All ETDs from UAB

Advisory Committee Chair

Janet Yother

Advisory Committee Members

Kevin Dybvig

Michael Niederweis

David Pritchard

Charles Turnbough

Hui Wu

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The principle objective of this dissertation is to develop a thorough understanding of the genetics and biochemistry of capsule synthesis. Specifically, it involves the identification and characterization of the capsular glycosyltransferases in Streptococcus pneumoniae serotype 2, with investigations into their regulation. S. pneumoniae (the pneumococcus) is a significant human pathogen that has the ability to persist as a commensal and then transition to a virulent state, the latter of which is a major contributor to global morbidity and mortality. The capsular polysaccharide (CPS) of S. pneumoniae is a protective surface structure that plays an integral role in this disease process. The vast majority of pneumococcal serotypes synthesize capsule via the Wzy-dependent pathway, which requires the coordinated activity of multiple glycosyltransferases. Apart from the initiating capsular glycosyltransferase (Cps2E for serotype 2), few reports have functionally identified or characterized the other glycosyltransferases required to complete capsule repeat unit synthesis. Here, we utilize genetic, biochemical, and bioinformatic approaches to demonstrate that Cps2T, Cps2F, Cps2G, and Cps2I are glycosyltransferases that catalyze the remaining glycosidic linkages in the S. pneumoniae serotype 2 capsule repeat unit. Cps2T catalyzes the second and committed step in repeat unit biosynthesis. Cps2F is a novel rhamnosyltransferase catalyzing the third step with the sequential addition of two sugar residues. Cps2G catalyzes the fourth step, and our evidence suggests that Cps2I catalyzes the final step in repeat unit biosynthesis. An additional aim of this dissertation is to investigate whether glycosyltransferase activity may be regulated to influence capsule levels and/or distribution. Here, we generate and analyze defined mutations in the initiating capsular glycosyltransferase Cps2E and analyze the effects on capsule initiation. We identified 5 residues (A162, D167, V196, L200, H258) in the extracytoplasmic domain of Cps2E and demonstrate that these residues are highly conserved among S. pneumoniae serotypes and required for efficient cytoplasmic sugar transfer in vitro. In addition, we analyze the data generated from both aims to propose mechanisms of how glycosyltransferase activity may be regulated to influence capsule in S. pneumoniae.

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