Abstract
Staphylococcus aureus infections present an enormous global health concern complicated by an alarming increase in antibiotic resistance. S. aureus is among the few bacterial species that express nitric-oxide synthase (bNOS) and thus can catalyze NO production from L-arginine. Here we generate an isogenic bNOS-deficient mutant in the epidemic community-acquired methicillin-resistant S. aureus (MRSA) USA300 clone to study its contribution to virulence and antibiotic susceptibility. Loss of bNOS increased MRSA susceptibility to reactive oxygen species and host cathelicidin antimicrobial peptides, which correlated with increased MRSA killing by human neutrophils and within neutrophil extracellular traps. bNOS also promoted resistance to the pharmaceutical antibiotics that act on the cell envelope such as vancomycin and daptomycin. Surprisingly, bNOS-deficient strains gained resistance to aminoglycosides, suggesting that the role of bNOS in antibiotic susceptibility is more complex than previously observed in Bacillus species. Finally, the MRSA bNOS mutant showed reduced virulence with decreased survival and smaller abscess generation in a mouse subcutaneous infection model. Together, these data indicate that bNOS contributes to MRSA innate immune and antibiotic resistance phenotypes. Future development of specific bNOS inhibitors could be an attractive option to simultaneously reduce MRSA pathology and enhance its susceptibility to commonly used antibiotics.
Highlights
Methicillin-resistant Staphylococcus aureus (MRSA) generates NO through bacterial NO synthase
Loss of bacterial NO synthase (bNOS) increased MRSA susceptibility to reactive oxygen species and host cathelicidin antimicrobial peptides, which correlated with increased MRSA killing by human neutrophils and within neutrophil extracellular traps. bNOS promoted resistance to the pharmaceutical antibiotics that act on the cell envelope such as vancomycin and daptomycin
We found the ⌬NO synthase (NOS) mutant was much more sensitive to H2O2 killing, with 2 log fewer surviving bacteria after 30 min of H2O2 exposure compared with the WT MRSA strain (Fig. 1B). ⌬NOS mutant H2O2 resistance could be completely restored to WT levels by in trans complementation of nos on a plasmid vector or by addition of exogenous NO (Fig. 1B), which the bacteria could encounter in activated phagocytes in the host
Summary
Methicillin-resistant Staphylococcus aureus (MRSA) generates NO through bacterial NO synthase (bNOS). Results: Loss of bNOS increases MRSA sensitivity to host neutrophils, cathelicidin antimicrobial peptides, and cell envelopeactive antibiotics. We generate an isogenic bNOS-deficient mutant in the epidemic community-acquired methicillin-resistant S. aureus (MRSA) USA300 clone to study its contribution to virulence and antibiotic susceptibility. The MRSA bNOS mutant showed reduced virulence with decreased survival and smaller abscess generation in a mouse subcutaneous infection model Together, these data indicate that bNOS contributes to MRSA innate immune and antibiotic resistance phenotypes. Generation of reactive oxygen species (ROS) through respiratory burst is a central element of normal neutrophil killing activity, as evidenced by the marked susceptibility of patients with genetic defects in NADPH oxidase (chronic granulomatous disease) to recurrent severe S. aureus infections [17]. Our analysis was conducted using a representative isolate of the epidemic USA300 CA-MRSA strain, the most common agent of serious bacterial infections in recent epidemiologic surveillance in the United States [12]
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