Role of rocA Polymorphisms on Virulence and Pathogenesis of Serotype M28 Group A Streptococcus
Abstract
Group A Streptococcus (Streptococcus pyogenes, GAS) is a human-specific pathogen that causes a wide range of diseases and significant morbidity and mortality worldwide. Despite over a century of research, no licensed human vaccine exists to protect against GAS infection, which may be due, in part, to the complex regulation of its many virulence factors and surface exposed proteins. Several virulence regulators have been well described for GAS, including the CovRS (control of virulence regulator/sensor) two-component system. CovRS is a negative regulator of virulence in GAS. That is, CovS phosphorylates CovR to repress virulence gene expression. Whereas CovRS has been extensively studied over the past two decades, my research efforts have focused on RocA (regulator of Cov), an accessory protein to the CovRS two-component system. RocA is believed to interact with CovS to increase CovR phosphorylation, although the molecular mechanism of this phenomenon remains unknown.
Recent whole-genome population-based sequencing studies of serotype M28 GAS invasive disease clinical isolates identified an unusually high number of polymorphisms in rocA compared to other serotypes. Thus, I hypothesized that amino acid changes in RocA result in altered RocA-RocA and RocA-CovS protein interactions, giving rise to an altered global transcriptome and increased virulence in serotype M28 GAS. I used naturally occurring clinical isolates and constructed isogenic rocA GAS mutant strains for transcriptome sequencing (RNA-seq), in vitro virulence factor assays, and in vivo animal infection model studies. Additionally, I performed substituted cysteine accessibility method as applied to transmembrane orientation (SCAMTM) to determine the membrane topology of RocA and bacterial adenylate cyclase-based two-hybrid (BACTH) assays to study RocA protein-protein interactions.
My results demonstrate that deletion of rocA results in a significantly altered GAS transcriptome and significantly increased virulence in vivo. Naturally-occurring polymorphisms in rocA also altered the global GAS transcriptome and increased strain virulence. The observed virulence phenotypes are the result of altered RocA-RocA and RocA-CovS protein interactions. Taken together, the data add important new understanding of the molecular pathogenesis of RocA in serotype M28 GAS.
Citation
Bernard, Paul Edward (2022). Role of rocA Polymorphisms on Virulence and Pathogenesis of Serotype M28 Group A Streptococcus. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /197080.