Species-specific components of the Helicobacter pylori Cag type IV secretion system

Abstract

Helicobacter pylori strains containing the cag 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 cag 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 Δcag3 mutant strain (lacking peripheral OMCC components), but not from a ΔcagX mutant strain (defective in OMCC assembly). Negative stain electron microscopy analysis indicated that OMCC preparations isolated from ΔcagW, cagL::kan, ΔcagI, and ΔcagH 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.

Keywords: Helicobacter pylori; bacterial protein secretion; bacterial secretion systems; proteomics.

Fig 1

Volcano plot of HA-CagF interactions compared with protein interactions of HA-tagged control proteins. Immunopurified preparations from an HA-CagF-producing strain and from strains producing HA-tagged non-Cag control proteins (HP0179-HA, HP0838-HA) were analyzed by mass spectrometry, and the results were compared using SAINTexpress. Differences in the detected protein-protein interactions were assessed based on analysis of the Bayesian false discovery rate (−log(BFDR)) and fold change (log₂(Fold Change)), with average interaction probability (AvgP) represented by dot size. A value of 0.001 was added to BFDR values calculated as 0 by SAINTexpress to allow for graphical analysis. Specific groups of interacting proteins are identified based on the color code. Twelve Cag proteins (CagA, the five OMCC components, and six newly isolated Cag proteins) had BFDR scores of 0. Detailed data pertaining to individual Cag proteins are shown in Table 1, and the complete data for all immunopurifications are shown in Table S1.

Fig 2

Cag proteins enriched in the HA-CagF immunopurification elution compared with unprocessed H. pylori lysate. The protein contents of H. pylori lysates and elution samples from HA-CagF immunopurifications (three biologically independent replicates) were analyzed by mass spectrometry. For each replicate, the spectral counts of the lysate and the immunopurification elution sample were normalized; data were log₂-transformed, and averages for each protein were plotted. A value of 1 was added to the spectral count for each protein prior to log₂ transformation to allow for graphical analysis of samples where no spectral counts were detected. Statistical significance was determined by comparing lysate and elution spectral counts for each Cag protein (paired t-test). *, P < 0.05; **, P < 0.01. OMCC components, blue; CagA and CagF, pink; other Cag proteins, gray.

Fig 3

Size exclusion chromatography of the HA-CagF immunopurified sample. An HA-CagF immunopurification was performed, and the elution sample (labeled “Load”) was fractionated by size exclusion chromatography. (A) Fractions were analyzed by Western blotting using anti-CagY, CagA, CagX, CagF, or CagT antisera. (B) Cag T4SS outer membrane core complexes from fraction 4 were visualized by negative stain electron microscopy. Scale bar, 100 nm.

Fig 4

Mass spectrometry analysis of size exclusion chromatography fractions. An HA-CagF immunopurification was performed, and the elution sample was fractionated by size exclusion chromatography. Fractions were pooled as indicated for mass spectrometry analysis. (A) Percentages of total spectral counts for the five outer membrane core complex proteins (blue) or CagA and CagF (pink) in pooled size exclusion chromatography fractions are shown. (B) Percentages of total spectral counts for newly isolated Cag proteins (CagW, CagL, CagI, and CagH) in pooled size exclusion chromatography fractions are shown.

Fig 5

Functional properties of CagW, CagL, CagI, and CagH. (A) The indicated strains were co-cultured with AGS cells, and an IL-8 induction assay was performed as described in the Methods section. Statistically significant differences in the IL-8-inducing activities of mutant strains compared with the WT/HA-CagF strains were determined by a one-way ANOVA with Dunnett’s multiple comparison test. Results are from two biologically independent experiments. ***, P < 0.001. (B) Cag T4SS outer membrane core complexes were isolated from the indicated strains via immunopurification as described in the Methods section. Complexes were visualized by negative stain electron microscopy. Scale bar, 100 nm.