Doctor of Philosophy
School of Biological Sciences, Faculty of Science
Hollands, Andrew, Initiation of invasive disease in M1T1 group A streptococcus, Doctor of Philosophy thesis, School of Biological Sciences, Faculty of Science, University of Wollongong, 2009. https://ro.uow.edu.au/theses/3070
Streptococcus pyogenes (group A streptococcus; GAS) is an important human pathogen that colonizes epithelial and mucosal surfaces. Group A streptococcal disease can be relatively minor, such as streptococcal pharyngitis, or severe and life-threatening, such as necrotizing fasciitis. There has been a resurgence of severe infection with GAS since the mid-1980s that has been paralleled be the emergence of a globally disseminated clone, M1T1. The M1T1 clone of GAS presents as the most common cause of streptococcal pharyngitis in developed countries and are also overrepresented in cases of severe infection. Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal or no symptoms. Although phenotypic or genetic correlates underlying a bacterium’s shift to enhanced virulence potential have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of GAS is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the group A streptococcal control of virulence regulatory sensor kinase (covR/S) operon are associated with severe invasive disease, abolishing expression of a broad spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. This study describes how a bacteriophage-encoded group A streptococcal DNase (Sda1), which facilitates the pathogen’s escape from neutrophil extracellular traps (NETs), can serve as a selective force for covR/S mutation. The results provide a paradigm whereby horizontal gene transfer and natural selection exerted by the innate immune system iv generate hypervirulent bacterial variants with increased risk of systemic dissemination. This study sought to investigate if there was a cost of fitness associated with covR/S mutation that counterbalances the dramatic increase in virulence. It was found that covR/S mutant bacteria had reduced capacity to bind fibronectin and collagen, both components of the extracellular matrix bound by streptococcal adhesins. The covR/S mutant strain examined in this study also showed reduced capacity to bind to epithelial cell layers as a consequence of increased capsule expression. This mutant strain displayed reduced capacity to form biofilms. An animal model of skin colonization was used to show that the covR/S mutant strain has a colonization defect. This reduced capacity to colonize presents an explanation as to why hypervirulent covR/S mutant M1T1 group A streptococci are not rapidly spread amongst the community. The role of SpeB in the course of infection is still unclear. This study utilized a SpeB-negative M1T1 clinical isolate, 5628, with a naturally occurring mutation in the gene encoding the regulator RopB, to elucidate the role of RopB and SpeB in systemic virulence. Allelic exchange mutagenesis was used to replace the mutated ropB allele in 5628 with the intact allele from the well characterized isolate 5448. The inverse allelic exchange was also performed to replace the intact ropB in 5448 with the mutated allele from 5628. An intact ropB was found to be essential for SpeB expression. While the ropB mutation was shown to have no effect on haemolysis of RBCs, extracellular DNase activity or survival in the presence of neutrophils, strains with the mutated ropB allele were less virulent in murine systemic models of v infection. An isogenic SpeB knockout strain containing an intact RopB showed similarly reduced virulence. Microarray analysis found genes of the SpeB operon to be the primary target of RopB regulation. These data show that an intact RopB and efficient SpeB production are necessary for systemic infection with GAS.
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