Protein-Protein Interactions Required for CagA Recruitment and Secretion by the Helicobacter pylori Cag Type IV Secretion System

Abstract

Helicobacter pylori strains that contain the cag pathogenicity island (PAI) utilize the Cag type IV secretion system (T4SS) to deliver a bacterial effector protein (CagA) and non-protein substrates into human gastric cells. The Cag T4SS outer membrane core complex (OMCC) contains multiple copies of five proteins, two of which are species-specific proteins. Additional species-specific proteins encoded by the cag PAI are required for Cag T4SS activity, but their localization within the Cag T4SS is undefined. By using optimized mass spectrometric methods and modifications of a previously described OMCC immunopurification method, we have identified four cag PAI-encoded proteins (CagW, CagL, CagI, and CagH) that co-purify with the Cag T4SS OMCC. Analysis of immunopurified samples by size exclusion chromatography revealed that CagW, CagL, CagI and CagH co-elute with OMCC components. These four Cag proteins are copurified with the OMCC in immunopurifications from a Δcag3 mutant strain (lacking peripheral OMCC components), but not from a ΔcagX mutant strain (defective in OMCC assembly). Mutant strains with deletions of cagW, cagL, cagI, or cagH lacked T4SS activity but retained the ability to assemble OMCCs. Furthermore, by targeting several different Cag proteins and adding crosslinkers to the bacteria prior to immunopurification, we improved our ability to isolate multiple Cag proteins, providing further insight into protein-protein interactions among cag PAI-encoded proteins. We also generated H. pylori strains that produce CagA N-terminal fusion proteins (APEX2-CagA, GFP-CagA) and found that these fusion proteins retained the ability to interact with the Cag T4SS OMCC. In summary, the experiments described in my dissertation provide new insights into protein-protein interactions relevant to the structural organization of the Cag T4SS.