Defense Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Dennis Ohman

Abstract

The opportunistic pathogen P. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis patients. During chronic infection of the cystic fibrosis lung, P. aeruginosa undergoes conversion to a mucoid phenotype, constitutively producing the exopolysaccharide alginate, composed of the uronic acids D-mannuronate and L-guluronate. This alginate production contributes significantly to virulence in the cystic fibrosis lung. Evidence suggests that the acetylation state of the mannuronate component of the alginate influences the ability of components of the immune system to phagocytose the organism. To garner new and relevant information regarding the mechanism of alginate acetylation in Pseudomonas aeruginosa, a variety of approaches were undertaken. Analysis of the alginate produced by algX, algG, and algK alginate biosynthesis mutants revealed that the small oligouronides they produced were unacetylated. This strongly supports the hypothesis that the mannuronates are acetylated in periplasm, and that a polymer of at least some specific size is required. While three alginate biosynthesis gene products (AlgI, AlgJ, and AlgF) have been shown to be involved in alginate acetylation, another gene in the cluster, algX, shares 30% identity with one of them and thus generates speculation as to its potential involvement in the process. To test this possibility, an algX mutant was complemented with a plasmid carrying a mutation at a conserved residue shown to be required for alginate acetylation in the homologous protein. Analysis of alginate from this construct suggested that AlgX is not involved in alginate acetylation. To determine if changes in levels of alginate acetylation are accomplished at the transcriptional level, transcript levels of several alginate biosynthesis genes in different media were determined by real-time PCR. As qRT-PCR had not been previously performed on any of the alginate biosynthesis genes, this yielded important information about the transcription of the operon. In addition, beta-galactosidase assays on upstream regions of several biosynthesis genes identified two previously unrecognized promoters, one upstream of algG and one upstream of algI. The remaining approach was to examine protein interactions of AlgF, the protein product of one of the three acetylation genes. 2-D redox SDS-PAGE gels indicated that disulfide bonding may be important for interactions with this protein. While mass spectrometry was unable to identify the binding partners of AlgF, efforts are ongoing to create a mutation in the P. aeruginosa genome that changes the cysteine residue in AlgF to a serine residue. This would be a definitive method for determining the importance of disulfide bonding in AlgF.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

May 2010

Available for download on Tuesday, May 12, 2020

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