Kinetic characterization of novel protein-protein interactions in Helicobacter pylori flagellar assembly
Disciplines
Molecular Biology
Abstract (300 words maximum)
Helicobacter pylori is an ε-proteobacterium that colonizes the stomach in half of the world’s population. It is the primary cause of peptic ulcers and chronic gastritis, and is a major risk factor for gastric cancer and mucosa-associated lymphoma. H. pylori cells generally possess 2 to 6 polar flagella that are required for host colonization. The assembly of flagella occurs via a dedicated Type III Secretion System (T3SS). Although studies of flagellar gene regulation in Escherichia coli and Salmonella have shed much light on the mechanism by which gene expression is coupled to flagellar assembly, little is known with regard to how H. pylori coordinates such activities and there are a number of striking differences between pylori and other bacteria in structure, regulation and genes involved. Using known flagellar genes and gene products as well as leads from yeast two-hybrid interactome ‘hits’, the goal of the present study was to validate, kinetically characterize and develop biological context for flagellar protein-protein interactions in H. pylori. Proteins were expressed in and purified from E. coli. Biolayer interferometry (BLI), an optical biosensing technique was used to screen an array of candidate proteins for interaction. Hits from that screen were fully characterized in association-then-dissociation experiments to determine kinetic and affinity constants. Studies in progress at the time of submission are seeking to characterize the context of interactions in vivo via knock-out and mutagenesis experiments. The results of these studies will provide a valuable framework for gene regulation studies in H. pylori and other ε-Proteobacteria.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Dr. Jonathan McMurry
Kinetic characterization of novel protein-protein interactions in Helicobacter pylori flagellar assembly
Helicobacter pylori is an ε-proteobacterium that colonizes the stomach in half of the world’s population. It is the primary cause of peptic ulcers and chronic gastritis, and is a major risk factor for gastric cancer and mucosa-associated lymphoma. H. pylori cells generally possess 2 to 6 polar flagella that are required for host colonization. The assembly of flagella occurs via a dedicated Type III Secretion System (T3SS). Although studies of flagellar gene regulation in Escherichia coli and Salmonella have shed much light on the mechanism by which gene expression is coupled to flagellar assembly, little is known with regard to how H. pylori coordinates such activities and there are a number of striking differences between pylori and other bacteria in structure, regulation and genes involved. Using known flagellar genes and gene products as well as leads from yeast two-hybrid interactome ‘hits’, the goal of the present study was to validate, kinetically characterize and develop biological context for flagellar protein-protein interactions in H. pylori. Proteins were expressed in and purified from E. coli. Biolayer interferometry (BLI), an optical biosensing technique was used to screen an array of candidate proteins for interaction. Hits from that screen were fully characterized in association-then-dissociation experiments to determine kinetic and affinity constants. Studies in progress at the time of submission are seeking to characterize the context of interactions in vivo via knock-out and mutagenesis experiments. The results of these studies will provide a valuable framework for gene regulation studies in H. pylori and other ε-Proteobacteria.