Steroid Drugs Inhibit Bacterial Respiratory Oxidases and Are Lethal Toward Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: Cytochrome bd complexes are respiratory oxidases found exclusively in prokaryotes that are important during infection for numerous bacterial pathogens. METHODS: In silico docking was employed to screen approved drugs for their ability to bind to the quinol site of Escherichia coli cytochrome bd-I. Respiratory inhibition was assessed with oxygen electrodes using membranes isolated from E. coli and methicillin-resistant Staphylococcus aureus strains expressing single respiratory oxidases (ie, cytochromes bd, bo', or aa3). Growth/viability assays were used to measure bacteriostatic and bactericidal effects. RESULTS: The steroid drugs ethinylestradiol and quinestrol inhibited E. coli bd-I activity with median inhibitory concentration (IC50) values of 47 ± 28.9 µg/mL (158 ± 97.2 µM) and 0.2 ± 0.04 µg/mL (0.5 ± 0.1 µM), respectively. Quinestrol inhibited growth of an E. coli "bd-I only" strain with an IC50 of 0.06 ± 0.02 µg/mL (0.2 ± 0.07 µM). Growth of an S. aureus "bd only" strain was inhibited by quinestrol with an IC50 of 2.2 ± 0.43 µg/mL (6.0 ± 1.2 µM). Quinestrol exhibited potent bactericidal effects against S. aureus but not E. coli. CONCLUSIONS: Quinestrol inhibits cytochrome bd in E. coli and S. aureus membranes and inhibits the growth of both species, yet is only bactericidal toward S. aureus.

The identification of two M20B family peptidases required for full virulence in Staphylococcus aureus.

We have previously demonstrated that deletion of an intracellular leucine aminopeptidase results in attenuated virulence of S. aureus. Herein we explore the role of 10 other aminopeptidases in S. aureus pathogenesis. Using a human blood survival assay we identified mutations in two enzymes from the M20B family (PepT1 and PepT2) as having markedly decreased survival compared to the parent. We further reveal that pepT1, pepT2 and pepT1/2 mutant strains are impaired in their ability to resist phagocytosis by, and engender survival within, human macrophages. Using a co-infection model of murine sepsis, we demonstrate impairment of dissemination and survival for both single mutants that is even more pronounced in the double mutant. We show that these enzymes are localized to the cytosol and membrane but are not necessary for peptide-based nutrition, a hallmark of cell-associated aminopeptidases. Furthermore, none of the survival defects appear to be the result of altered virulence factor production. An exploration of their regulation reveals that both are controlled by known regulators of the S. aureus virulence process, including Agr, Rot and/or SarA, and that this cascade may be mediated by FarR. Structural modeling of PepT1 reveals it bears all the hallmarks of a tripeptidase, whilst PepT2 differs significantly in its catalytic pocket, suggesting a broader substrate preference. In sum, we have identified two M20B aminopeptidases that are integral to S. aureus pathogenesis. The future identification of protein and/or peptide targets for these proteases will be critical to understanding their important virulence impacting functions.