Menadione reduces the expression of virulence- and colonization-associated genes in Helicobacter pylori.

Abstract

Novel treatment options are needed for the gastric pathogen Helicobacter pylori due to its increasing antibiotic resistance. The vitamin K analogue menadione has been extensively studied due to interest in its anti-bacterial and anti-cancer properties. Here, we investigated the effects of menadione on H. pylori growth, viability, antibiotic resistance, motility and gene expression using clinical isolates. The MIC of menadione was 313 µM for 11/13 isolates and 156 µM for 2/13 isolates. The minimum bactericidal concentrations were 1.25-2.5 mM, indicating that concentrations in the micromolar range were bacteriostatic rather than bactericidal. We were not able to experimentally evolve resistance to menadione in vitro. Sub-MIC menadione (16 µM for 24 h) did not significantly inhibit bacterial growth but significantly (P<0.05) changed the expression of 1291/1615 (79.9%) genes encoded by strain 322A. The expression of the virulence factor genes cagA and vacA was downregulated in the presence of sub-MIC menadione, while genes involved in stress responses were upregulated. Sub-MIC menadione significantly (P<0.0001) inhibited the motility of H. pylori, consistent with the predicted effects of the observed significant (P<0.05) downregulation of cheY, upregulation of rpoN and changes in the expression of flagellar assembly pathway genes seen in the transcriptomic analysis. Through in-depth interrogation of transcriptomic data, we concluded that sub-MIC menadione elicits a general stress response in H. pylori with survival in the stationary phase likely mediated by the upregulation of surE and rpoN. Sub-MIC menadione caused some modest increases in H. pylori susceptibility to antibiotics, but the effect was variable with strain and antibiotic type and did not reach statistical significance. Menadione (78 µM) was minimally cytotoxic to human gastric adenocarcinoma (AGS) cells after 4 h but caused a significant loss of cell viability after 24 h. Given its inhibitory effects on bacterial growth, motility and expression of virulence- and colonization-associated genes, menadione at low micromolar concentrations may have potential utility as a virulence-attenuating agent against H. pylori.