Exploring the role of scaffold proteins in controlling helical cell shape in Helicobacter pylori

Thesis (Ph.D.)--University of Washington, 2022Helicobacter pylori infects the stomachs of 50% of the world’s population and can cause gastric cancer and ulcers. There are many factors that contribute to H. pylori’s ability to colonize the stomach including the helical shape of H. pylori cells. The helical shape of H. pylori cells is required for robust infection of the stomach and promotes pathology, however, how the helical shape of H. pylori cells is patterned and maintained is unresolved. Additionally, how the helical cell shape of H. pylori enhances colonization and increases disease severity is not completely understood. Previous work identified helical- cell-shape-promoting protein complexes containing the peptidoglycan-hydrolase (Csd1), a peptidoglycan precursor synthesis enzyme (MurF), a non-enzymatic homologue of Csd1 (Csd2), non-enzymatic transmembrane proteins (Csd5 and Csd7), and a cytoplasmic bactofilin (CcmA). Bactofilins are highly conserved, spontaneously polymerizing cytoskeletal proteins in bacteria. During my investigations, I sought to understand specifically how two of the scaffold proteins, CcmA and Csd5, were involved in generating the helical shape of H. pylori cells.Using a combination of CcmA deletion analysis, in vitro polymerization, and in vivo co- immunoprecipitation experiments we identified that the bactofilin domain and N-terminal region of CcmA are required for helical cell shape and the bactofilin domain of CcmA is sufficient for polymerization and interactions with transmembrane proteins Csd5 and Csd7. We also found that CcmA’s N-terminal region inhibits interaction with Csd7. Deletion of the N-terminal region of CcmA increases CcmA-Csd7 interactions and destabilizes the peptidoglycan-hydrolase Csd1. Using super-resolution microscopy, we found that Csd5 recruits CcmA to the cell envelope and promotes CcmA enrichment at the major helical axis. Additionally, we identified that in WT H. pylori and surprisingly, in ∆csd5 cells, tetrapeptides in the cell wall are organized in a helical band that wraps around the cell. The SH3 domain of Csd5 binds to tetrapeptides to direct Csd5 and CcmA to the region of the cell wall with increased levels of tetrapeptides. We hypothesize that together Csd5 and CcmA organize several other cell-shape-determining proteins and peptidoglycan synthesis machinery to coordinate increased levels of cell wall modification and synthesis where tetrapeptides are enriched to promote the curvature required to build a helical cell