How H. pylori gets its shape
Helicobacter pylori is the primary cause of stomach cancer, which is the third leading cause of cancer deaths worldwide. The helical shape of the bacterium is necessary for efficient stomach colonization. Thus, if researchers can understand the mechanisms by which H. pylori achieves its shape, they may be able to identify novel therapeutic strategies.
Previously, Fred Hutchinson Cancer Research Center faculty member Dr. Nina Salama and her team used genetic screening to identify a set of proteins that, when deleted, resulted in distinctly non-helical cell shapes.
Salama and her team wanted to find other proteins that interact with one of the shape-forming proteins they’d identified in their earlier work, called Csd5. Mass spectrometry-based proteomics is the primary technology for identifying protein-protein interactions. But the Salama Lab is not well-versed in the complexities of the technology or how to obtain optimal results.
The Proteomics & Metabolomics shared resource at Fred Hutch offered the technological expertise the Salama Lab needed to answer their questions about Csd5. Salama’s team worked in conjunction with the core to design and carry out immunoprecipitation mass spectrometry, or IP-MS, experiments using Csd5 fusion constructs to identify proteins that interact with Csd5. The core provided high-resolution mass spectrometry experiments and data analysis to identify protein-protein interactors. This research would not have possible without access to an experienced proteomics facility.
These experiments identified MurF, a cytoplasmic proteoglycan synthesis enzyme, and CcmA, a known H. pylori cell-shape protein, and they showed enrichment for nearly all the components of the F1F0 ATP synthase. The team validated these interactions using reciprocal IPs with western blotting detection.
The researchers also conducted IP-MS experiments using domain deletions of Csd5. Those results strongly suggested that the N-terminal and transmembrane domains of Csd5 are required for interactions with MurF, CcmA and ATP synthase.
Finally, the team’s IP-MS experiments in mutant strain backgrounds strongly suggested Csd5, MurF and CcmA interact individually and/or together with ATP synthase.
Ultimately, this research has built a model in which Csd5 promotes helical shape as part of a membrane-associated, multiprotein complex that includes interactions with the periplasmic cell wall, a proteoglycan precursor synthesis enzyme, the bacterial cytoskeleton and ATP synthase. Moreover, it has led to additional proteomic work to identify more of CcmA’s interaction partners — suggesting that these bacteria have an additional subcomplex for generating a helical cell shape that, together with the Csd5 complex, form a larger “shapeosome.”
Read the team’s papers on these findings:
Blair KM, Mears KS, Taylor JA, et al. The Helicobacter pylori cell shape promoting protein Csd5 interacts with the cell wall, MurF, and the bacterial cytoskeleton. Mol. Microbiol. 2018;110:114-127. doi:10.1111/mmi.14087
Yang DC, Blair KM, Taylor JA, et al. A genome-wide Helicobacter pylori morphology screen uncovers a membrane-spanning helical cell shape complex. J. Bacteriol. 2019;201(14):e00724-18. doi: 10.1128/JB.00724-18