Year

2006

Degree Name

Masters by Research

Department

Department of Biological Sciences - Faculty of Science

Abstract

Streptococcus pyogenes (group A streptococcus; GAS) is a Gram-positive bacterium that causes invasive and non invasive infections in human skin and mucosal tissue. Since the mid-1980s, a conspicuous increase in the incidence and severity of invasive infections caused by GAS has been observed worldwide. S. pyogenes infections are endemic amongst many Aboriginal communities of Northern Australia. With rates of infection in these communities being amongst the highest in the world, there is an urgent need for identifying the key virulence factors involved in the initiation and progression of invasive GAS diseases. Streptokinase, a secreted group A streptococcal protein, activates host plasminogen converting it to the broad spectrum protease plasmin. Several plasmin(ogen) binding receptors present on the GAS cell surface facilitate the acquisition of cell surface plasmin activity. The gene encoding encoding streptokinase (ska) is present in all GAS isolates. In this study, allelic variants of ska have been characterised in various GAS isolates from the Northern Territory of Australia. The region encoding the � domain of ska in these isolates was sequenced and phylogenetically analysed to identify the specific allelic variant genotype for each isolate. Culture supernatants of these strains were examined for the presence of streptokinase and the cysteine protease, SpeB, via immunoblotting using rabbit polyclonal antisera directed against these proteins. Two GAS strains (ALAB49 and 5448) and their isogenic ska and speB mutants were used to demonstrate the specificity and applicability of this technique for detecting the presence of streptokinase and SpeB in the culture supernatants. Using this methodology, streptokinase expression in culture supernatants of NT GAS isolates was investigated. Streptokinase was found to be expressed in mid-log, late-log and early stationary growth phases for all NT isolates examined in this study. It was also found that different ska alleles expressed streptokinase proteins of variable molecular weights. The streptokinase expressed by strains possessing alleles of ska 1 cluster (NS53, NS730 and NS931) and strains possessing alleles of ska 2a sub-cluster (NS696, 5448) express a 48 kDa streptokinase protein and strains possessing alleles of the ska 2b sub-cluster (NS13, ALAB49, NS88.2 and NS455) express a 50 kDa streptokinase protein. Culture supernatants from all GAS strains in early stationary growth phase were found to contain pro-SpeB (inactive precursor). Mature SpeB was only detected in the early stationary phase culture supernatants of strain NS53. Examples of the three known allelic ska variants were cloned from strains NS931, 5448 and ALAB49. DNA sequence analysis of these clones showed a high percentage of identity with previously published ska sequences. The recombinant plasmid pSka1 (containing the ska insert from NS931) was expressed and purified. The yield of the purified protein obtained was 1.16 mg/ml. Future work will determine the functional differences between the recombinant proteins expressed by these clones and it is hoped that this will help gain a better understanding of the role of the allelic variation of streptokinase in GAS disease virulence.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.