Date of Award

5-2004

Document Type

Thesis

Department

Biology

First Advisor

William McShan

Second Advisor

Sonya Williams

Third Advisor

Rosemary Harkins

Abstract

Group A streptococci (Streptococcus pyogenes) is one of the most frequent pathogens of humans. It is estimated that between 5-15% of normal individuals harbor the bacterium, usually in the upper respiratory tract, without signs of disease. S. pyogenes can initiate an active infection when defenses are compromised or when the organism is able to penetrate the host's defenses. When the bacteria invade vulnerable tissues, a variety of suppurative infections can occur. Acute S. pyogenes infection may be present as pharyngitis ("strep throat"), scarlet fever (rash), impetigo (infection of the superficial layers of the skin) or cellulitis (infection of the deep layers of the skin). Invasive, severe infections can result in necrotizing fasciitis ("flesh-eating bacteria"), myositis (muscle inflammation and soreness) and streptococcal toxic shock syndrome. Patients may also develop immune-mediated post-streptococcal sequelae, such as acute rheumatic fever and acute glomerulonephritis, following other infections caused by S. pyogenes.

Recently, the S. pyogenes strain SF370 genome sequencing project discovered a number of possible regulator genes. The specific regulator gene that we are observing is Spy0146 and its upstream companion gene, Spy0145. Spy0146 is homologous to the putative perfringolysin O regulator (pfoR) from Clostridium perfringens, while Spy0145 is a member of a family of transcriptional regulators. Our analysis of the region of the streptococcal chromosome containing gene Spy 0146 suggested that the upstream gene, Spy 0145 might be linked to Spy0146 as an operon. If Spy0145 and Spy0146 are cotranscribed, then both may play an important coordinated role in global gene regulation of S. pyogenes.

Previously in the laboratory, a knockout of one of these genes (Spy0146) had been demonstrated to alter the mRNA expression of many S. pyogenes genes, including the virulence factors streptolysin O and NADase. This mutation in Spy0146, in fact, both increased and decreased transcription activity, depending upon the particular genes analyzed.

The research being conducted here has two main goals. We would like 1) to determine whether the genes Spy0145 and Spy0146 are co-transcribed on the same polycistronic message and 2) to replace the inactivated copy of gene Spy0146 with a cloned copy in an integration vector to return the cell to wild type phenotype (natural state).

To achieve our first goal, we isolated the streptococcal RNA using the hot phenol method and synthesized cDNA. Using polymerase chain reaction (PCR), we demonstrated that both Spy0145 and Spy0146 were found on the same polycistronic message. Preliminary results furthel suggested that this polycistronic message is more rapidly turned over during logarithmic growth as compared to cells in stationary phase. Further, for our second goal, we isolated the gene by method of using PCR, cloned it into the E. coli bacteria, then transferred to a streptococcal shuttle vector (p7INT), reintroducing the gene in the mutant strain of S. pyogenes. This arrangement of two transcriptional factors (Spy0145 & Spy0146) on one mRNA is very unusual in bacteria. A preliminary review of the scientific literature has failed to find a similar example of regulators being co-transcribed. This unusual arrangement may suggest that these two genes may work in a coordinated fashion in the global gene regulation of S. pyogenes.

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