Date of Award

Summer 2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Waynes Hynes

Committee Member

Christopher Osgood

Committee Member

Fred Dobbs

Committee Member

Alex Greenwood

Abstract

S. pyogenes expresses many virulence factors, controlled by a complex regulatory network. These include hyaluronate lyase, an enzyme that degrades hyaluronic acid, a major component of the human extracellular matrix. Paradoxically, hyaluronic acid is also the sole component of the bacterium's capsule, a primary defense against the host immune response. The hylA gene, which encodes the enzyme, has been shown to take three structural forms: a full-length gene, one containing a 3' deletion and a gene that produces a prematurely truncated protein. This work was intended to show that the structure of hylA is associated with serotype, and therefore, disease pattern of S. pyogenes.

The structure of hylA was determined to have two regions where changes that influence amino acids, and therefore protein structure, result in a loss of enzymatic activity. The 3' region contains the deletion and the premature stop codon, and an area of the 5' region contains a one-amino acid change that leads to a loss of activity. Site-directed mutagenesis of the amino acid in the 5' region resulted in a restoration of activity in a truncated strain but not in a deleted strain, suggesting a relationship between the 3' and 5' domains.

Epidemiological studies of a number of strains showed that serotypes containing the full-length hylA gene produce an active enzyme, while other serotypes have one of the other two gene structures and do not produce an active enzyme. Enzyme-producing serotypes of S. pyogenes correlate with non-invasive infection, whereas those serotypes causing more invasive and life-threatening infections tend to be enzyme-negative.

Mutagenesis of proteins associated with other virulence factors in S. pyogenes gene failed to identify the regulator of hylA expression. The mga gene, the covR gene and other two-component response regulators were knocked out, but none were shown to affect hylA expression. Further investigations will explore other regulators controlling virulence factors and metabolic processes.

DOI

10.25777/kyym-g105

ISBN

9780549218272

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