Mechanisms of Bacterial Cell-Cell and Cell-Host Communication
Bacterial cells use secretion processes to deliver protein and DNA molecules to other bacterial, plant or animal cells. E. Zechner’s research addresses the molecular mechanisms used by a particular class of secretion systems, the Type IV group, which contributes to virulence of several prominent pathogens and the cell to cell gene transfer process known as bacterial conjugation. The initiation stage of conjugative DNA transfer is studied using biochemical, biophysical, and molecular genetic approaches to gain insights to regulation of the initial cell contact, the nature and mechanisms of exocellular signal perception, enzymatic processing of DNA molecules destined for transfer, and activation of the multiprotein transport machinery ATPases.
Our research activities expanded in Funding Period 3 to include production, secretion and cytotoxicity of the small toxic metabolite tilivalline produced by a Gram-negative member of the human microbiota. This work aims to understand the non-ribosomal peptide synthase-mediated biosynthesis of tilivalline, its host cell receptor, its bioactivity in in vitro models of intestinal epithelial cells, and its direct role in the development of antibiotic associated hemorrhagic colitis in animal models of disease.
Laboratory know-how and infrastructureThis laboratory’s strength is in nucleic acid enzymology, particularly in the enzymology of DNA replication and molecular mechanisms of bacterial horizontal gene transfer. Molecular genetics for structure-function studies, as well as the overexpression and purification of proteins from bacterial cells are routine procedures. The ZMB is well equipped for these tasks including French press, ultracentrifuge, FPLC protein purification systems, electrophoresis facilities for protein analysis and immunochemistry. Biochemical analysis of reconstituted protein-ligand binding, DNA supercoiling, DNA transesterase, DNA unwinding, ATPase, and protein AMPylation activities are performed with and without radiolabeled substrates. Fluorescence microscopy is available in house and electron microscopy is accessible on campus. Recent work with pathogen secretion systems dedicated to human and animal infection, as well as toxin production, has expanded competence in pathogenomics, cell biology, and models for bacterial adherence, colonization and infection. A superb animal facility maximizes the quality of our in vivo models.