All ETDs from UAB

Advisory Committee Chair

Janet L Yother

Advisory Committee Members

Kevin Dybvig

Michael E Niederweis

Peter E Prevelige

David G Pritchard

Document Type

Dissertation

Date of Award

2012

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Streptococcus pneumoniae is a Gram-positive pathogen and an important cause of community-acquired pneumonia and meningitis, and it can also cause otitis media and bacteremia. A major virulence factor of S. pneumoniae is its polysaccharide capsule which functions to prevent complement-mediated opsonophagocytosis. At least 93 distinct serotypes that vary in sugar composition and structure exist. S. pneumoniae can asymptomatically colonize the nasopharynx and transition to other body sites such as the lungs and blood causing invasive disease. The ability to regulate capsule production in these vastly different environments is critical for survival as low levels of capsule are necessary for colonization, while high levels of capsule are needed to prevent complement-mediated opsonophagocytosis. In serotype 3 S. pneumoniae, [-4)-&beta-D-Glc-(1-3)-&beta-D-GlcUA-(1-], capsule is synthesized by the type 3 synthase, encoded by cps3S, using the UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) sugar precursors on a phosphatidylglycerol membrane anchor. The polysaccharide capsule may remain anchored to the membrane or released from the cell. Previous work has shown that [UDP-GlcUA] plays a critical role in modulating capsule production, but little is known how the concentration of this nucleotide sugar is regulated within the cell. In this study, we found that increased NADH/NAD+ ratios inhibited in vitro activity of purified Cps3D, the type 3 UDP-Glc dehydrogenase that catalyzes the NAD+-dependent oxidation of UDP-Glc to UDP-GlcUA. Analysis of conditions with altered capsule production correlated with increased NADH/NAD+ ratios, suggesting that the NADH/NAD+ ratio may alter Cps3D activity. Shortened capsule chain-length in some of the conditions with altered capsule production was also observed, suggesting that Cps3D activity was altered by the NADH/NAD+ ratio. An NADH oxidase mutant was constructed and found to have significantly increased NADH/NAD+ ratio that also correlated with reduced capsule production and shortened chain-lengths. The data presented here suggest a metabolic model where the NADH/NAD+ ratio alters Cps3D activity, the enzyme responsible for synthesizing UDP-GlcUA, and alterations in Cps3D activity ultimately lead to changes in capsule production. Another aspect of serotype 3 capsule synthesis: release of capsule from the cell was examined. We characterized released capsule from the cell and found that it was of similar size to cell-associated capsule. In addition, capsule release was found to occur steadily over time. However, we were unable to determine the exact mechanism of capsule release.

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