Cell surface biogenesis in Streptococcus pneumoniae

PROJECT SUMMARYThe cell surface of pathogenic bacteria contains many key virulence factors that are used to interface with thehost. Cell surface polymers also contain the molecular signatures recognized by the innate immune system toactivate a defensive response, and surface molecules or their biogenesis pathways serve as important targetsfor many of our most effective vaccine and antibiotic therapies. A better understanding of the mechanismsresponsible for bacterial surface assembly will therefore impact virtually all areas of pathogenesis research andinform the development of new treatments for infections. Although some aspects of cell surface assembly canbe inferred from studies of non-pathogenic organisms, results from the models will never be entirely predictive.Departures from the model are likely to be especially pronounced for pathogens like Streptococcuspneumoniae (Sp) that adopt a different (ovoid) morphology and grow via distinct mechanisms from rod-shapedorganisms like Escherichia coli and Bacillus subtilis where studies of cell surface assembly have traditionallybeen investigated. Sp is a major cause of life-threatening disease in young children and older adults, and theincidence of drug-resistant infections with this organism is on the rise. The efficacy of the polyvalent Spvaccine is also declining due to the emergence of strains with altered surface polysaccharides that escapevaccine-induced immunity. It is therefore important to identify new ways of disabling Sp growth. To do so, wehave initiated a program to investigate cell surface assembly in Sp that leverages the joint expertise of theRudner and Bernhardt laboratories in cell wall biogenesis, gram-positive biology, microscopy, biochemistry,and genetics. Importantly, our approach is not limited to the characterization of homologs of well-studied cellmorphogenesis factors from the rod-shaped model organisms. Instead we are taking advantage of forwardgenetic screens powered by modern sequencing methods to discover new players and biological mechanismsinvolved in Sp growth. Our preliminary genetic analyses have uncovered two novel regulators of the penicillin-binding proteins (PBPs) of Sp. These are the first set of factors identified in gram-positive bacteria that controlthe activity of these critical cell wall synthases. The first aim of the project will investigate the mechanism bywhich these factors modulate PBP activity and connect the function of these enzymes with other componentsof the morphogenetic system. In addition to controlling PBP activity, proper surface assembly also requires theregulation of enzymes that cleave the cell wall. The factors governing the activity of these enzymes are poorlyunderstood in all bacteria. Our second aim will build on promising results where we have identified a regulatoryrole for surface polymers called lipoteichoic acids (LTAs) in controlling the activity of the cell wall hydrolaseLytA responsible for Sp cell lysis following beta-lactam treatment. Overall, our results promise to uncover newways of either blocking cell wall assembly or triggering autolysis for therapeutic development.