Point Mutations in Helicobacter pylori's fur Regulatory Gene that Alter Resistance to Metronidazole, a Prodrug Activated by Chemical Reduction

Sung Sook Choi,Douglas E Berg,Peter T Chivers,Niyaz Ahmed

doi:10.1371/journal.pone.0018236

Sung Sook Choi, Douglas E Berg + Show 2 more

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https://doi.org/10.1371/journal.pone.0018236

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Abstract

Background Helicobacter pylori's Fur regulatory protein controls transcription of dozens of genes in response to iron availability, acidity and oxidative stress, and affects the vigor of infection and severity of disease. It is unusual among Fur family proteins in being active both when iron-loaded and iron-free.Metholodolgy/Principal FindingsWe tested if H. pylori fur mutations could affect resistance to metronidazole (Mtz), an anti-H. pylori prodrug rendered bactericidal by chemical reduction. Point mutations were made by PCR in DNA containing fur and a downstream chloramphenicol resistance gene, and were placed in the H. pylori chromosome by transformation of a fur-deletion (Δfur) strain. Several substitutions affecting H. pylori Fur's ∼10 residue N terminal arm, which has no counterpart in prototype (E. coli-type) Fur proteins, increased Mtz resistance, as did mutations affecting the region between DNA binding and dimerization domains. Three types of mutations decreased resistance more than did Δfur: substitutions affecting the N-terminal arm; substitutions affecting the metal binding pocket; and nonsense mutations that resulted in a truncated Fur protein with no C-terminal dimerization domain. Most metal binding pocket mutations were obtained only in fur genes with additional inactivating mutations, and thus seemed deleterious or lethal because they.Conclusions/SignificanceThese results establish that H. pylori Fur's distinctive N terminal arm is functional, and more generally illustrate that point mutations can confer informative phenotypes, distinct from those conferred by null mutations. We propose that fur mutations can affect Mtz susceptibility by altering the balance among Fur's several competing activities, and thereby the expression of genes that control cellular redox potential or elimination of bactericidal Mtz activation products. Further analyses of selected mutants should provide insights into Fur interactions with other cellular components, metabolic circuitry, and how H. pylori thrives in its special gastric niche.

Highlights

The gastric pathogen Helicobacter pylori chronically infects the stomachs of billions of people worldwide
Persistent infection is thought to depend on a constellation of quantitative factors – prominent among them, H. pylori’s abilities: (i) to provoke low level tissue damage and inflammation and the release of nourishing metabolites, without destroying the gastric epithelium on which H. pylori depends; (ii) to cope with inflammation-associated oxidative stresses and transient exposure to stomach acid; (iii) to acquire iron and other micronutrients needed as metalloprotein cofactors and protein structural components, while avoiding the toxicity of these metals when they are in excess or not properly sequestered; and (iv) to swim away from acidic environments and toward the near-neutral, nourishing epithelial surface [1,2,3,4,5,6,7]
Each of these activities is affected or controlled in part by the H. pylori Fur protein, which belongs to a widespread family of transcription regulators whose members have been most studied in terms of controlling iron uptake and utilization [4,5,6,8]

Summary

Introduction

The gastric pathogen Helicobacter pylori chronically infects the stomachs of billions of people worldwide. Persistent infection is thought to depend on a constellation of quantitative factors – prominent among them, H. pylori’s abilities: (i) to provoke low level tissue damage and inflammation and the release of nourishing metabolites, without destroying the gastric epithelium on which H. pylori depends; (ii) to cope with inflammation-associated oxidative stresses and transient exposure to stomach acid; (iii) to acquire iron and other micronutrients needed as metalloprotein cofactors and protein structural components, while avoiding the toxicity of these metals when they are in excess or not properly sequestered; and (iv) to swim away from acidic environments and toward the near-neutral, nourishing epithelial surface [1,2,3,4,5,6,7] Each of these activities is affected or controlled in part by the H. pylori Fur protein, which belongs to a widespread family of transcription regulators whose members have been most studied in terms of controlling iron uptake and utilization [4,5,6,8]. Most metal binding pocket mutations were obtained only in fur genes with additional inactivating mutations, and seemed deleterious or lethal because they

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