HP1581 (WecA)-dependent O-antigen biosynthesis governs adhesion, virulence, and biofilm dynamics in Helicobacter pylori

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Background Helicobacter pylori lipopolysaccharide (LPS) contributes to bacterial fitness, immune evasion, host colonization, and virulence. The O-antigen region contains Lewis antigens and other glycans that facilitate adaptation to the gastric environment. HP1581 is predicted to encode a WecA-like UDP- N -acetylglucosamine-1-phosphate transferase that catalyzes the first step of O-antigen biosynthesis. However, its role in LPS assembly and H. pylori pathogenesis remains unclear. Methods An HP1581 ( wecA ) knockout mutant and a complemented strain were constructed in H. pylori strain 26695 to investigate the role of HP1581 in LPS biosynthesis, bacterial physiology, outer membrane vesicle (OMV) composition, host cell interactions, and virulence. Biofilm formation and swimming motility were examined using O-antigen-deficient mutants of the motile H. pylori G27 strain, including HP1581 ( wecA ), HP1206 ( wzk ), and HP1039 ( waaL ) mutants. Results Disruption of HP1581 abolished O-antigen biosynthesis and eliminated Lewis X and Lewis Y antigen expression, resulting in a truncated LPS structure lacking the O-antigen region. The mutant displayed increased sensitivity to sodium dodecyl sulfate and novobiocin, elevated surface hydrophobicity and autoaggregation, and reduced survival during late-stage growth. Loss of HP1581 significantly impaired bacterial adhesion and internalization in AGS cells and attenuated the induction of the elongated (hummingbird) phenotype. In addition, OMVs from the mutant contained substantially lower levels of CagA and VacA. In the Galleria mellonella infection model, the mutant exhibited reduced persistence and attenuated virulence. Furthermore, O-antigen-deficient mutants of the motile H. pylori G27 strain, including HP1581 ( wecA ), HP1206 ( wzk ), and HP1039 ( waaL ) mutants, exhibited enhanced biofilm formation despite reduced swimming motility. Biofilm stability was found to depend primarily on extracellular proteins rather than extracellular DNA. Conclusion HP1581 (WecA) is essential for O-antigen biosynthesis and plays critical roles in maintaining outer membrane integrity, regulating bacterial surface properties, promoting host cell interactions, modulating virulence-associated factors, and influencing biofilm development. These findings identify HP1581 (WecA)-dependent O-antigen biosynthesis as a key determinant of H. pylori virulence and persistence and highlight HP1581 as a potential therapeutic target.

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