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Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

A. S. Bayer,^1,2* L. I. Kupferwasser,^1, M. H. Brown,³ R. A. Skurray,³ S. Grkovic,^3, T. Jones,¹ K. Mukhopadhay,¹ and M. R. Yeaman^1,2

Division of Infectious Diseases, Harbor-UCLA Medical Center, Torrance, California, and LA Biomedical Research Institute at Harbor-UCLA, Torrance, California,¹ Geffen School of Medicine at UCLA, Los Angeles, California,² School of Biological Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia³

Received 8 January 2006/ Returned for modification 13 February 2006/ Accepted 12 April 2006

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

Current address: Division of Cardiology, Cedars-Sinai Medical Center, Los Angeles, CA.

Current address: Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, NSW, Australia.

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