The helix is a popular shape in engineering. It is used in our screws to ensure a strong hold, in our springs to store mechanical energy, even in our pastas to capture the right amount of sauce. It is perhaps no surprise that nature, too, has discovered many uses for the helix. The most famous is the DNA double helix, but some organisms have gone so far as to evolve helical anatomy. Take, for example, the bacteria Helicobacter pylori. This little corkscrew of a cell is an extremely successful human pathogen – it’s infected the stomachs of a few billion people worldwide and is the major cause of gastric ulcers and cancers. And the helical shape of H. pylori is no mere curiosity – in fact, it is key to the bacteria’s infectiousness. Despite its importance, little is known of how H. pylori twists itself into this shape. Dr. Nina Salama, Professor in the Human Biology, Basic Sciences, and Public Health Sciences divisions, Dr. Penny E. Petersen Memorial Chair for Lymphoma Research at Fred Hutch, and Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member, is driven to understand how this bacterium gets its shape and how it underlies disease. In a new research article in eLife, Dr. Salama, former graduate student Dr. Sophie Sichel, and collaborator Dr. Benjamin Bratton of Vanderbilt University examined the molecular basis of H. pylori helix formation.
Several interacting proteins are known to be required for H. pylori helicity; most of them localize to the cell envelope and are involved in forming or modifying the peptidoglycan (PG) cell wall. However, how these proteins shape the cell remains mysterious. Dr. Salama’s group chose to focus on one of these proteins: the bactofilin CcmA. “Bactofilins are cytoskeletal elements that, through mutational analysis, are known to regulate changes in cell morphology or synthesis of cell appendages formed by the cell wall,” explains Dr. Salama. To dissect how this protein regulates cell shape, the authors first broke the protein down into three domains – the N-terminal, C-terminal, and Bactofilin domains – and asked which was necessary for this process. They found that removing the N-terminal domain resulted in straight cells, while removing the C-terminal domain did not affect cell shape – pointing to the N-terminus as housing critical protein segments. Next, they examined the roles of these domains in another key property of the protein – the ability to polymerize into large filament bundles. Removing the N-terminal and C-terminal domains also had quite different effects on polymerization: loss of the N-terminal domain strongly disrupted filament bundling, while loss of the C-terminal domain appeared to increase bundling. These results suggest that the ability to properly polymerize may be important to CcmA’s function.
Next, the authors examined whether CcmA’s N-terminal and Bactofilin domains interact with other proteins implicated in helix formation. Using co-immunoprecipitation, they identified interactions with two such proteins, Csd5 and Csd7. The Bactofilin domain interacts with both proteins, while the N-terminal domain modifies these interactions by inhibiting the association with Csd7, suggesting that striking the right balance of association with these two factors might influence cell shape. Why might this be the case? Importantly, the group also identified an indirect effect of CcmA on another key protein in shaping the cell – the PG degradation factor Csd1. They found that when the CcmA-Csd7 interaction is allowed it prevents Csd7 from interacting with Csd1 and ultimately destabilizes the Csd1 protein, which likely contributes to shape defects in this condition.
Finally, the group asked how these interactions affect the localization of CcmA. Using a sensitive, high-resolution microscopy approach, they mapped the localization of CcmA in individual bacterial cells and showed that it is normally found on the outside of the helix in a region called the major axis. Deleting CcmA’s binding partners disrupted this localization, suggesting that getting CcmA and its interacting proteins to the correct place is key to its proper function. Perhaps, the authors speculate, the localization of CcmA creates differential regulation of PG synthesis and degradation along the major and minor helical axes, twisting the cell into shape.
While previous genetic data has implicated many proteins in H. pylori helix formation, Dr. Salama’s work is beginning the unravel how the complex interactions between these proteins work to ensure that they are present at the right place and time within the cell to regulate cell shape. Important questions regarding the functions of these proteins remain, she says. Among them, she notes, are: “[if] CcmA is recruited to the cell envelope and the major helical axis by Csd5…how does Csd5 get to this location?” and “how does spatial/temporal regulation of Csd1 hydrolase activity [by CcmA] promote helical cell shape? We hypothesize that this could allow for sustained PG insertion at the helical axis, but more work needs to be done to show that is true.”
This work was supported by the National Institutes of Health, the GO-MAP Graduate Opportunity Program, and the VUMC Discovery Scholars in Health and Medicine Program.
Fred Hutch/UW Cancer Consortium member Nina Salama contributed to this work.
Sichel SR, Bratton BP, Salama NR. Distinct regions of H. pylori's bactofilin CcmA regulate protein-protein interactions to control helical cell shape. Elife. 2022 Sep 8;11:e80111. doi: 10.7554/eLife.80111. PMID: 36073778; PMCID: PMC9507126.