subsequently (>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.

Global analysis of the zinc homeostasis network in Pseudomonas aeruginosa and its gene expression dynamics

“…During this time scale, the amount of the CadA P-type ATPase strongly decreased. This is consistent with our previous observations on CadA acting like a sentinel and patrolling Zn excess (28). This second period was also marked by the increase of the CzcD protein.…”

“…This second period was also marked by the increase of the CzcD protein. This very late induction took place after the CzcCBA efflux, that may explain why this CDF was not essential for Zn resistance (28). Overall, these results reveal a clear dynamics for the export systems, while regulation of Zn import systems appears as slow-acting.…”

“…In P. aeruginosa , CadA was first described as a system involved in Cd resistance (27). Similar to the E. coli ZntA, this protein has recently been shown to be essential for Zn resistance (28). CadA is regulated by CadR that belongs to the MerR family of response regulators.…”

“…CadR is constitutively expressed and permanently located on the cadA promoter. The activity of this regulator is directly linked to its ability to bind Zn since it plays the dual role of repressor and activator of cadA transcription, depending on whether it is in conditions of Zn limitation or excess respectively (28).…”

“…Our lab previously highlighted a dynamic in the expression of Zn export systems in P. aeruginosa . Indeed, we had shown that the P-type ATPase CadA was not only the first bulwark against a boost in Zn, but also facilitated the induction of the major export system, the CzcCBA efflux pump (28). These results support the hypothesis that the different systems involved in Zn resistance are not redundant, but follow a precise strategic plan, guaranteeing a rapid adaptation of the cell to variations in Zn concentrations.…”