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

Christy L Ventura,Scott D Kobayashi,Mary Ann Robinson,Glenn A Nardone,Natalia Malachowa,Frank R Deleo,Carl H Hammer,Malcolm James Horsburgh

doi:10.1371/journal.pone.0011634

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.

Highlights

Staphylococcus aureus is a leading cause of human bacterial infections worldwide
Methicillin-resistant S. aureus (MRSA) remains a significant problem for healthcare facilities in most industrialized countries, an methicillin-resistant S. aureus (MRSA) strain known as USA300 is the most abundant cause of bacterial infections outside of healthcare facilities in the United States [2,3,4]
The ability of Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 to produce relatively high levels of secreted molecules such as phenol-soluble modulins (PSMs) provides an explanation in part for the enhanced ability of these pathogens to avoid destruction by neutrophils [6,7]

Summary

Introduction

Staphylococcus aureus is a leading cause of human bacterial infections worldwide. The high prevalence of infections is confounded by the ability of the pathogen to readily acquire genetic elements that confer resistance to antibiotics. Methicillin-resistant S. aureus (MRSA) remains a significant problem for healthcare facilities in most industrialized countries, an MRSA strain known as USA300 is the most abundant cause of bacterial infections outside of healthcare facilities in the United States [2,3,4]. The ability of USA300 to cause infections in otherwise healthy individuals suggests the strain has enhanced capacity to circumvent killing by the innate immune system—a notion confirmed by studies with human neutrophils [5]. The ability of CA-MRSA strains such as USA300 to produce relatively high levels of secreted molecules such as phenol-soluble modulins (PSMs) provides an explanation in part for the enhanced ability of these pathogens to avoid destruction by neutrophils [6,7]

Methods

Results

Conclusion