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

Abstract

Staphylococcus aureus is a prominent human pathogen and leading cause 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, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin–herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.

Staphylococcus aureusLukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1

“…Despite the lack of overlap in cellular targets identified thus far, the staphylococcal leukotoxins have been shown to exhibit synergistic effects (26). The identification of CD11b as a cellular target for LukAB provides an explanation for the previously reported synergism between LukAB and PVL toward human PMNs (10,11), as sublytic concentrations of PVL increase CD11b surface levels on these cells (27). From an evolutionary standpoint, the use of multiple, nonoverlapping cellular targets provides an explanation for why S. aureus has such a large arsenal of cytotoxins and is such a welladapted pathogen when it comes to evading host PMNs.…”

“…A single S. aureus strain can produce up to five different leukotoxins including two versions of γ-hemolysin (HlgAB and HlgCB), leukocidin E/D (LukED), Panton-Valentine leukocidin (PVL), and leukocidin A/B (LukAB, also known as LukGH) (4)(5)(6). The sequence similarity among these leukocidins ranges from 60% to 80% with the exception of LukAB, which is only 30-40% similar to the others (9,10). LukAB is the most recently identified member of the bicomponent leukocidin family (9,10).…”

“…The sequence similarity among these leukocidins ranges from 60% to 80% with the exception of LukAB, which is only 30-40% similar to the others (9,10). LukAB is the most recently identified member of the bicomponent leukocidin family (9,10). This toxin contributes to the cytotoxicity of clinical isolates toward innate immune cells and has been shown to play an important role in the success of S. aureus in both ex vivo and in vivo models of infection (9,11).…”

“…This toxin contributes to the cytotoxicity of clinical isolates toward innate immune cells and has been shown to play an important role in the success of S. aureus in both ex vivo and in vivo models of infection (9,11). LukAB specifically targets phagocytes such as polymorphonuclear cells (PMNs or neutrophils) (9)(10)(11)(12), which are an integral part of the host innate immune response to S. aureus (13). The capacity to kill PMNs is conserved among all leukocidins (4,5), although the actions of LukED (14) and Hlg (15) are not limited to phagocytes (5).…”

Differential regulation of staphylococcal virulence by the sensor kinase SaeS in response to neutrophil-derived stimuli

“…SaeRS regulates transcription of multiple toxins, including γ-toxin (hlgA, hlgB, hlgC), LukSF-PVL, and LukAB/LukGH, which have been shown to contribute to PMN lysis (26)(27)(28)(29)(30)(31). Additionally, transcription of the saePQRS operon and SaeR target genes are activated in response to PMN phagocytosis and PMN components (32)(33)(34).…”