Roles of the lipopolysaccharide biosynthesis-related gene in the fitness of and its virulence in .

is a pathogenic bacterium associated with the development of gastric cancer and other gastric disorders. One of its major virulence factors, lipopolysaccharide (LPS), plays a crucial role in maintaining bacterial integrity, mediating host adhesion, and modulating the immune response. Recent studies have indicated that ADP-heptose, an intermediate in the heptose biosynthetic pathway involved in the LPS synthesis cascade, is a novel pathogen-associated molecular pattern for . This study focuses on the gene, which is predicted to encode RfaE/HldE, an enzyme with kinase and ADP-transferase activities essential for heptose production. An gene-disrupted mutant was first generated, and the resulting mutant exhibited a truncated LPS structure, confirming its role in LPS biosynthesis. The -deficient mutant displayed increased sensitivity to the detergent SDS and the antibiotic novobiocin, heightened surface hydrophobicity, and a propensity for autoaggregation. Additionally, the mutant exhibited reduced adhesion and internalization capabilities, a diminished elongation phenotype, and failed to induce IL-8 secretion in infected gastric AGS cells. In an infection model, the knockout mutant showed significantly attenuated virulence, as no bacterial load was detectable in the larvae's hemolymph 48 h post-infection, unlike the wild-type strain. Finally, we provided evidence that the enzyme encoded by is involved in a general protein glycosylation system linked to LPS biosynthesis, specifically glycosylating the adhesin AlpA. These findings highlight the essential role of RfaE/HldE/HP0858 in LPS biosynthesis and bacterial virulence, making it a promising target for future therapeutic interventions against infections.