Helicobacter pylori Exploits a Unique Repertoire of Type IV Secretion System Components for Pilus Assembly at the Bacteria-Host Cell Interface
Figure 2
Functional analysis of CagH and CagI.
(A) A metronidazole-resistant (MetR) strain of H. pylori was generated by transforming the WT strain with pMM672 (p-ΔrdxA); metronidazole-resistant transformants contained a deletion of the rdxA locus. The cagI chromosomal locus was deleted by transforming the metronidazole-resistant H. pylori with p-cagI::rdxA/cat; this resulted in chloramphenicol-resistant (CatR), metronidazole-sensitive (MetS) colonies, designated cagI::rdxA/cat. To generate a mutant containing an unmarked cagI deletion, H. pylori cagI::rdxA/cat was transformed with pΔcagI5-1, and metronidazole-resistant colonies were selected. (B) Schematic illustration of the chromosomal cagH-cagI-cagL region in the WT strain, cagI::rdxA-cat mutant, and ΔcagI unmarked mutant. A similar approach was used to generate an unmarked ΔcagH mutant strain. (C) Schematic of plasmids encoding epitope-tagged forms of CagH and CagI. PureAB, ureAB promoter; cat, chloramphenicol resistance mediated by chloramphenicol acetyltransferase gene of C. coli. The hemagglutinin (HA) epitope sequence was inserted into CagH at the N-terminus (CagH-HA), and the FLAG epitope was inserted at residue 51 of CagI (CagI-FLAG). Numbers indicate position of residues in the epitope tagged proteins. These plasmids were introduced into ΔcagH and ΔcagI mutants, respectively, and the resulting complemented strains contained sequences encoding CagH-HA or CagI-FLAG, inserted into the ureA locus. (D) Immunoblot analysis of CagH-HA and CagI-FLAG in the complemented ΔcagH and ΔcagI mutant strains, using monoclonal antibodies directed against the epitope tags. (E) WT H. pylori or the indicated mutants were co-cultured with AGS cells for 4.5 h, and IL-8 expression was analyzed by ELISA. ΔcagH and ΔcagI mutants were defective in ability to induce IL-8 secretion, and complementation corrected the defect. Values from six replicate samples were compared to the wild-type control by ANOVA followed by Dunnett's post hoc correction; # indicates p<0.05. CagL remained detectable in all strains throughout the assay, as demonstrated by anti-CagL immunoblot (inset). (F) AGS cells were co-cultured with WT H. pylori or the indicated mutants, and tyrosine-phosphorylated CagA was detected with an anti-phosphotyrosine antibody (α-PY99). Samples also were immunoblotted with an anti-CagA antibody and antiserum reactive with multiple H. pylori proteins (α-Hp). CagA translocation was impaired in the ΔcagH and ΔcagI mutants.