Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Joseph W. St. Geme, III

Abstract

The gram-negative coccobacillus Kingella kingae is an emerging pediatric pathogen and is increasingly recognized as a common etiological agent of osteoarticular infections and bacteremia in young children. The pathogenesis of K. kingae disease involves colonization of the posterior pharynx, invasion into the bloodstream, and dissemination to distant sites of infection. Previous studies have revealed that K. kingae produces a number of surface factors that may contribute to the pathogenic process, including a polysaccharide capsule and an exopolysaccharide. The purpose of this work was to determine the role of the K. kingae polysaccharide capsule and exopolysaccharide in promoting resistance to complement-mediated and neutrophil-mediated killing. We determined that both the K. kingae capsule and exopolysaccharide prevented efficient binding of IgG, IgM, C4b, and C3b to the bacterial surface and inhibited complement-mediated killing. Abrogation of the classical complement pathway using EGTA-treated human serum restored survival of capsule-deficient, exopolysaccharide- deficient K. kingae to wild-type levels, demonstrating that capsule and exopolysaccharide promote resistance to the classical complement pathway. Consistent with these results, the capsule and exopolysaccharide enhanced K. kingae pathogenicity in juvenile rats with an intact complement system. Loss of the capsule and the exopolysaccharide resulted in avirulence, however not in rats lacking complement. Experiments using primary human neutrophils and a series of isogenic K. kingae mutants demonstrated the critical role of the capsule and the conditional role of the exopolysaccharide in preventing the neutrophil oxidative burst response and neutrophil- mediated killing of K. kingae. In the absence of capsule, the exopolysaccharide prevented K. kingae opsonization and subsequently, reduced ROS production. The loss of capsule promoted neutrophil binding of K. kingae but had no effect on neutrophil phagocytosis and bacterial internalization of K. kingae. In contrast, the exopolysaccharide efficiently blocked neutrophil phagocytosis of K. kingae and resisted the bactericidal effects of antimicrobial peptides. This works establishes that the K. kingae surface polysaccharides are multi-functional and play a critical role in K. kingae pathogenicity, facilitating evasion of host immunity. Our studies suggest that the K. kingae capsule and exopolysaccharide have potential as targets for vaccine development.

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Microbiology Commons

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