Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released\ud from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to\ud initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo.\ud In S. aureus, the plasmid-carried qacA gene encodes a multi-drug transporter, conferring resistance to\ud organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously\ud showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study\ud evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer\ud tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r\ud qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1\ud susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not\ud tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did\ud not affect the susceptibility of Et (ruling out Et–tPMP-1 cotransport). The following cytoplasmic membrane\ud parameters did not differ significantly between the qacA-bearing and parental strains: contents of\ud the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol;\ud fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited\ud greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among\ud these families of efflux pumps, only the multi-drug transporter encoded by qacA conferred a tPMP-1r\ud phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon\ud membrane structure or function unrelated to PMF-dependent peptide efflux
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