Sunday, May 5, 2024
5/5/2024
PROJECT SUMMARY Bacteria come in many shapes, which may enhance motility, biofilm formation, nutrient uptake, and pathogenesis. However, the functional consequences of shape have not been well studied, owing in part to a paucity of tools to manipulate bacterial cell shape. To probe how form (cell shape) drives function (radiation to diverse niches), we must first understand how shape is generated. Bacterial shapes, varying from spheres to rods to helices, all arise from the same cell wall polymer: peptidoglycan (PG). The PG wall surrounds the cell to contain turgor pressure. The major hypothesis in the field holds that diverse shapes arise from different patterns of PG synthesis. Indeed, Caulobacter crescentus and Vibrio cholerae, curved rods, require fixed cell spanning cytoskeletal proteins that bias PG synthesis to the opposite side of the cell to generate curvature. Helicobacter pylori has emerged as a useful model to study of helical shape. This bacterium persistently colonizes the human stomach causing chronic inflammation and clinical pathologies ranging from peptic ulcers to gastric cancer, the world’s fourth leading cause of cancer mortality. We isolated mutants with stable non- helical shapes, and our work demonstrating their defects in stomach colonization presented the first experimental evidence for a link between cell curvature and bacterial infectivity that has now been extended to other bacteria (Vibrio, Campylobacter). H. pylori’s strategy for maintaining helical shape differs significantly from other studied bacteria. We showed that two distinct cytoskeletal proteins, MreB and the bactofilin CcmA, promote higher relative rates of new cell wall incorporation at the major and minor helical axes, respectively, and neither form cell spanning filaments. We found that CcmA requires the transmembrane protein Csd5 to localize to the major helical axis and the Csd5-CcmA complex also contains the MurF PG precursor synthesis enzyme, suggesting a possible mechanism for promoting PG synthesis. Our analyses of H. pylori morphology during chronic infection revealed shape diversification (including changes in helical pitch and even loss of helical curvature) that genetically maps to previously identified cell shape genes. In this renewal application we propose to define mechanisms by which the Csd5-CcmA complex is localized the major helical axis (Aim 1), define mechanisms of enhanced PG synthesis at the major and minor helical axes and the role of spatially variation in relative PG synthesis rates and/or endopeptidase activity in maintaining helical cell shape (Aim 2), and determine how cytoskeletal protein dynamics and natural sequence polymorphisms modify helical pitch and stomach colonization (Aim3). A more complete understanding of the mechanisms H. pylori uses to maintain and vary helical cell shape is needed to identify vulnerabilities that could be targeted to augment current treatment regimens and to probe the mechanisms by which H. pylori persists in the stomach to cause disease.
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