Species-specific components of the Cag type IV secretion system.

strains containing the pathogenicity island (PAI) deliver an effector protein (CagA) and non-protein substrates into gastric cells through a process that requires the Cag type IV secretion system (T4SS). The Cag T4SS outer membrane core complex (OMCC) contains multiple copies of five proteins, two of which are species-specific proteins. By using modifications of a previously described OMCC immunopurification method and optimized mass spectrometric methods, we have now isolated additional PAI-encoded proteins that are present in lower relative abundance. Four of these proteins (CagW, CagL, CagI, and CagH) do not exhibit sequence relatedness to T4SS components in other bacterial species. Size exclusion chromatography analysis of immunopurified samples 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 Δ mutant strain (lacking peripheral OMCC components), but not from a Δ mutant strain (defective in OMCC assembly). Negative stain electron microscopy analysis indicated that OMCC preparations isolated from Δ Δ Δ mutant strains are indistinguishable from wild-type OMCCs. In summary, by using several complementary methods, we have identified multiple species-specific Cag proteins that are associated with the Cag T4SS OMCC and are required for T4SS activity.