A periplasmic metallochaperone of the Cation Diffusion Facilitator YiiP is required for Zn2+ sensing in Pseudomonas aeruginosaript

Pseudomonas aeruginosa thrive to survive in harsh conditions imposed by the host. We have previously described two Zn2+-transporting members of the Cation Diffusion Facilitator family, YiiP and CzcD, inhibiting susceptibility to imipenem by decreasing the expression of the outer membrane porin OprD (A. Salusso and D. Raimunda, Frontiers in cellular and infection microbiology 7:84, 2017, https://doi.org/10.3389/fcimb.2017.00084). Here we provide evidence that a protein encoded in yiiPs operon, PA3962, is fundamental to the coupling of these processes. Immunodetection assay indicates that PA3962 locates in membranes. Supporting a role in oprD regulation, a PA3962 insertional mutant has a significant increase of oprD expression levels and imipenem sensitivity, which is suppressed by gene complementation but not in presence of Zn2+, as opposed to the YiiP mutant. We identified 2 pairs of conserved acidic residues in a hydrophobic juxtamembrane domain. Metal binding specificity and stoichiometry was explored in wild-type and mutant versions of these. Zn2+ appears as the cognate metal of PA3962, with residues D40, D47 and D65 required for its coordination. As the periplasmic Zn2+-sensor CzcS regulates oprD expression, the interaction with it was analyzed in vitro. Interaction was Zn2+-dependent, and mutations of D47A or D65A abolished it. We propose a role for PA3962, hereafter periplasmic metallochaperone of YiiP (PmcY), in the context of Zn2+ signaling pathways in P. aeruginosa. The relay YiiP-PmcY, supplies Zn2+ and activates CzcS/CzcR, down-regulating the transcription of the imipenem-permeable OprD. This mechanism would allow P. aeruginosa to put a brake on unspecific mechanisms for micronutrient uptake, with potential xenobiotic entry, while the cytosolic Zn2+ quota is still sufficient.