Molecular Biophysics of Dynamic Bacterial Nanomachines - PROJECT SUMMARY Interdisciplinary research in the Shaffer lab centers on discovering fundamental molecular mechanisms underpinning unilateral cargo delivery across biologically diverse membranes. Using the Helicobacter pylori cag type IV secretion system (cag T4SS) as a robust model system, our studies focus on understanding the biology and biophysics of intercellular DNA translocation through dynamic bacterial nanomachines assembled by evolutionarily divergent microbes. Although H. pylori is a significant carcinogenic bacterium that stimulates the development of infection-associated gastric malignancies, the projects outlined in this MIRA application do not seek to study determinants of bacterial pathogenesis or aspects of microbial carcinogenesis. Rather, our studies leverage the versatile cag T4SS nanomachine as a paradigm for understanding how a single molecular device transports disparate microbial payload into target eukaryotic cells in a physiologically relevant context. Whereas conjugative T4SS machineries secrete nucleoprotein complexes into recipient prokaryotes, the capacity to translocate a bacterial oncoprotein and an expanded repertoire of fragmented nucleic acids, lipopolysaccharide biosynthetic metabolites, and peptidoglycan substrates into human cells distinguishes the cag T4SS from other nanomachines. Our current and future studies are uniquely poised to fill long-standing knowledge gaps in T4SS biology to strengthen our understanding of mechanisms underscoring interbacterial and trans-kingdom cargo delivery though diversified machineries. Studies performed by my group over the past five years yielded novel molecular details of cag T4SS substrate translocation and led to the discovery of mechanisms underlying trans- kingdom DNA conjugation in the context of human infection. In the next five years, we will address three major deficiencies in our understanding of T4SS architecture, biogenesis, and function. First, despite many decades of research, we do not understand how the cag T4SS apparatus orchestrates cargo selection and secretion, and the mechanisms by which discordant payload is coupled to the translocation channel are unresolved. Second, while multiple bacterial nanomachines exhibit substructure symmetry mismatch, the biological significance of T4SS architectural asymmetry is unknown. Third, while DNA processing has been extensively investigated in prototypical interbacterial conjugation systems, the mechanisms governing trans-kingdom DNA transfer across donor and recipient membranes have not been elucidated in the context of human infection. Our studies will employ powerful chemical biology, genetic, and biochemical approaches in combination with in situ cryo-electron tomography (cryo-ET) and high-resolution fluorescence microscopy to extend our fundamental understanding of T4SS biology and eliminate numerous ‘black boxes’ that obscure the inner workings of these dynamic machines. Collectively, our multidisciplinary approach and collaborative efforts will continue to generate novel insights into T4SS structural and functional diversity and will provide a mechanistic framework for the development of innovative biotherapeutics for precision medicine.
