All ETDs from UAB

Advisory Committee Chair

David G Pritchard

Advisory Committee Members

Susan K Hollingshead

John F Kearney

Kirill M Popov

Charles L Turnbough, Jr

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Fatal systemic anthrax is caused by exposure to spores of Bacillus anthracis. The outermost layer of the B. anthracis spore is called the exosporium. It consists of a paracrystalline basal layer and an external hair-like nap. The filaments of the hair-like nap are primarily composed of the glycoprotein BclA. Our previous studies showed that a 715-Da tetrasaccharide and a 324-Da disaccharide are attached to BclA through GalNAc. We named the novel nonreducing terminal sugar of the 715-Da tetrasaccharide anthrose. We subsequently proposed a plausible anthrose biosynthetic pathway and identified a gene cluster of four continuous genes that appeared to encode anthrose biosynthetic enzymes. These genes, bas3322 to bas3319, encode an enoyl-CoA hydratase, a glycosyltransferase, an aminotransferase, and an O-acyltransferase. We devised a novel microhydrazinolysis procedure that greatly facilitated our studies of the oligosaccharides. We knew that the oligosaccharides were linked to BclA through GalNAc residues and we attempted to identify the gene that encoded the enzyme for GalNAc biosynthesis. We subsequently identified the gene, bas5304. We found it encodes a bifunctional UDP-Glu/GlcNAc 4-epimerase, which converts UDP-GlcNAc to UDP-GalNAc. Surprisingly, a ∆bas5304 mutant still made oligosaccharides. However, monosaccharide analysis of oligosaccharides of the mutant revealed that GalNAc had been replaced by GlcNAc. Thus, while GalNAc appears to be the preferred amino sugar for the linkage of oligosaccharides to the BclA protein backbone, in its absence, GlcNAc can serve as a substitute linker. We also examined BclA oligosaccharides of mutant spores in which individual genes in anthrose operon had been deleted. Deletion of the first gene of the operon, bas3322, resulted in the production of abnormal pentasaccharides containing anthrose analogs. Deletion of either gene bas3320 or gene bas3319 resulted in the disappearance of the pentasaccharide and the appearance of a new tetrasaccharide with a nonreducing terminal 3-O-methyl rhamnose. Deletion of gene bas3321 resulted in BclA being substituted only with the trisaccharide, evidence that the gene encodes a dTDP-ß-L-rhamnose α-1,3-L-rhamnosyl-transferase. To further confirm their roles in anthrose biosynthesis, we cloned and expressed three of the genes of the anthrose operon and experimentally demonstrated that the proteins exhibited the predicted activities.

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