Hyaluronate Lyase Gene of <i>Streptococcus pyogenes</i>: Molecular Characterization and Regulation

<p><em>Streptococcus pyogenes</em> 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 <em>hyl</em>A 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 <em>hyl</em>A in the enzymatically inactive strains, 71698 and SF370, was determined to have a different pattern of expression than that of the active strain 10403.</p> <p>The effect of various environmental conditions on <em>hyl</em>A expression was evaluated. Temperature had little effect on the relative amount of <em>hyl</em>A transcript for strain 10403. However, <em>hyl </em>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 <em>hyl</em>A expression. However, no difference was detected in the titer of enzymatic activity.</p> <p>Expression of <em>hyl</em>A and <em>hasA</em> were examined for the hyaluronate lyase non-producing strain 71698; and indicated the relative amount of <em>hasA</em> transcript was considerably greater than that of <em>hyl</em>A.</p> <p>Also, 176 strains of <em>S. pyogenes</em> were screened by PCR for <em>hyl</em>A 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.</p> <p>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:<em>hyl</em>A from a HylA-negative to a HylA-positive phenotype.</p> <p>For future virulence studies, an isogenic mutant strain 10403ΔhylA was created by replacing an internal portion of <em>hyl</em>A with erythromycin resistance gene (<em>ermR</em>). Inactivation of hyaluronate lyase did not affect growth or protease or hemolytic activities.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Year dc:date.available

2003

Author dc:creator

• Walton, Sheryl Lynne

Contributors dc:contributor

• Wayne Hynes
• Keith Carson
• Christopher Osgood
• Robert Ratzlaff

• Hyaluronate lyase
• Streptococcus pyogenes
• hylA
• Microbiology
• Molecular Biology

Identifier

9780496385485

OAI identifier oai:identifier

oai:digitalcommons.odu.edu:biomedicalsciences_etds-1091