How Helicobacter pylori stays in shape

From the Salama lab, Human Biology Division
Illustration of bacterium Helicobacter pylori (H. pylori)

The bacterium Helicobacter pylori (H. pylori) colonizes the human stomach and is responsible for a significant global cancer burden as the primary cause of stomach cancer.  H. pylori is shaped like a corkscrew, or helix, and the bacterium’s helical shape has been shown to be crucial for its ability to thrive in the stomach. This discovery was made by Dr. Nina Salama (Human Biology Division) and her colleagues in 2010 showing for the first time that the bacterium’s helical shape helps it colonize the stomach.  H. pylori colonization of the stomach is present in about half of the world’s population, and in a subset of infected people, causes chronic inflammation accountable for a variety of gastric disorders.  Since then, the Salama lab has sought to better delineate the mechanisms that define how H. pylori maintains its shape, with the hope that novel therapies may come in the form of disrupting the helical conformation to prevent infection.

In most bacteria, the shape of the organism is determined by the peptidoglycan (PG) cell wall, comprised of a meshwork of glycan chains held together by peptide crosslinks that surrounds the cytoplasmic membrane to provide protection from osmolysis. Since bacteria are encased in a cell wall, any form of bacterial growth, division, and change in cell shape requires an intricate coordination of PG hydrolases that cleave PG, as well as PG synthases that facilitates the rebuilding of the cell wall. Genetic screens in H. pylori have also reinforced these findings and identified proteins that function as PG hydrolases give rise to distinctly non-helical cell shapes when deleted. One of the non-enzymatic proteins, Csd5, was also identified in these screens. Csd5 is of interest because it is found exclusively in H. pylori and Helicobacter acinonychis, and while its deletion results in straight cells, there were no significant alterations to PG composition, leaving the mechanism by which Csd5 promotes helical shape a mystery.

To tackle this mystery and get at the mechanistic underpinnings of how the Csd5 protein promotes H. pylori helicity, members of Dr. Nina Salama’s lab and their collaborators performed a structure-function analysis of H. pylori Csd5.  Spearheaded by graduate student Kris Blair, the authors show that specific domains of Csd5 mediate its interactions with the cytoskeleton, cell wall, and PG precursor synthesis enzyme. The results of their study were published in a recent issue of Molecular Microbiology.

As a starting point, the authors took an in silico approach by mapping predicted Csd5 secondary structure features onto a multiple sequence alignment of diverse Csd5 variants from a selected list of H. pylori sequences to find distinct regions of high sequence conservation in the N and C-terminal regions of Csd5 most likely to have functional roles.  To assess the role of these domains on the overall function of Csd5, the authors generated a series of deletion variants and found that the SH3 domain was required for generating the helical shape of H. pylori, while the N-terminal and transmembrane domains are required to maintain Csd5 protein stability and function, and facilitate correct subcellular localization. 


Image of Schematic model of the Csd5 H
Schematic model of the Csd5 H. pylori cell shape promoting protein complex Figure from publication

Csd5 is unlikely to be a lone player in the orchestration of H. pylori cell shape.  As such, the authors looked for Csd5 protein interaction partners using immunoprecipitation (IP) and mass spectrometry in two new, independent Csd5 protein fusions. The top unique hit in the proteomic screen turned out to be MurF, a cytoplasmic PG precursor synthesis enzyme. Ccsd5 also interacts with CcmA, a bactofilin homolog previously shown to contribute to H. pylori cell shape. Components of the F1F0 ATP synthase were also identified in the proteomic screen to be interacting partners of Csd5. The authors proceeded to validate these candidate interactions by performing reciprocal IPs followed by western blotting to detect the presence of the candidate proteins. They also used the domain deletions to determine that the N-terminal and transmembrane domains are required for interaction. Taken together, Csd5, MurF, CcmA and ATP synthase indeed interact as part of a complex to maintain helical cell shape. The authors speculate that this shape-promoting protein complex spans the cytoplasmic membrane and connects the cell wall with the cytoskeleton.  

Given the association between PG synthesis, cell shape and intermediate filament proteins in curved organisms, the identification of Csd5 proteins with CcmA, a known cell shape protein and bactofilin, and with MurF, a known cell elongation factor, are not surprising.  Nonetheless, from this work, the authors discovered the first example of a gram negative SH3 domain interaction with PG and its essential role within a protein complex responsible for maintaining helical cell shape in H. pylori. Future work to further dissect these interactions will be crucial in gaining more insight into the variety of mechanisms H. pylori uses to stay in shape.

Blair KM, Mears KS, Taylor JA, Fero J, Jones LA, Gafken PR, Whitney JC, Salama NR. 2018. The Helicobacter pylori cell shape promoting protein Csd5 interacts with the cell wall, MurF, and the bacterial cytoskeleton. Molecular Microbiology. [Epub ahead of print]

Funding was provided by the National Institutes of Health and the National Science Foundation.