Characterization of a waaF mutant of Helicobacter pylori strain 26695 provides evidence that an extended lipopolysaccharide structure has a limited role in the invasion of gastric cancer cells

Abstract

An ld-heptosyltransferase gene, HP1191 (waaF), involved in biosynthesis of the inner-core region of Helicobacter pylori strain 26695 lipopolysaccharide (LPS), has been cloned and its function established by complementation of Salmonella enterica serovar Typhimurium waaF mutant strain, strain 3789. Insertional inactivation of the HP1191 open reading frame in strain 26695 resulted in the formation of a deeply truncated LPS molecule, as observed using SDS–PAGE. Subsequent compositional and fatty acid analyses, followed by capillary electrophoresis – mass spectrometry and nuclear magnetic resonance studies established its structure as the following: PE→7)-l-α-d-Hepp-(1→5)-α-Kdop-(2→6)-Lipid A, where PE represents a phosphoethanolamine group, ld-Hep represents l-glycero-d-manno-heptose, and Kdo represents 3-deoxy-d-manno-oct-2-ulosonic acid. This structural analysis identifies the activity of HP1191 as a heptosyltransferase and a waaF homolog. In vitro invasion assays using human cultured gastric adenocarcinoma cells as a host cell model confirmed that the level of invasion was unaffected for an H. pylori HP1191::Kan deep-rough mutant strain compared with the wild-type strain 26695 expressing the O-chain polysaccharide, providing evidence that LPS is not a critical factor for invasion.