Identification of a Novel Staphylococcus aureus Two-Component Leukotoxin Using Cell Surface Proteomics

Staphylococcus aureus is a prominent human pathogen and leadingcause of bacterial infection in hospitals and the community.Community-associated methicillin-resistant S. aureus (CA-MRSA)strains such as USA300 are highly virulent and, unlike hospital strains, oftencause disease in otherwise healthy individuals. The enhanced virulence ofCA-MRSA is based in part on increased ability to produce high levels of secretedmolecules that facilitate evasion of the innate immune response. Althoughprogress has been made, the factors that contribute to CA-MRSA virulence areincompletely defined. We analyzed the cell surface proteome (surfome) of USA300strain LAC to better understand extracellular factors that contribute to theenhanced virulence phenotype. A total of 113 identified proteins were associatedwith the surface of USA300 during the late-exponential phase of growthin vitro. Protein A was the most abundant surface moleculeof USA300, as indicated by combined Mascot score following analysis of peptidesby tandem mass spectrometry. Unexpectedly, we identified a previouslyuncharacterized two-component leukotoxin–herein named LukS-H andLukF-G (LukGH)-as two of the most abundant surface-associated proteins ofUSA300. Rabbit antibody specific for LukG indicated it was also freely secretedby USA300 into culture media. We used wild-type and isogeniclukGH deletion strains of USA300 in combination with humanPMN pore formation and lysis assays to identify this molecule as a leukotoxin.Moreover, LukGH synergized with PVL to enhance lysis of human PMNs invitro, and contributed to lysis of PMNs after phagocytosis. Weconclude LukGH is a novel two-component leukotoxin with cytolytic activitytoward neutrophils, and thus potentially contributes to S.aureus virulence.