All ETDs from UAB

Advisory Committee Chair

Kevin Dybvig

Advisory Committee Members

Stephen Barnes

David E Briles

John F Kearney

David G Pritchard

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Capsule has been associated with virulence in numerous bacterial pathogens. The Mollicutes are a host-specific group of pathogens that cause a variety of economically relevant diseases important to man. Mycoplasma pneumoniae is responsible for 2 million cases of atypical pneumonia and 100,000 hospitalizations every year in the United States (Atkinson et al., 2008) (Krause and Taylor-Robinson, 1992). There was a toxic galactan molecule described as early as 1958 in Mycoplasma mycoides subsp. mycoides (Plackett and Buttery, 1958), although no evidence was presented that it was produced by the bacteria. Over the intervening fifty years the evidence to support the production of exopolysaccharides by Mycoplasma species has been limited to anecdotal and visual evidence (electron micrographs). This work attempted to show definitively that Mycoplasma pulmonis produces an exopolysaccharide that functions as a capsule in the traditional sense of the word. We identified four distinct monosaccharides associated with the M. pulmonis exopolysaccharide fraction by gas chromatography: glucose, galactose, rhamnose, and N-acetylglucosamine (GlcNAc). These are incorporated into two exopolysaccharides, that we designated EPS-I and EPS-II. Our data suggests that EPS-I is a linear polysaccharide composed of equimolar concentrations of galactose and glucose, with the galactose terminal linked through a beta configuration. EPS-II contains GlcNAc, perhaps in equimolar ratio with the monosaccharide rhamnose. A screen conducted on a transposon library (French et al., 2008) identified two mutants of M. pulmonis that failed to produce EPS-I. The genes interrupted by the transposon are contained within the overlapping genes MYPU_7410 and MYPU_7420, an apparent operon that codes for a heterodimeric pair of annotated ABC transporter permeases. These two genes were inserted into a transposon vector and used to complement the mutants, restoring production of EPS-I. This transposon was cloned into M. pneumoniae and Mycoplasma gallisepticum, inducing the production of EPS-I in these related species. These data suggests that the enzymes encoded by the genes MYPU_7410 and MYPU_7420 function as glycosyltransferases, where synthesis and transport to the extracellular environment are linked.

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