Alpha-Toxin Induces Programmed Cell Death of Human T cells, B cells, and Monocytes during USA300 Infection

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.

MsaB activates capsule production at the transcription level in Staphylococcus aureus

“…The survival of S. aureus in whole human blood was measured with an in vitro method (Nygaard et al, 2012) with minor modifications. In brief, heparinized venous blood samples were collected from healthy donors according to a protocol approved by the Institutional Review Board for Human Subjects at the University of Southern Mississippi.…”

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

“…All procedures were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects at Montana State University. S. aureus-induced cytotoxicity assays were performed as previously described with minor adjustments (47). PMNs (1 × 10 6 ) were resuspended in RPMI + 5 mM Hepes and plated in 96-well plates precoated with 20% (vol/vol) human serum diluted in Dulbecco's phosphate buffered saline (DPBS).…”

“…Diluted supernatants were added to plated PMNs, synchronized (400 × g, 8 min, 4°C), and then incubated statically at 37°C with 5% CO 2 . Membrane permeability and cytotoxicity caused by whole bacteria were evaluated as described using flow cytometry (11,47) or the CytoTox 96 Non-Radioactive Cytotoxicity assay (Promega) according to the manufacturer's instructions (31,33).…”

Infectious Spondylitis-Associated <i>Staphylococcus aureus</i> with Virulence Gene <i>pvl</i> or <i>tst</i> Causes More Necrosis than Apoptosis in Human Alveolar Basal Epithelial Cell Line A549

“…Additionally, PVL, staphylococcal protein A and coagulase inhibited proliferation and induced apoptosis of osteoblasts [37]. A recent finding has demonstrated that α-toxin (Hla) is an important virulence factor that causes membrane damage and induces apoptosis of human peripheral blood mononuclear cells (PBMCs) during USA300 infection [38].…”