The functional and catalytic landscape of urease reveals a conserved target against Helicobacter pylori.

Nearly half of the global population is infected with Helicobacter pylori. Antibiotic use has led to substantial antimicrobial resistance and unintended gut microbiota depletion, creating an urgent need for alternative therapeutic strategies. Here, we demonstrate that urease, a key enzyme that enables H. pylori survival by hydrolysing urea to neutralize stomach acid, is a conserved antibacterial target with low risk of resistance development. Using comprehensive deep mutational scanning coupled with phage-based functional screening, we systematically evaluated how mutations in core residues affect urease expression, enzymatic activity, bacterial colonization, and virulence, uncovering the catalytic nature of H. pylori urease. We found exceptional evolutionary conservation within the urease catalytic pocket, and potential mutation sites that affect urease activity are not close to the core of this pocket. Analysis of existing urease inhibitors revealed that their binding sites are not typically in these potential mutation sites, which indicates that the potential for resistance development is low. In addition, we show that targeting urease alone is effective in eradicating H. pylori and synergistically boosts the efficacy of antibiotics. Notably, the incorporation of urease inhibitors into antibiotic-based therapeutic regimens effectively preserves gut microbiota diversity and microbial genomic stability, thereby lowering the risk of antibiotic resistance. Collectively, our study elucidate the inherent resistance-resistant property of urease and establish the clinical value of combining urease inhibitors with antibiotics to reduce antibiotic resistance.