Calprotectin-mediated zinc chelation inhibitsPseudomonas aeruginosaprotease activity in cystic fibrosis sputum

Abstract

Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to control cultures, but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc-starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to post-translationally inhibit zinc metalloprotease activity and thereby impact the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment.

The human innate immune protein calprotectin elicits a multi-metal starvation response inPseudomonas aeruginosa

“…The shared response to both Zn limitation and CP treatment was consistent with the known Zn-starvation response and included the downregulation of Zn(II)-containing ribosomal proteins (RpmJ and RpmE) ( Fig 2A, Fig S3 ) and the upregulation of Znu transport proteins (ZnuA, ZnuC), pseudopaline biosynthesis and transport (CntM, CntO) proteins, the Zn(II)-independent ribosomal protein RpmE1, and the Zn(II)-independent paralog to DksA, DksA2 ( Fig 2B, Fig S3 ). Notably, several Zn(II)-dependent proteases (LasA, LasB, ImpA, AprA) were upregulated by both CP and Zn(II) limitation, seemingly contrary to the observed Zn-starvation response and a recent report on decreased protease activity in response to CP (39) ( Fig 2B ). The upregulated proteins that were specific to the Zn-limitation proteome included Zn(II)-independent proteins (CynT2, FolE2) that were not significantly changed in response to CP.…”

“…Consistent with a recent study (39), CP-treated cells showed a robust Zn-starvation response. This response included increased levels of proteins for Zn(II)-uptake systems, including ZnuACD and proteins for the synthesis and uptake of pseudopaline (CntM and CntO, respectively) (22, 25, 26).…”

“…Also consistent with the previously described Zn-starvation response (23), Zn(II)-containing proteins RpmE and RpmJ were downregulated and Zn(II)-independent proteins RpmE1, DksA2, and PyrC2 were upregulated upon CP treatment ( Fig S2 ). Despite the robust Zn-starvation response to CP observed here and in previous work (39), only negligible changes in cell-associated Zn levels were observed for PAO1 and PA14 grown in metal-replete CDM supplemented with 10 μM CP (10, 46).…”

“…In this work, we investigated how CP-dependent metal depletion affects P. aeruginosa physiology and virulence capacity using quantitative, label-free proteomics. In agreement with prior studies (10, 39), our analyses show that CP causes both Fe- and Zn-starvation responses in P. aeruginosa . Moreover, we identified increases in the expression of Zn(II)-containing proteases and membrane-modifying enzymes in response to CP.…”

“…Our study also demonstrated a change in virulence factor production in response to CP with the downregulation of the phenazines, which we determined to be a consequence of Fe limitation (10). Moreover, a recent investigation demonstrated that CP withholds Zn(II) from P. aeruginosa and induces a Zn-starvation response (39). This study also reported decreased Zn(II) protease activity caused by CP, indicating attenuated virulence potential.…”