Abstract
This investigation examines the influence of alpha-toxin (Hla) during USA300 infection of human leukocytes. Survival of an USA300 isogenic deletion mutant of hla (USA300Δhla) in human blood was comparable to the parental wild-type strain and polymorphonuclear leukocyte (PMN) plasma membrane permeability caused by USA300 did not require Hla. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) following infection by USA300, USA300Δhla, and USA300Δhla transformed with a plasmid over-expressing Hla (USA300Δhla Comp) demonstrated this toxin plays a significant role inducing plasma membrane permeability of CD14+, CD3+, and CD19+ PBMCs. Rapid plasma membrane permeability independent of Hla was observed for PMNs, CD14+ and CD19+ PBMCs following intoxication with USA300 supernatant while the majority of CD3+ PBMC plasma membrane permeability induced by USA300 required Hla. Addition of recombinant Hla to USA300Δhla supernatant rescued CD3+ and CD19+ PBMC plasma membrane permeability generated by USA300 supernatant. An observed delay in plasma membrane permeability caused by Hla in conjunction with Annexin V binding and ApoBrdU Tunel assays examining PBMCs intoxicated with recombinant Hla or infected with USA300, USA300Δhla, USA300Δhla Comp, and USA300ΔsaeR/S suggest Hla induces programmed cell death of monocytes, B cells, and T cells that results in plasma membrane permeability. Together these findings underscore the importance of Hla during S. aureus infection of human tissue and specifically demonstrate Hla activity during USA300 infection triggers programmed cell death of human monocytes, T cells and B cells that leads to plasma membrane permeability.
Highlights
Staphylococcus aureus is a common Gram-positive bacterial pathogen that can produce a wide spectrum of disease in humans, ranging from superficial skin abscesses to invasive life-threatening disease
This investigation demonstrates Hla is the principle component produced by USA300 generating T cell plasma membrane permeability and significantly compromises B cell and monocyte plasma membrane integrity
Factors other than Hla in USA300 supernatant contributed to substantial polymorphonuclear leukocyte (PMN) and CD14+ peripheral blood mononuclear cells (PBMCs) plasma membrane permeability within 15 minutes post-intoxication and caused the majority of observed plasma membrane permeability to these cell types
Summary
Staphylococcus aureus is a common Gram-positive bacterial pathogen that can produce a wide spectrum of disease in humans, ranging from superficial skin abscesses to invasive life-threatening disease. Strains of community-associated MRSA (CA-MRSA) distinct from previously characterized hospital-associated MRSA (HA-MRSA) are a prominent cause of skin and soft-tissue infections and are noted for their enhanced capacity to produce disease in humans [2,3]. The CA-MRSA strain identified by pulse-field gel electrophoresis (PFGE) as type USA300 is a leading cause of softtissue infections in the United States [2,4]. This strain exhibits a heightened ability to elicit human polymorphonuclear leukocyte (PMN) destruction in vitro as well as produce more severe dermonecrotic soft-tissue infections in mice relative to other S. aureus strains [5,6,7]. Elevated expression of virulence gene regulators agr, saeR/S, and sarA as well as the pore-forming toxin hla in USA300 relative to USA400 is thought to contribute to the increased pathogenicity of this strain during rat models of pneumonia [11]
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