ROS-induced allosteric regulation of NikR coordinates HP0910-mediated OMP2 methylation to modulate H. pylori biofilm dynamics and therapeutic targeting

Abstract

Biofilm formation represents a critical survival strategy for Helicobacter pylori (H. pylori), facilitating antibiotic resistance and chronic colonization. In this study, we demonstrate that reactive oxygen species (ROS) released by macrophages enhance H. pylori biofilm formation through a novel epigenetic pathway. Transcriptomic and genetic analyses revealed that the nickel-responsive regulator NikR is allosterically activated by ROS, transitioning from its apo to holo conformation. This conformational shift markedly represses the expression of the DNA methyltransferase HP0910, resulting in hypomethylation of the omp2 gene, which encodes an outer membrane protein (OMP), and subsequent OMP2 overexpression, as validated by quantitative reverse transcription PCR (qRT-PCR) and reporter assays. Elevated OMP2 levels enhance extracellular polymeric substance (EPS) production, leading to a significant increase in biofilm biomass and thickness, as quantified by crystal violet staining and confocal laser scanning microscopy (CLSM). ROS scavenging reverses this phenotype, promoting biofilm dispersal. Furthermore, molecular docking and functional assays identified flopropione, a small-molecule compound targeting OMP2, led to an approximately 80 % reduction in biofilm biomass in vitro. When combined with standard triple therapy (omeprazole, amoxicillin, and clarithromycin), flopropione significantly improved bacterial clearance (>2-log₁₀ reduction) in a murine infection model. Collectively, our findings elucidate the ROS–NikR–HP0910–OMP2 signaling axis that regulates H. pylori biofilm dynamics and identify flopropione as a promising anti-biofilm therapeutic candidate against multidrug-resistant infections.