Year of Award

2011

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Other Degree Name/Area of Focus

Biomolecular Structure and Dynamics

Department or School/College

Division of Biological Sciences

Committee Chair

Jesse C. Hay

Commitee Members

Frank R. DeLeo, J. Stephen Lodmell, Scott Samuels, Scott A. Wetzel, J. B. Alexander Ross, James M. Musser

Keywords

innate immunity, neutrophil, Staphylococcus aureus, toxin

Publisher

University of Montana

Abstract

Community-associated methicillin-resistant S. aureus (CA-MRSA) infections are the leading cause of community bacterial infections in the United States. Genes encoding Panton-Valentine leukocidin (PVL) are present in most Staphylococcus aureus strains that cause CA-MRSA infections. Despite an association of PVL with the most abundant CA-MRSA strains, its role in pathogenesis--if any--remains unknown. PVL is a pore-forming toxin with specificity for myeloid cells, including polymorphonuclear leukocytes (PMNs or neutrophils). It is widely assumed that PVL-mediated pore formation in the plasma membrane of PMNs leads ultimately to cytolysis. However, this hypothesis has not been tested and previous studies indicate that levels of PVL in vivo are likely insufficient to cause host cell lysis. On the other hand, sublytic concentrations of PVL prime neutrophils for enhanced production of reactive oxygen species (ROS), and cause secretion of proinflammatory molecules and granule exocytosis. The molecular basis of PVL-mediated priming is unknown. To better understand the role played by PVL during S. aureus infection, we used human PMNs to investigate the correlation between PVL-mediated pore formation and cytolysis, and the molecular basis of PVL-mediated priming. Using S. aureus culture conditions that favor high production of PVL, there was on average more neutrophil plasma membrane permeability and cell lysis caused by supernatants derived from wild-type (PVL-positive) CA-MRSA strains compared to those from isogenic lukS/F-PV negative (PVL-negative) strains. Unexpectedly, there was not always a correlation between pore formation and cell lysis caused by S. aureus culture supernatants, and these findings were confirmed using purified PVL. Consistent with published studies, sublytic concentrations of PVL primed neutrophils for enhanced release of superoxide, caused upregulation of CD11b, and accelerated apoptosis. Microarray analysis revealed changes in the PMN transcriptome following exposure to PVL, including upregulation of molecules involved in the proinflammatory response. Consistent with microarray data, multiple proinflammatory molecules were released from neutrophils after stimulation with PVL. We propose that the primary function of PVL is distinct from leukocyte lysis--namely, that it elicits a proinflammatory response that can enhance the host innate immune response to S. aureus infection.

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© Copyright 2011 Shawna Graves