Characterization of the Pilin Ortholog of theHelicobacter pyloriType IVcagPathogenicity Apparatus, a Surface-Associated Protein Expressed during Infection

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Abstract
TheHelicobacter pylori cagpathogenicity island (cagPAI) encodes components of a type IV secretion system (T4SS) involved in host interaction and pathogenicity. Previously, sevencagPAI proteins were identified as homologs ofAgrobacterium tumefaciensVir proteins, which form a paradigm T4SS. The T pilus composed of the processed VirB2 pilin is an external structural part of theA. tumefaciensT4SS. InH. pylori,cag-dependent assembly of pili has not been observed so far, nor has a pilin (VirB2) ortholog been characterized. We have here identified, using a motif-based search, anH. pylori cagisland protein (HP0546) that possesses sequence and predicted structural similarities to VirB2-like pilins of other T4SSs. The HP0546 protein displays interstrain variability in its terminal domains. HP0546 was expressed as a FLAG-tagged fusion protein inEscherichia coli,A. tumefaciens, andH. pyloriand was detected as either two or three bands of different molecular masses in the insoluble fraction, indicating protein processing. As reported previously, isogenicH. pylorimutants in the putativecagpilin gene had reduced abilities to inducecagPAI-dependent interleukin-8 secretion in gastric epithelial cells. Fractionation analysis ofH. pylori, using a specific antiserum raised against an N-terminal HP0546 peptide, showed that the protein is partially surface exposed and that its surface localization depended upon an intactcagsystem. By immunoelectron microscopy, HP0546 was localized in surface appendages, with surface exposure of an N-terminal epitope. Pronounced strain-to-strain variability of this predicted surface-exposed part of HP0546 indicates a strong selective pressure for variation in vivo.

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