134. Necrotizing S. aureus Pneumonia: Alpha-hemolysin Induces Platelet/Neutrophil Aggregate Formation and Neutrophil Activation
Session: Oral Abstract Session: Bacterial Pathogenesis and Virulence
Friday, October 21, 2011: 9:15 AM
Room: 151AB
Background:  Mechanisms of alveolar destruction in MRSA necrotizing pneumonia are not well defined, though exotoxins, e.g. Panton−Valentine Leukocidin (PVL) and/or alpha hemolysin, likely play important roles.  Platelet/neutrophil aggregates (PNA) and activated neutrophils (PMNL) contribute to lung injury and microcirculation deficits in other infections.  We have demonstrated that at least one MRSA exotoxin in culture supernatants, other than PVL, stimulated these activities in human whole blood ex vivo.  In this report, we identify alpha hemolysin as the sole mediator of both effects.  

Methods:  Proteins from stationary phase (20 hr) culture supernatant of the PVL-deficient MRSA strain, LACDpvl, were fractionated by isoelectric focusing (IEF) and fractions screened for the ability to stimulate PNA formation as assessed by dual color flow cytometry.  Both the % of PMNLs binding to more than 1 platelets and the mean fluorescence intensity (MFI) of the platelet signal (CD42b) were followed using blood from 2 different donors.  PMNL activation was assessed by CD11b MFI.  Fractionated uninoculated culture media served as negative control.  Proteins in fractions of interest were separated by SDS-PAGE and identified by LC-MS/MS.   Recombinant forms of the identified proteins were prepared using standard techniques.

Results:  Mean baseline PNA (%) and CD11b MFI were 7.7 ± 0.3% and 13.95 ± 0.25, respectively.  Eight fractions of LACDpvl culture supernatant (pH 8.0-9.5) induced both PNA activity and PMNL activation; maximum activity (PNA: 67.9 ± 7.2%; MFI CD42b: 1.8 ± 0.07; MFI CD11b: 99.1 ± 4.6) was found at pH 8.5 and correlated with a 37 kDa band on SDS-PAGE.  Mass spectrometry identified alpha-hemolysin and phosphatidylinositol-specific phospholipase C (PI-PLC) in this 37 kDa band.  Recombinant alpha-hemolysin (0.0625-0.125 µg) dose-dependently stimulated both PNA formation (48.4 to 55.2 ± 1.5 to 2.3%) and CD11b upregulation (MFI: 44.7 to 71.2 ± 0.52 to 0.2), whereas rPI-PLC did not elicit these activities at any dose tested up to 50 µg.

Conclusion: S. aureus alpha-hemolysin is the sole extracellular toxin mediating both PNA formation and PMNL activation ex vivo.  These activities may contribute to lung destruction in MRSA necrotizing pneumonia.


Subject Category: B. Bacterial pathogenesis, studies in animal models, molecular pathogenicity

Tanyalak Parimon, MD, Medicine, Pulmonary and Critical Care Section and Infectious Disease Laboratory, Medicine Department, Boise Veterans Affairs Medical Center, Boise, ID, Zhi Li, PhD, Research and Development and Infectious Disease Research Laboratory, Boise Veterans Affairs Medical Center, Boise, ID, Amy Bryant, PhD, Research and Development, Infectious Disease Research Laboratory, Boise Venterans Affairs Medical Center, Boise, ID, Eric McIndoo, BS, Research and Development, Infectious Disease Section, Boise Veterans Affairs Medical Center, Boise, ID and Dennis Stevens, MD, PhD, FIDSA, VA Medical Center, Boise, ID

Disclosures:

T. Parimon, None

Z. Li, None

A. Bryant, None

E. McIndoo, None

D. Stevens, None

Findings in the abstracts are embargoed until 12:01 a.m. EST Thursday, Oct. 20 with the exception of research findings presented at IDSA press conferences.