Helicobacter pylori is a Gram-negative helical rod-shaped bacterium that infects the stomach of 50% of all humans. H. pylori is an extracellular pathogen that is found in close proximity to the mucosal epithelial cells lining the stomach 6,7. H pylori express polar flagella on its surface, and both flagellar-based motility and chemotaxis are required for infection of the gastric mucosa 13-16. Helical cell shape is thought to enhance H. pylori's flagellar based-motility through the viscous mucus layer to allow for efficient colonization7. Our lab discovered several genes that promote normal helical cell shape in H. pylori and some of these genes are required for efficient colonization of the gastric mucosa in a mouse infection model17. Deletion of these genes individually or in tandem affect the bacteria's curvature and twist through alterations in peptidoglycan (PG) cross-linking17, resulting in distinc non-helical rod shapes (i.e. curved, "c"-shaped, and straight rods). Our preliminary studies suggest that cell shape plays a role in H. pylori's swimming velocity or some other aspect of swimming behavior. We hypothesize that helical cell shape is important for H. pylori motility and by affecting velocity and/or chemosensory directional switching, cell shape impacts H. pylori's localization within different gastric mucosal niches of the stomach.
Aim 1 will characterize the effect of cell shape on motility. We will test whether helical cell shape enhances H. pylori's velocity and swimming behavior by comparing the different cell shape mutants to the wild-type helical bacteria. Using live video microscopy, we will record the swimming behavior and determine the directional switching frequency of the bacteria in purified gastric mucin, the relevant polymer for H. pylori in the stomach (aim 1A). We will also directly probe the effect of cell shape on translational motion by measuring flagellar and cell body rotation for bacterial cell immobilized in gastric mucin gel at low pH (aim 1B).
Aim 2 will determine the effect of bacterial cell shape on localization of H. pylori to specific gastric niches within the stomach.
Aim 3 will investigate the mechanisms by which PG fragments are delivered to host gastric epithelial cells and modulate innate immune detection. H. pylori delivers PG fragments to host gastric epithelial cells via the cag-type IV secretion apparatus (cagT4SS) to activate a major pro- inflammatory pathway through the intracellular pattern recognition molecule NOD118. NOD1-expressing cells can respond to digested PG released from H. pylori cells that accumulate muro-tripeptides18, a NOD1 agonist. Some of our cell shape mutants accumulate high levels of this agonist in the PG sacculus. Thus, the cell wall modification events that drive cell shape changes may impact the synthesis and release of PG fragments that are delivered to host gastric epithelial cells. We will generate strains that overexpress cell wall modifications enzymes to determine if perturbations in PG degradation affect delivery to host cells via the cagT4SS or by cagT4SS-independent mechanisms (aim 3A). We will also investigate the mechanisms by which PG recycling mutants cause elevated pro-inflammatory cytokine release (aim 3B).

Public Health Relevance

Helicobacter pylori is a motile helical rod shaped bacterium and its characteristic helical cell shape is thought to enhance its flagellar based-motility throug the viscous mucus layer to allow for efficient colonization of the human stomach. Our lab discovered genes that promote H. pylori's helical cell shape by altering peptidoglycan crosslinking in the cell wall. We will test the hypothesis that helical cell shape is important forH. pylori motility and by affecting velocity and/or chemosensory directional switching, cell shape impacts H. pylori's localization within different gastric mucosal niches of the stomach;and we will characterize the effect of H. pylori cell shape perturbation on innate immune detection of cel wall components.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31AI098424-03
Application #
8617793
Study Section
Special Emphasis Panel (ZRG1-F13-C (20))
Program Officer
Adger-Johnson, Diane S
Project Start
2012-03-16
Project End
2016-03-15
Budget Start
2014-03-16
Budget End
2015-03-15
Support Year
3
Fiscal Year
2014
Total Cost
$35,134
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109