All ETDs from UAB

Advisory Committee Chair

Kevin Dybvig

Advisory Committee Members

David Briles

Daniel Bullard

Hui Wu

Janet Yother

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Public Health (DrPH) School of Public Health

Abstract

Mycoplasmas are host-specific human and animal pathogens within the class of wall-deficient bacteria named Mollicutes and have smaller sizes and genomes than the walled bacteria. Mycoplasma genome sequences reveal few if any genes for synthesis of a glycocalyx. Nevertheless, we find that mycoplasmas produce glycolipids, glycoprotein, and polysaccharides. Using gas chromatography and mass spectrometry, glucose, mannose, rhamnose, and galactose were detected in the glycolytic species Mycoplasma pneumoniae and Mycoplasma pulmonis. All of these sugars except galactose was found in Mycoplasma arthritidis. Rhamnose was in the rare D configuration in the glycolytic species and in both D and L forms in M. arthritidis. Since there is no known source of rhamnose in the culture media or animal hosts, we speculated that mycoplasmas synthesize rhamnose and pursued this idea further by tracing rhamnose labeling with 13C isotopes. Surprisingly, 13C-glucose did not label glycoconjugates in any species. However, 13C-labeled starch, a glucose polymer, labeled rhamnose, glucose, N-acetylglucosamine, and galactose. Mycoplasmas indeed synthesize rhamnose. Supporting this claim are observations that supplementing growth medium with starch increased the amount of rhamnose produced by the mycoplasmas over 10-fold and resulted in a higher L-rhamnose to D-rhamnose ratio. Methanol/chloroform extraction showed that the majority of rhamnose and glucose partitioned in the aqueous fraction or the protein-concentrated interphase, suggesting the presence of glycoprotein. All species tested reacted lightly with a glycoprotein stain, and heavily stained bands were observed in concentrated lipoprotein extracts from M. arthritidis. Using high resolution mass spectrometry, threonine and serine residues that were O-glycosylated with a hexose were identified. Using 13C-labeled starch, we confirmed that glucose was attached to protein. This is the first definitive characterization of any glycan or glycosylation site in any mollicutes. These studies show that although mycoplasmas do not have common glycoconjugate-producing machinery, they do produce glycoconjugates. Identifying and characterizing this novel machinery would reveal protein functions that could extend well beyond this group of bacteria.

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