Abstract
Metronidazole (MTZ) is often used in combination therapies to treat infections caused by the gastric pathogen Helicobacter pylori. Resistance to MTZ results from loss-of-function mutations in genes encoding RdxA and FrxA nitroreductases. MTZ-resistant strains, when cultured at sub-MICs of MTZ (5 to 20 μg/ml), show dose-dependent defects in bacterial growth; depressed activities of many Krebs cycle enzymes, including pyruvate:ferredoxin oxidoreductase (PFOR); and low transcript levels of porGDAB (primer extension), phenotypes consistent with an involvement of a transcriptional regulator. Using a combination of protein purification steps, electrophoretic mobility shift assays (EMSAs), and mass spectrometry analyses of proteins bound to porG promoter sequences, we identified HP1043, an essential homeostatic global regulator (HsrA [for homeostatic stress regulator]). Competition EMSAs and supershift analyses with HsrA-enriched protein fractions confirmed specific binding to porGDAB and hsrA promoter sequences. Exposure to MTZ resulted in >10-fold decreases in levels of HsrA and in levels of the HsrA-regulated gene products PFOR and TlpB. Exposure to paraquat (PQ), hydrogen peroxide, or organic peroxides showed near equivalence with MTZ, revealing a common oxidative stress response pathway. Finally, direct superoxide dismutase (SOD) assays showed an inverse relationship between HsrA levels and SOD activity, suggesting that HsrA may serve as a repressor of sodB. As a homeostatic sentinel, HsrA appears to be ideally positioned to enable rapid shutdown of genes associated with metabolism and growth while activating (directly or indirectly) oxidative defense genes in response to low levels of toxic metabolites (MTZ or oxygen) before they reach DNA-damaging levels.