Lancefield group A streptococci (Streptococcus pyogenes) are one of the most prevalent human pathogens (25 million infection/year) capable of causing a variety of severe suppurative infections, as well as non-suppurative sequelae such as post-streptococcal glomerulonephritis and rheumatic fever. In addition, group A streptococci were shown to induce a lethal disease that resembles toxic shock syndrome. Within the last decade, an increase in the number of invasive group A streptococcal infections associated with bacteremia deep soft-tissue infection, shock, and multi- organ failure were observed in patients with no prior underlying disease. Group A and C streptococci have the ability to surround themselves with a capsule formed by hyaluronic acid which serves as a virulence factor. The structure of streptococcal hyaluronic acid is identical to mammalian hyaluronic acid where it is an ubiquitous component of connective tissue. The overall objective of this research program is to continue a systematic study of the Streptococcus pyogenes hyaluronic acid synthase complex in order to better understand the mechanism of expression of this capsulae in the human host. Towards this objective they plan to : 1) Isolate and sequence the hyaluronate synthase (has) capsule operon. Determine which genes of the operon are required for production of capsule in group A streptococci and analyze group A and non-group A streptococci for these genes; 2) determine the function of the genes required for hyaluronic acid capsule biosynthesis. Overexpress and purify the has A product to determine whether it is the intact synthase or a component thereof; 3) determine whether the has operon transcript is regulated via transcriptional or post- transcriptional mechanisms. Dot-blot analyses, Northern blots, and RNAase protection assays will be used to resolve the amount and size of has operon mRNA present in strains of group A streptococci during various stages of growth; and, 4) analyze the cis and trans elements which regulate the has operon. Promoter sequence(s) and transcription accessory factors will be determined by sequencing of the 1 kb region upstream of the has A promoter, and DNAase 1 footprinting of the promoter region. If post-transcriptional mechanisms regulate the expression of the hyaluronic capsule, factors such as mRNA stability, the presences of inhibitory enzymes, or mutations within the operon will be investigated. The completion of this aims will also provide investigators with a biochemical analysis of the synthase complex and a better understanding of the expression of this important bacterial and mammalian mucopolysaccharide.
