Date of Award

Spring 2003

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Wayne Hynes

Committee Member

Keith Carson

Committee Member

Christopher Osgood

Committee Member

Robert Ratzlaff

Abstract

Streptococcus pyogenes causes an assortment of diseases ranging from pharyngitis to necrotizing fasciitis. This bacterium has the ability to elaborate several extracellular products capable of causing tissue damage; one of which is a hyaluronate lyase. Little information is available regarding the regulation of streptococcal hyaluronidase. As such, the expression of hylA in the hyaluronate lyase-producing strain ATCC 10403 and in strains that do not produce detectable enzyme was analyzed by RT-PCR and quantitative PCR. Hyaluronate lyase transcript was detected throughout growth for all strains; however, expression of hylA in the enzymatically inactive strains, 71698 and SF370, was determined to have a different pattern of expression than that of the active strain 10403.

The effect of various environmental conditions on hylA expression was evaluated. Temperature had little effect on the relative amount of hylA transcript for strain 10403. However, hyl A expression was slightly decreased after growth in pH 5.0 and increased under anaerobic and increased carbon dioxide conditions. The presence of hyaluronic acid in the growth media resulted in a two-fold increase in hylA expression. However, no difference was detected in the titer of enzymatic activity.

Expression of hylA and hasA were examined for the hyaluronate lyase non-producing strain 71698; and indicated the relative amount of hasA transcript was considerably greater than that of hylA.

Also, 176 strains of S. pyogenes were screened by PCR for hylA and showed all contained the gene; however, 34 strains showed a decrease in the size of the 3′region. The 3 ′ region was PCR amplified, cloned, and the sequence determined from six of these 34 strains. Comparison of the sequences revealed a deletion of 183 bp in the same location in all six strains.

A preliminary analysis of the role of hyaluronate lyase in streptococcal infection was evaluated. Preliminary data suggests hyaluronate lyase to be an important virulence factor as was determined by a 5.7% reversion of single-recombinant mutant strain 10403::pHAS:hylA from a HylA-negative to a HylA-positive phenotype.

For future virulence studies, an isogenic mutant strain 10403ΔhylA was created by replacing an internal portion of hylA with erythromycin resistance gene (ermR). Inactivation of hyaluronate lyase did not affect growth or protease or hemolytic activities.

DOI

10.25777/rg0g-b962

ISBN

9780496385485

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