All ETDs from UAB

Advisory Committee Chair

Kevin Dybvig

Advisory Committee Members

Daniel Bullard

David E Briles

Janet Yother

Trenton R Schoeb

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Mycoplasmas cause a group of diseases that are characterized by their chronicity and resistance to treatment. Diseases caused by the murine pathogens Mycoplasma pulmonis and Mycoplasma arthritidis are often studied as models of human diseases caused by a variety of chronic pathogens that induce a similar pathology. Among several candidate pathogenic factors, superantigens and degradative enzymes such as glycosidases are potentially important but not well characterized. The M. arthritidis mitogen (MAM) is a superantigen secreted by M. arthritidis. Its role with respect to arthritogenicity and toxicity is unclear. To improve the efficiency of transformation and hence enhance efforts to generate MAM mutants through transposon mutagenesis, the function of Marth_orf138, a candidate DNA methyltransferase-coding gene that could be a component of a restriction and modification system that served as a barrier to gene transfer, was studied. The expression of Marth_orf138 in E. coli protected DNA from digestion by the HhaI restriction enzyme. However, the transformation efficiency of M. arthritidis was not significantly affected by modification of plasmids by the HhaI DNA methyltransferase, and existing methods had to be used to obtain MAM mutants. Mutants that overproduced MAM and failed to produce MAM were generated. Mitogenic activity and lethal toxicity in DBA/2J mice correlated with the amount of MAM produced. However, there was no correlation between the severity of arthritis developed as determined by histopathological examination of joint tissue and the amount of MAM produced. Our results demonstrated that MAM is associated with lethal toxicity but not arthritis in mice. Glycosidases are potentially important virulence factors but have not been carefully studied in mycoplasmas. By studying the genome sequence of M. pulmonis, five genes coding for glycosidases were identified. Cellular localization studies demonstrated that the MYPU_4630 protein was secreted. The enzymatic assay of purified glutathione S-transferase-MYPU_4630 recombinant protein demonstrated that the protein had glycosidic activity for &alpha-linked N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). MYPU_4630 protein is the first identified glycosidase with activity for GlcNAc and GalNAc in any mycoplasma. Future studies will examine whether mycoplasmal polysaccharides are a substrate for the enzyme and the relevance of the enzyme to pathogenesis.

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