In Situ Architecture of Specialized Bacterial Secretion Systems

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Bacteria have evolved specialized nanomachines functioning as secretion systems to deliver proteins or DNA from the bacterial cytoplasm to the surrounding milieu or into other eukaryotic or bacterial target cells. To date, nine different types of bacterial secretion systems have been identified. The most widely-distributed and versatile of these, the type IV secretion systems (T4SSs), traverse the cell envelopes of many Gram-negative and -positive species. Members of one large subfamily, the DNA transfer or conjugation systems, are medically problematic because they deliver mobile genetic elements (MGEs) and their cargoes of antibiotic resistance genes and virulence determinants among bacterial populations; these systems also elaborate conjugative pili or other surface adhesins that promote establishment of robust, antibiotic-resistant biofilm communities. A second T4SS subfamily, the ‘effector translocators’ are deployed by many medically- important pathogens to deliver protein effectors across the cell envelope either to the surrounding milieu or into eukaryotic host cells to incite infection. By use of in situ cryo-electron tomography (Cryo-ET), I have recently solved the structures of three different T4SSs, the Legionella pneumophila Dot/Icm, Escherichia coli F plasmid Tra, and Helicobacter pylori Cag systems within their natural cell envelopes. These new structures are changing existing paradigms for how T4SSs are architecturally configured, they present the first clear views of central substrate translocation channels, and they identify novel F-encoded structures configured as basal platforms for F pili. Type IX secretion systems (T9SSs), which are found mainly in the phylum Bacteroidetes, also play critical roles in infection. Porphyromonas gingivalis, for example, deploys its T9SS to secrete gingipain proteinases and virulence factors to incite periodontal disease. Very recently, I solved the structure of this T9SS in its natural cellular context by in situ Cryo-ET. This large (~50 nm diameter), envelope-spanning nanomachine differs markedly from any other bacterial secretion systems visualized to date. In this MIRA proposal, I seek to comprehensively define the structures and subunit compositions of the F plasmid Tra and H. pylori Cag T4SSs and the P. gingivalis T9SS by addressing key unresolved questions that are ideally or uniquely approachable using in situ Cryo-ET. We will i) solve in situ structures with emphasis on regions of these nanomachines such as the inner membrane complexes, translocation channels, and machine - pilus junctions that have not been amenable to structural analyses using in vitro approaches, ii) leverage our resources through collaborations with experts in the T4SS and T9SS fields to place our structural findings in broader mechanistic and biological contexts, and iii) refine methods for data collection and processing to improve the resolution limits of in situ Cryo-ET. Our studies will generate important new insights into the architectures, biogenesis, and mechanisms of action of bacterial secretion nanomachines, and set the stage for design of intervention therapies.