Global Changes in Staphylococcus aureus Gene Expression in Human Blood

Natalia Malachowa,Frank R Deleo,Adam D Kennedy,Kevin R Braughton,Duncan W Shabb,Henry F Chambers,Scott D Kobayashi,Daniel E Sturdevant,Binh An Diep,Adeline R Whitney,Michael Otto

doi:10.1371/journal.pone.0018617

Abstract

Staphylococcus aureus is a leading cause of bloodstream infections worldwide. In the United States, many of these infections are caused by a strain known as USA300. Although progress has been made, our understanding of the S. aureus molecules that promote survival in human blood and ultimately facilitate metastases is incomplete. To that end, we analyzed the USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB), a standard laboratory culture media. Notably, genes encoding several cytolytic toxins were up-regulated in human blood over time, and hlgA, hlgB, and hlgC (encoding gamma-hemolysin subunits HlgA, HlgB, and HlgC) were among the most highly up-regulated genes at all time points. Compared to culture supernatants from a wild-type USA300 strain (LAC), those derived from an isogenic hlgABC-deletion strain (LACΔhlgABC) had significantly reduced capacity to form pores in human neutrophils and ultimately cause neutrophil lysis. Moreover, LACΔhlgABC had modestly reduced ability to cause mortality in a mouse bacteremia model. On the other hand, wild-type and LACΔhlgABC strains caused virtually identical abscesses in a mouse skin infection model, and bacterial survival and neutrophil lysis after phagocytosis in vitro was similar between these strains. Comparison of the cytolytic capacity of culture supernatants from wild-type and isogenic deletion strains lacking hlgABC, lukS/F-PV (encoding PVL), and/or lukDE revealed functional redundancy among two-component leukotoxins in vitro. These findings, along with a requirement of specific growth conditions for leukotoxin expression, may explain the apparent limited contribution of any single two-component leukotoxin to USA300 immune evasion and virulence.

Highlights

Staphylococcus aureus is a leading cause of human bacterial infections worldwide, many of which are caused by methicillinresistant S. aureus (MRSA)
We determined that the USA300 strain Los Angeles County clone (LAC) was ingested rapidly by phagocytic leukocytes—largely neutrophils—in human blood (Fig. 1A)
Within 90 min, some of the LACcontaining neutrophils had lysed and by 120 min there were few remaining intact neutrophils (Fig. 1A). These findings are consistent with the known ability of USA300 to cause rapid neutrophil lysis after phagocytosis [8,9]

Summary

Introduction

Staphylococcus aureus is a leading cause of human bacterial infections worldwide, many of which are caused by methicillinresistant S. aureus (MRSA) (reviewed in refs. [1,2]). Klevens et al reported that 75.2% of all invasive MRSA infections, including those that originate from community- or healthcare settings, are bacteremias [3]. A strain known as pulsed-field type USA300 is a leading cause of community-associated MRSA (CA-MRSA) infections and an abundant cause of healthcare-associated bloodstream infections in the United State and Canada [3,4,5,6,7]. The molecules that contribute to the ability of USA300 (and S. aureus in general) to survive in human blood and cause significant human disease remain incompletely defined. Inasmuch as the relative contribution of gamma-hemolysin to USA300 virulence is not known, we compared the ability of USA300 wild-type and isogenic hlgABC deletion strains to circumvent killing by human polymorphonuclear leukocytes and cause host cell lysis. Isogenic deletion of genes encoding multiple USA300 leukotoxins (hlgABC, lukSF-PV, and lukDE) singly and in combination indicated functional redundancy exists among these molecules

Methods

Results

Conclusion