title: Transposon disruption of the complex I NADH oxidoreductase gene (snoD) in Staphylococcus aureus is associated with reduced susceptibility to the microbicidal activity of thrombin-induced platelet microbicidal protein 1.
abstract: The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against Staphylococcus aureus. We previously reported that a Tn551 S. aureus transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1(r)) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun. 65:3293-3299, 1997). In the current study, the genetic basis for tPMP-1(r) in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn551 mutant strains showed that the fourth gene in the staphylococcal mnh operon (mnhABCDEFG) was insertionally inactivated. This operon was previously reported to encode a Na(+)/H(+) antiporter involved in pH tolerance and halotolerance. However, the capacity of ISP479R to grow at pH extremes and in high NaCl concentrations (1 to 3 M), coupled with its loss of transmembrane potential (DeltaPsi) during postexponential growth, suggested that the mnh gene products are not functioning as a secondary (i.e., passive) Na(+)/H(+) antiporter. Moreover, we identified protein homologies between mnhD and the nuo genes of Escherichia coli that encode components of a complex I NADH:ubiquinone oxidoreductase. Consistent with these data, exposures of tPMP-1-susceptible (tPMP-1(s)) parental strains (both clinical and laboratory derived) with either CCCP (a proton ionophore which collapses the proton motive force) or pieracidin A (a specific complex I enzyme inhibitor) significantly reduced tPMP-induced killing to levels seen in the tPMP-1(r) mutants. To reflect the energization of the gene products encoded by the mnh operon, we have renamed the locus sno (S. aureus nuo orthologue). These novel findings indicate that disruption of a complex I enzyme locus can confer reduced in vitro susceptibility to tPMP-1 in S. aureus.
authors.affiliation: Department of Medical Microbiology & Immunology, University of Wisconsin, 1300 University Avenue, Biochemistry Building, Room 250, Madison, WI 53706, USA.
authors.forename: Arnold S
authors.initials: AS
authors.lastname: Bayer
authors.forename: Peter
authors.initials: P
authors.lastname: McNamara
authors.forename: Michael R
authors.initials: MR
authors.lastname: Yeaman
authors.forename: Natalie
authors.initials: N
authors.lastname: Lucindo
authors.forename: Tiffanny
authors.initials: T
authors.lastname: Jones
authors.forename: Ambrose L
authors.initials: AL
authors.lastname: Cheung
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Sahl
authors.forename: Richard A
authors.initials: RA
authors.lastname: Proctor
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001798:Blood Proteins / Q000494:pharmacology*; D004251:DNA Transposable Elements; D024881:Drug Resistance, Bacterial; D042967:Electron Transport Complex I / Q000235:genetics* / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D008560:Membrane Fluidity; D008564:Membrane Potentials; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D015539:Platelet Activation; D011817:Rabbits; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001798:Blood Proteins; D004251:DNA Transposable Elements; C077150:platelet microbicidal protein, Oryctolagus cuniculus; D042967:Electron Transport Complex I