Year

2006

Degree Name

Doctor of Philosophy (PhD)

Department

School of Biological Sciences - Faculty of Science

Abstract

Streptococcus pyogenes (group A Streptococcus; GAS) is a Gram-positive human pathogen responsible for numerous life-threatening diseases, including necrotising fasciitis and streptococcal toxic shock syndrome (STSS). The non-suppurative sequelae of acute rhematic fever (ARF) and acute post-streptococcal glomerulonephritis (APSGN) may develop upon repeated exposure to GAS. Over the last two decades, there has been a world-wide resurgence in GAS infection. In this study, a proteomic analysis was undertaken to identify the major cell wall-associated proteins of GAS. Mutanolysin cell wall extracts from GAS strain NS931 (serotype M69), NS13 (serotype M53) and S43 (serotype M6) were separated by two-dimensional gel electrophoresis and the landmark proteins identified by matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. A total of 155 protein spots representing 74 distinct cell wall-associated proteins were identified, with molecular masses ranging from 14.4 to 77.5-kDa and a pI range of 4.4 to 7.9. Of these 74 proteins, nine putative virulence determinants (including M protein, SpeB, SpeM, FcrA, arginine deiminase, superoxide dismutase, C3-degrading protease and the two plasminogen-binding proteins SEN and Plr/GAPDH), eight glycolytic enzymes, eight carbohydrate metabolism enzymes, two chaperonins and four proteins with transporter function were detected in one or more of the GAS strains examined. Two-dimensional immunoblot analysis with pooled GAS-reactive human antisera revealed that thirty-three of these proteins were immunoreactive. Biotinylation of the GAS cell surface prior to two-dimensional western blotting identified a subset of twenty-three cell wall-associated proteins that are surface-exposed. These data illustrate the usefulness of proteomics in analysing the cell surface topology of GAS, which lays the foundation for future work identifying new GAS virulence determinants and vaccine antigens. The multifunctional chaperone and serine protease HtrA (DegP) is involved in the refolding and degradation of aberrant proteins destined for secretion. In numerous pathogenic organisms HtrA is required for thermostability, resistance to oxidative stress and full virulence. The HtrA of human pathogen S. pyogenes influences the maturation of secreted virulence factors streptolysin S and streptococcal pyrogenic exotoxin B (SpeB), and is localised at the ExPortal secretory microdomain. In this study, we examined the role of HtrA in regulating expression of GAS virulence phenotypes. An in-frame htrA deletion mutant displayed similar growth kinetics to the wild-type (wt) in the presence of paraquat-induced oxidative stress. Lack of HtrA did not influence capsule production or GAS adherence and invasion of human epithelial cells. Two-dimensional gel electrophoresis detected elevated levels of cleaved M protein fragments in the htrA mutant culture supernatant. The HtrA-mutant also had higher levels of cell wall-associated M protein than the wt. Time-course analysis of culture supernatant showed that the degradation rate of several virulence factors, including M protein, streptokinase, streptococcal enolase, superoxide dismutase A and glyceraldehyde-3-phosphate dehydrogenase, was reduced in the htrA mutant. This degradation corresponded to a lag in the processing of the 40-kDa SpeB zymogen to the active 28-kDa form. In vitro protease assays demonstrated that proteolytically active HtrA was unable to process SpeB zymogen to the active protease. These data indicate that HtrA is essential for the efficient maturation of SpeB zymogen into the active protease and the subsequent SpeB-mediated degradation of surface and secreted GAS virulence factors. The globally disseminated S. pyogenes M1T1 clone causes a number of highly invasive human diseases. The transition from local to systemic infection occurs by an unknown mechanism; however invasive M1T1 clinical isolates are known to express significantly less of the cysteine protease SpeB than M1T1 isolates from local infections. Here, we show that in comparison to the M1T1 strain 5448, the isogenic mutant ∆speB accumulated 75-fold more human plasmin activity on the bacterial surface following incubation in human plasma. SpeB was shown to degrade several bacterial and host molecules that contribute to the accumulation of bacterial surface plasmin activity. Human plasminogen was an absolute requirement for M1T1 strain 5448 virulence following subcutaneous infection of humanised plasminogen transgenic mice. S. pyogenes M1T1 isolates from the blood of infected humanised plasminogen transgenic mice expressed reduced levels of SpeB in comparison with the parental 5448 used as inoculum. The M1T1 mutant ∆speB displayed markedly reduced virulence in this transgenic model, demonstrating a requirement for SpeB at the site of infection. We propose that the human plasminogen system plays a critical role in group A streptococcal M1T1 systemic disease initiation. Although SpeB is required for S. pyogenes M1T1 survival at the site of local infection, it also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system. Therefore, loss of SpeB activity in a sub-population of S. pyogenes M1T1 at the site of infection results in accumulation of surface plasmin activity thus triggering systemic spread.

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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.