Date of Award

1-1-2009

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (ix, 200 pages) : PDF file, illustrations (some color)

Dissertation/Thesis Chair

Nicholas Mantis

Committee Members

Harry Taber, Ronald Limberger, Keith Derbyshire, Dennis Metzger

Keywords

IgA, Mucosal, Salmonella, Shigella, Immunoglobulin A, Salmonella typhimurium, Shigella flexneri, Endotoxins

Subject Categories

Microbiology

Abstract

Secretory IgA (SIgA) antibodies directed against the serotype-specific, O-antigen of lipopolysaccharide (LPS) are the primary determinants of mucosal immunity to enteric bacterial pathogens, including of Salmonella enterica serovar Typhimurium and Shigella flexneri. While the singular importance of SIgA in preventing enteric infections is recognized, the underlying mechanisms by which these antibodies protect the mucosal epithelium remains poorly understood. In this study, I demonstrated that Sal4, a protective, anti-LPS specific monoclonal IgA, is a potent inhibitor of S. Typhimurium flagella-based motility and type three secretion (T3S) mediated entry into epithelial cells. Sal4's effects on motility and invasion occurred rapidly (<15 min), and were independent of agglutination. I also present evidence the protective anti-LPS, monoclonal IgA, IgAC5, reduced S. flexneri T3S of IpaB and IpaC. Concurrent with the IgA-induced reduction in T3S, there was a decrease in both the proton motive force and ATP levels in both bacterial species examined. I also report that Sal4 and IgAC5 have effects on the bacterial envelope. Scanning, transmission, and cyro-electron microscopy revealed dramatic antibody-mediated alterations in the topology of the outer membrane (OM) of S.Typhimurium and S. flexneri. Furthermore, Sal4-treatment caused a ~5 fold increase in O-antigen, and elevated levels of lipid A, released into culture supernatants, consistent with the loss of LPS. Correspondingly, the OM of antibody-treated bacteria was ~50% more permeable than control cells. Based on these data, I proposed that Sal4 binding to the LPS destabilizes the outer leaflet of the OM of S. Typhimurium, thereby compromising the integrity of the bacterial envelope, disrupting bioenergetics by physical and/or mechanical stress, and arresting both flagella-based motility and T3S. This study reveals a previously unrecognized capacity of SIgA to "disarm" enteric pathogens in mucosal secretions, thereby preventing colonization and invasion of the intestinal epithelium.

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