Helicobacter pylori (HP) is a major cause of peptic ulcer disease and an early risk factor for gastric cancer. The two most widely used types of therapies against HP depend on metronidazole (Mtz) or clarithromycin (Cla), in combination with other agents, but resistance (R) to Mtz is common, and CIaR is also known. The resistance mechanisms that predominate in American and European populations have been identified: inactivation of a nitroreductase gene, rdxA (MtzR); and sequence changes in one segment of 23S rRNA. Our analyses of MtzR indicated that Mtz is a prodrug, activated (metabolized) by MtzS HP to hydroxylamine, a mutagen and a bacteriocidal agent. HP is an extremely diverse species, with each isolate readily distinguishable from most others. There are numerous duplicate and divergent genes in the HP genome. We suggest that recombination among these genes, gene transfer among different HP strains, and mutation, can all contribute to bacterial adaptation to different human hosts and in changing gastric environments, and more generally in the evolution of virulence. My long term goal is to understand how HP colonizes its human host, establishes infections that persist for years or decades, and in some cases cause overt disease; how it evolves as a human commensal and pathogen; and how best to combat the infections that it causes. Four sets of experiments are proposed: First, we will seek to better understand the emergence and persistence of drug resistance in HP populations. In these experiments, we will test the idea that mutations resulting in MtzR and/or CIaR diminish bacterial fitness (vigor of growth) in culture and in vivo in mouse infection models. Second, we will examine factors affecting the formation of recombinants in HP, with a special focus on duplicate and divergent genes. This will be modeled using alleles for CIaR, which occur in the 23S rRNA gene; there are two copies of the 23S rRNA gene per HP genome. We will examine transformation of HP to CIaR, and test factors determining if a given HP strain can exhibit a CIaR phenotype while being heterozygous for cIaR and cIaS alleles, vs. if if it must be cIaR/cIaR homozygous. Recombination will also be examined using """"""""synthetic merozygotes"""""""", in which a cIaR allele is present in truncated (inactive) 23S rRNA gene in a plasmid vector, along with the duplicate expressed chromosomal 23S rRNA genes. Genetic exchange between different HP strains, and the importance of a mutS-like possible mismatch correction function will also be studied. Third, we will seek to better understand the control of mutation in HP, and test whether Mtz therapy itself is mutagenic. Sensitive genetic tests will be used to assess the mutagenic potency of Mtz in HP in culture and in mouse models. Fourth, we will seek to more fully understand mechanisms of drug resistance in HP. We will assess whether any significant fraction of MtzR and CIaR HP isolates from as yet unstudied human populations (India, China, minorities in the US) acquire resistance by mechanisms that are distinct from rdxA inactivation (MtzR) or 23S rRNA mutation (CIaR), the mechanisms found to underly resistance in the mainstream US population.
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