Oral Presentation MedVetPATHOGENS 2018

In Vivo Structures of a Bacterial Nanomachine Revealed by Electron Cryotomography (#34)

Carrie Shaffer 1
  1. University of Kentucky, Lexington, KY, United States

Numerous bacteria use sophisticated molecular machines to deliver microbial cargo to target cells. One versatile apparatus, the type IV secretion system (T4SS), is a cell-surface nanomachine that traffics diverse macromolecular substrates between microbes and into eukaryotic cells. T4SS-dependent mechanisms play a pivotal role in bacterial pathogenesis and survival within distinct host niches, and we have only recently begun to decipher the extraordinary structural and functional diversity that exists among T4SS machineries. The cag T4SS is deployed by carcinogenic Helicobacter pylori to inject a diverse repertoire of immunostimulatory substrates into gastric epithelial cells, including the oncoprotein CagA, chromosomally-derived nucleic acid, fragments of peptidoglycan, and lipopolysaccharide biosynthesis metabolites. Translocated cargo activates components of the innate immune system and dysregulates signaling pathways that influence the development of gastric disease. Electron cryotomography (ECT) has emerged as a powerful technology that enables the three-dimensional resolution of intact cellular structures in a near-native state. Using ECT to visualize the host-pathogen interface, we resolved the molecular architecture of the H. pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. We discovered that when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as barrel-like densities within the cytoplasm. Collectively, these studies unveiled the structure of a dynamic molecular machine that evolved to function in the human stomach. Current research efforts focus on mapping the topology of components within the cag T4SS apparatus and applying ECT to visualize the ultrastructure of nanomachines assembled by veterinary pathogens with the potential for zoonotic transmission.