Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Secretion of toxins into host cells is an important component of microbial pathogenesis. In order to gain access to the host cell cytosol, toxins must cross the plasma membrane, traverse the membrane of endocytic vesicles, or cross the membrane of organelles. The process cytolysin-mediated translocation: CMT) in Streptococcus pyogenes uses the pore-forming protein streptolysin O: SLO) to translocate the S. pyogenes NAD+-glycohydrolase: SPN) effector into the cytosol of eukaryotic host cells. Although, the fundamentals of this protein translocation process are established the details of the mechanism remain elusive. The current studies illustrate that the translocation process is more complex than initially projected. It was thought that the effector protein, SPN, entered the host cell cytosol through the lumen of the translocator, SLO. In this thesis, mutational analysis demonstrates that SPN translocation into the host cell does not require SLO pore formation. Furthermore, the SLO pore-forming mutant and wild type strains use an indistinguishable pathway to translocate SPN. While the pore-forming mutant can translocate SPN, the cytotoxic affects of CMT occur only when SLO forms pores and translocates SPN. These studies illustrate that SLO pore formation can occur independently of SPN secretion and that there is a synergistic effect between these two SLO activities. Further studies using pharmacological inhibitors to probe the involvement of the host cell during CMT indicate that clathrin-dependent endocytosis does not play a role. The carboxyl-terminus of SLO makes contact with the host cell membrane through an area denoted as Domain 4. Mutational studies indicate that expression of Domain 4 from the related pore-forming protein, perfringolysin O: PFO), is insufficient for CMT. Moreover, CMT is unaffected by extraction of cholesterol to levels that block membrane binding by the cholesterol-dependent PFO protein. Although cholesterol is unnecessary, mutations that interrupt cholesterol binding by SLO indicate the sterol does increase the efficiency of CMT. Furthermore, a loop region within Domain 4 is important and provides SLO with its specificity in CMT. Taken together, even though SLO binds to cholesterol, there might be an additional membrane receptor necessary for CMT. In total, the research described in this thesis furthers the knowledge of the CMT mechanism and provides opportunities for future investigation into the role of CMT in infections.
Magassa, N'Goundo, "Characterization Of Streptolysin O As The Translocator For The Cytolysin-Mediated Translocation Process In Streptococcus Pyogenes" (2010). All Theses and Dissertations (ETDs). 221.