Proteomics & Metabolomics Case Study

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. 

The Challenge

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 Approach

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.

Graphic illustration of the Salama Lab’s current model of how the shapeosome patterns H. pylori’s helical shape. It shows the role of structural protein Csd2, proteoglycan hydrolase Csd1, predicted bactofilin CcmA – a class of spontaneously polymerizing cytoskeletal proteins -- Csd5, structural protein Csd7, scaffolding protein SH3 which interacts directly with proteoglycan, and essential proteoglycan precursor enzyme MurF in the proteoglycan cell wall of the bacterium.
The current model of how the shapeosome patterns helical shape in H. pylori, in which Csd5 promotes helical shape as part of a membrane-associated, multiprotein complex that includes interactions with the periplasmic cell wall, a proteoglycan (PG) precursor synthesis enzyme, the bacterial cytoskeleton and ATP synthase. Graphic by the Salama Lab, Fred Hutch Click for high-resolution image

The Outcome

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 More

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

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“The great thing about working with Phil and the rest of the proteomics staff is they engaged at all levels: study design, optimization and data analysis. They also provided valuable training to trainees to both enhance their understanding of the data and to become proficient in doing independent analyses.”

– Dr. Nina Salama, Professor, Human Biology and Public Health Sciences divisions, and holder of the Dr. Penny E. Petersen Memorial Chair for Lymphoma Research, Fred Hutch