Pseudomonas Aeruginosa CDF Transporters CzcD and YiiP are Involved in Zn2+ Efflux, Outer Membrane Permeability and Antibiotic Resistance

Abstract

Transporters of the cation diffusion facilitator (CDF) family form dimers that export transition metals from the cytosol. The opportunistic pathogen Pseudomonas aeruginosa presents three homologous CDF genes, czcD, cotP and yiiP. CotP appears to have a role in Co2+ homeostasis as the deletion mutant strain ΔcotP is more sensitive and accumulate this ion. In inhibition halo experiments, ΔczcD and ΔyiiP strains showed low Zn2+ sensitivity. However, in iron-rich media and in the presence of Zn2+ the strains secreted higher levels of the iron chelator pyoverdine. The non-pyoverdine producer triple mutants, ΔzntA-ΔpvdA-ΔczcD and ΔzntA-ΔpvdA-ΔyiiP, displayed increased Zn2+ sensitivity and increased intracellular Zn2+ accumulation compared to the double mutant ΔzntA-ΔpvdA. Most importantly, the strains ΔczcD, ΔyiiP, ΔpvdA-ΔczcD and ΔpvdA-ΔyiiP showed a 4- to 8-fold increased sensitivity to imipenem and ciprofloxacin and a 2-fold increased sensitivity to chloramphenicol and gentamycin. Complemented strains attained similar levels of antibiotic resistance as the PAO1 parental strain. These observations correlated with increased sensitivity to EDTA-lysozyme treatment and overexpression of OprN and OprD porins in ΔczcD and ΔyiiP vs. WTand ΔcotP. We hypothesize that while CotP is a Co2+ efflux transporter CzcD and YiiP export Zn2+ to the periplasm and that their function is required for regulation of OprD synthesis, outer membrane stability and therefore antibiotic tolerance.