Identification of a D-glycero-D-manno-heptosyltransferase gene from Helicobacter pylori

Abstract

We have identified a Helicobacter pylori d-glycero-d-manno-heptosyltransferase gene, HP0479, which is involved in the biosynthesis of the outer core region of H. pylori lipopolysaccharide (LPS). Insertional inactivation of HP0479 resulted in formation of a truncated LPS molecule lacking an alpha-1,6-glucan-, dd-heptose-containing outer core region and O-chain polysaccharide. Detailed structural analysis of purified LPS from HP0479 mutants of strains SS1, 26695, O:3, and PJ1 by a combination of chemical and mass spectrometric methods showed that HP0479 likely encodes alpha-1,2-d-glycero-d-manno-heptosyltransferase, which adds a d-glycero-d-manno-heptose residue (DDHepII) to a distal dd-heptose of the core oligosaccharide backbone of H. pylori LPS. When the wild-type HP0479 gene was reintegrated into the chromosome of strain 26695 by using an "antibiotic cassette swapping" method, the complete LPS structure was restored. Introduction of the HP0479 mutation into the H. pylori mouse-colonizing Sydney (SS1) strain and the clinical isolate PJ1, which expresses dd-heptoglycan, resulted in the loss of colonization in a mouse model. This indicates that H. pylori expressing a deeply truncated LPS is unable to successfully colonize the murine stomach and provides evidence for a critical role of the outer core region of H. pylori LPS in colonization.

FIG. 1.

Chemical structures of l-glycero-d-manno-heptopyranose and d-glycero-d-manno-heptopyranose.

FIG. 2.

SDS-PAGE and immunoblot analysis of LPS from H. pylori HP0479 isogenic mutants. (A) Lane 1, H. pylori 26695; lane 2, H. pylori 26695::HP479kan; lane 3, H. pylori O:3; lane 4, H. pylori O:3::HP0479kan; lane 5, H. pylori strain SS1; lane 6, H. pylori SS1::HP0479kan; lane 7, H. pylori strain PJ1; lane 8, H. pylori PJ1::HP0479kan. (B) Immunoblot using anti-Le^X monoclonal antibody at a 1:250 dilution for lanes 1 to 6 from panel A. (C) Immunoblot using anti-Le^Y monoclonal antibody at a 1:500 dilution for lanes 1 to 6 from panel A.

FIG. 3.

SDS-PAGE and immunoblot analysis of LPS from complemented H. pylori HP0479 mutant. (A) Lane 1, H. pylori 26695; lane 2, H. pylori 26695::HP479kan; lane 3, H. pylori 26695::HP0479-HP0480::cam. (B) Corresponding immunoblot using anti-Le^X monoclonal antibody at a 1:250 dilution. (C) Corresponding immunoblot using anti-Le^Y monoclonal antibody at a 1:500 dilution.

FIG. 4.

FAB-MS spectra of permethylated H. pylori LPS in the positive mode, indicating characteristic primary and secondary fragments. (A) strain 26695; (B) 26695::HP0479kan mutant strain; (C) complemented 26695::HP0479-HP480::cam strain.

FIG. 5.

CE-MS and CE-MS/MS (positive-ion mode) analysis of delipidated LPSs from H. pylori SS1::HP0479kan, 26695::HP0479kan, and O:3::HP0479kan mutants. Separation conditions: 10 mM ammonium acetate containing 5% methanol, pH 9.0, 25 kV. (A) Extracted mass spectra at 14.6 min for strains SS1::HP0479kan, 26695::HP0479kan, and O:3::HP0479kan. (B) Tandem mass spectrum of precursor ions at m/z 1392. (C) Tandem mass spectrum of precursor ions at m/z 1612. Separation conditions were as for panel A except nitrogen collision gas, E_lab: 60 eV (laboratory frame of reference).

FIG. 6.

LPS structures of H. pylori strains 26695, SS1, O:3, and PJ1 and corresponding HP0479 mutants. (A) LPS structures from strains SS1, 26695, O:3, and PJ1. (B) HP0479 mutant LPS structure.

FIG. 7.

CE-IS-CID-MS (m/z 200 to1400) analysis of delipidated H. pylori LPS, using an orifice voltage of 200 V. (A) Extracted mass spectrum for strain 26695; (B) extracted mass spectrum for 26695::HP0479kan mutant strain; (C) extracted mass spectrum for the complemented 26695::HP0479-HP0480 strain; (D) extracted spectrum in the precursor ion scan for m/z 204. The collision energy was ramped from 40 to 60 V for the scan range of m/z 400 to 1400, with nitrogen as the collision gas.