Date of Award

Fall 2003

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Program/Concentration

Biology

Committee Director

Wayne L. Hynes

Committee Member

Christopher Osgood

Committee Member

Robert E. Ratzlaff

Call Number for Print

Special Collections LD4331.B46 B47 2003

Abstract

Group A Streptococci (GAS), also known as Streptococcus pyogenes, can cause a variety of human diseases ranging from asymptomatic to life threatening. Exactly how a single type of organism is able to inflict such a multitude of diseases remains to be fully understood. One possibility includes the large number of secreted virulence factors expressed by the organism. The recent sequencing of three streptococcal genomes has indicated the existence of several previously unknown genes, some of which may encode possible virulence factors. Among these is Spy1600, which based on its sequence similarities has been proposed to encode a hyaluronidase, a type of enzyme that is capable of breaking down the hyaluronic acid found in host connective tissue. According to the translated open reading frames (ORFs) of Spy1600, there is no signal peptide within the first forty amino acids of the N-terminus, which would suggest an intracellular product or a non-typical secretion mechanism. The aim of this project was to determine whether Spy1600 is conserved in other strains of GAS and if the gene encodes an active hyaluronidase. Primers were designed from a published sequence, and the gene was amplified by PCR. The gene was detected in all 50 strains tested, indicating a conserved genetic region in the genome. The Spy1600 genes from two different strains (M-type 1 and M-type 22) were cloned, transformed into E. coli, sequenced, and tested for hyaluronidase activity using a standard hyaluronidase assay. The gene was fused to a His­ tag containing leader peptide and expressed using an inducible promoter. Following expression, neither of the cloned genes showed activity, either intra- or extracellularly. The recombinant protein was also isolated by affinity chromatography and is, as anticipated, approximately 66.5 kDa. The purified protein did not show any hyaluronidase activity when assayed. Therefore it appears that Spy1600 does not seem to encode an active hyaluronidase, and the actual function of any protein produced by this gene remains to be established.

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DOI

10.25777/tf85-6r25

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