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Staphylococcus aureus (S. aureus) is a prominent Gram-positive bacterial pathogen that expresses numerous cytotoxins known to target human polymorphonuclear leukocytes (PMNs or neutrophils). These include leukocidin G/H (LukGH, also known as LukAB), the Panton–Valentine leukocidin (PVL), γ-hemolysin A/B (HlgAB), γ-hemolysin B/C (HlgBC), leukocidin E/D (LukED), α-hemolysin (Hla), and the phenol-soluble modulin-α peptides (PSMα). However, the relative contribution of each of these cytotoxins in causing human PMN lysis is not clear. In this study, we used a library of cytotoxin deletion mutants in the clinically relevant methicillin-resistant S. aureus (MRSA) isolate LAC (strain ST8:USA300) to determine the relative importance of each for causing human PMN lysis upon exposure to extracellular components as well as following phagocytosis. Using flow cytometry to examine plasma membrane permeability and assays quantifying lactose dehydrogenase release, we found that PVL was the dominant extracellular factor causing human PMN lysis produced by USA300. In contrast, LukGH was the most important cytotoxin causing human PMN lysis immediately following phagocytosis with contributions from the other bicomponent leukocidins only observed at later time points. These results not only clarify the relative importance of different USA300 cytotoxins for causing human PMN destruction but also demonstrate how two apparently redundant virulence factors play distinctive roles in promoting S. aureus pathogenesis.

The rise in bacterial resistance to common antibiotics has raised an increased need for alternative treatment strategies. The natural antibacterial product, 18β-glycyrrhetinic acid (GRA) has shown efficacy against community-associated methicillin-resistant Staphylococcus aureus (MRSA), although its interactions against planktonic and biofilm modes of growth remain poorly understood. This investigation utilized biochemical and metabolic approaches to further elucidate the effects of GRA on MRSA. Prolonged exposure of planktonic MRSA cell cultures to GRA resulted in increased production of staphyloxanthin, a pigment known to exhibit antioxidant and membrane-stabilizing functions. Then, 1D 1H NMR analyses of intracellular metabolite extracts from MRSA treated with GRA revealed significant changes in intracellular polar metabolite profiles, including increased levels of succinate and citrate, and significant reductions in several amino acids, including branch chain amino acids. These changes reflect the MRSA response to GRA exposure, including potentially altering its membrane composition, which consumes branched chain amino acids and leads to significant energy expenditure. Although GRA itself had no significant effect of biofilm viability, it seems to be an effective biofilm disruptor. This may be related to interference with cell–cell aggregation, as treatment of planktonic MRSA cultures with GRA leads to a significant reduction in micro-aggregation. The dispersive nature of GRA on MRSA biofilms may prove valuable for treatment of such infections and could be used to increase susceptibility to complementary antibiotic therapeutics.

Staphylococcus aureus is a common Gram-positive bacteria that is a major cause of human morbidity and mortality. The SaeR/S two-component sensory system of S. aureus is important for virulence gene transcription and pathogenesis. However, the influence of SaeR phosphorylation on virulence gene transcription is not clear. To determine the importance of potential SaeR phosphorylation sites for S. aureus virulence, we generated genomic alanine substitutions at conserved aspartic acid residues in the receiver domain of the SaeR response regulator in clinically significant S. aureus pulsed-field gel electrophoresis (PFGE) type USA300. Transcriptional analysis demonstrated a dramatic reduction in the transcript abundance of various toxins, adhesins, and immunomodulatory proteins for SaeR with an aspartic acid to alanine substitution at residue 51. These findings corresponded to a significant decrease in cytotoxicity against human erythrocytes and polymorphonuclear leukocytes, the ability to block human myeloperoxidase activity, and pathogenesis during murine soft-tissue infection. Analysis of SaeR sequences from over 8,000 draft S. aureus genomes revealed that aspartic acid residue 51 is 100% conserved. Collectively, these results demonstrate that aspartic acid residue 51 of SaeR is essential for S. aureus virulence and underscore a conserved target for novel antimicrobial strategies that treat infection caused by this pathogen.