Role of penicillin binding protein 1a in GBS virulence

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DESCRIPTION (provided by applicant): Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia and meningitis in the USA despite CDC-recommended chemoprophylaxis strategies for preventing infection due to this organism. Apart from the capsule, the factors required for survival of GBS in the host are not well defined. Recently, signature-tagged transposon mutagenesis (STM) was used to identify genes required for growth and survival of GBS in a neonatal rat sepsis infection model. A significant proportion of the avirulent mutants had transposon insertions in genes involved in cell surface metabolism emphasizing the significance of these functions for in vivo survival of GBS. We characterized the most attenuated mutant from the cell-surface metabolism group, which had a transposon insertion in a putative penicillin-binding protein gene (ponA) homologue. Based on sequence homology, the disrupted GBS gene is predicted to code for a class A, high molecular weight penicillin-binding protein (PBP1a), possessing both transglycosylase and transpeptidase activity. These bifunctional enzymes catalyze both the polymerization and cross-linking of bacterial peptidoglycan. The PBP1a gene mutant was significantly attenuated in both competitive index and 50 percent lethal dose assays of GBS virulence in neonatal rats. Additionally, the PBP1a gene mutant displayed a significant defect in resistance to opsonophagocytic killing as measured by in vitro bactericidal assays. Complementation analysis in vivo confirmed that the altered phenotypes observed in the mutant were due to the transposon insertion in ponA. The PBP1a gene mutant had a normal growth rate in vitro, produced wild-type levels of capsular polysaccharide and was otherwise phenotypically identical to the parent strain. We hypothesize that the GBS ponA gene is required for resistance to opsonophagocytic killing in vitro and virulence in vivo. Our investigation seeks to define the role of PBP1a in interactions with the host and virulence of GBS in vivo. Aim 1 will complete analysis of the ponA gene and the prfA gene (penicillin binding protein related factor A), a gene cotranscribed with ponA. A nonpolar prfA deletion mutant will be constructed and subjected to phenotypic analysis. Aim 2 will use genetic and biochemical approaches to define the structure and function of the PBP1a protein. Site-directed mutations that disrupt enzymatic activity of the protein will be introduced into PBP1a. Analysis of these mutants will allow us to evaluate whether enzymatic activity of the protein is required for virulence and resistance to opsonophagocytic killing. Aim 3 will determine the role of the PBP1a protein in resistance of GBS to opsonophagocytic killing by investigating the interaction of aponA mutant with serum opsonins and phagocytic cells. While bacterial cell-wall associated enzymes, including PBPs, have been reported to be required for virulence in numerous animal models of infection, no mechanism has been proposed to explain these observations. These studies will be the first to systematically investigate the role of PBP1a in virulence, will further our understanding of the pathogenesis of GBS infections, and may identify targets for preventative or therapeutic modalities.