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.
Highlights
Living cells require trace-metals, such as Zn, Fe, Cu, Co, Mg, Mo, and Ni as co-factors for enzymatic reactions, protein folding, and regulatory actions (Andreini et al, 2008)
The P-Type ATPase CadA Affects Zn Resistance In P. aeruginosa, four systems are known to participate in zinc export (Gonzalez et al, 2019): the CzcD and YiiP cation diffusion facilitators (CDFs), the CzcCBA Heavy metal Resistance Nodulation Division (HmRND) efflux pump and the CadA P-type ATPase
We focused on the export systems involved in resistance to Zn and we found that the two major systems are the CadA P-type ATPase and the CzcCBA efflux pump
Summary
Living cells require trace-metals, such as Zn, Fe, Cu, Co, Mg, Mo, and Ni as co-factors for enzymatic reactions, protein folding, and regulatory actions (Andreini et al, 2008). Bacteria lack of storage compartments they have evolved several metallostasis mechanisms allowing them to thrive under conditions of metal limitation or excess (Chandrangsu et al, 2017). Zn is sequestered by the host cells by a defense strategy called nutritional immunity, rendering the metal poorly accessible to bacterial pathogens (Kehl-Fie and Skaar, 2010). During this process, Zn is chelated by various proteins such as calprotectin secreted at the site of infection by neutrophils (Corbin et al, 2008), or psoriasin on the skin (Glaser et al, 2005), that inhibit pathogen proliferation. To ensure successful infection, bacteria must first deal with a phase of Zn starvation, rapidly followed by a toxic condition of Zn excess
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