DRUG RESISTANCE AND GENE TRANSFER IN H PYLORI

幽门螺杆菌（HP）是消化性溃疡疾病的主要原因，也是胃癌的早期危险因素。针对HP的两种最广泛使用的治疗方法依赖于甲硝唑（Mtz）或克拉霉素（Cla），与其他药物联合使用，但对Mtz的耐药性(R)很常见，CIaR也是已知的。已经确定了在美国和欧洲人群中占主导地位的耐药机制：一种硝基还原酶基因rdxA （MtzR）失活；23S rRNA的一个片段序列发生变化。我们对MtzR的分析表明，Mtz是一种前药，被MtzS HP激活（代谢）成羟胺，一种诱变剂和杀菌剂。HP是一个极其多样化的物种，每个分离物都很容易与大多数其他分离物区分开来。HP基因组中有许多重复和分化的基因。我们认为，这些基因之间的重组、不同HP菌株之间的基因转移和突变都有助于细菌适应不同的人类宿主和不断变化的胃环境，更广泛地说，有助于毒力的进化。我的长期目标是了解HP如何在其人类宿主中定居，建立持续数年或数十年的感染，并在某些情况下导致明显的疾病；它如何演变为人类共栖和病原体；以及如何最好地对抗它引起的感染。提出了四组实验：首先，我们将寻求更好地了解HP人群中耐药性的出现和持续。在这些实验中，我们将在小鼠感染模型中测试导致MtzR和/或CIaR的突变会降低细菌在培养和体内的适应性（生长活力）。其次，我们将研究影响HP中重组形成的因素，特别关注重复和分化基因。这将使用CIaR的等位基因进行建模，这些等位基因存在于23S rRNA基因中；每个HP基因组有两个23S rRNA基因拷贝。我们将研究HP向CIaR的转化，并测试确定特定HP菌株在CIaR和cIaS等位基因杂合时是否可以表现出CIaR表型，而如果它必须是CIaR / CIaR纯合。重组也将使用“合成分合子”进行检查，其中cIaR等位基因存在于质粒载体中截断（无活性）的23S rRNA基因中，以及重复表达的染色体23S rRNA基因。不同HP菌株之间的遗传交换，以及类似muts的可能错配校正功能的重要性也将被研究。第三，我们将寻求更好地了解突变在HP中的控制，并测试Mtz治疗本身是否具有诱变性。敏感的基因测试将用于评估Mtz在培养HP和小鼠模型中的诱变效力。第四，我们将寻求更全面地了解HP的耐药机制。我们将评估来自尚未研究的人群（印度、中国、美国少数民族）的MtzR和CIaR HP分离株是否通过不同于在美国主流人群中发现的潜在耐药机制的rdxA失活（MtzR）或23S rRNA突变（CIaR）的机制获得耐药性。

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.