DRUG RESISTANCE AND GENE TRANSFER IN H PYLORI

幽门螺杆菌(Hp)是消化性溃疡的主要病因，也是胃癌的早期危险因素。两种最广泛使用的HP疗法依赖于甲硝唑(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。在美洲和欧洲种群中占主导地位的抗性机制已被确定：硝基还原酶基因rdxA(MtzR)失活；23S rRNA的一段序列发生变化。我们对MtzR的分析表明，MTZ是一种前药，被MtzS HP激活(代谢)为羟胺、诱变剂和杀菌剂。幽门螺杆菌是一个极其多样化的物种，每个菌株都很容易与大多数其他菌株区分开来。幽门螺杆菌基因组中存在大量的重复基因和分化基因。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，以及突变，都可以有助于细菌适应不同的人类宿主和改变胃环境，更广泛地说，在毒力的进化中。我的长期目标是了解惠普如何在人类宿主上定居，建立持续数年或数十年的感染，在某些情况下还会导致公开的疾病；它是如何演变为人类共生和病原体的；以及如何最好地对抗它引起的感染。我们提出了四组实验：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)的想法。其次，我们将研究影响幽门螺杆菌重组子形成的因素，特别关注重复和分歧基因。这将使用发生在23S rRNA基因中的Ciar的等位基因来建模；每个HP基因组有两个23S rRNA基因的拷贝。我们将研究HP向Ciar的转化，并测试确定给定的HP菌株是否可以表现出Ciar表型，同时是Ciar和Cias等位基因的杂合子，以及是否必须是Ciar/Ciar纯合子。重组也将使用“合成合子”进行检测，在“合成合子”中，一个Ciar等位基因存在于一个被截断的(非活性的)23S rRNA基因中，同时也存在重复表达的染色体23S rRNA基因。不同幽门螺杆菌菌株之间的遗传交换，以及类似MutS的可能错配校正函数的重要性也将被研究。第三，我们将寻求更好地了解幽门螺杆菌突变的控制，并测试MTZ治疗本身是否具有突变性。敏感的基因测试将用于评估甲氨蝶呤在幽门螺杆菌、培养物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。我们将评估在尚未研究的人群(印度、中国，美国的少数民族)中是否有相当一部分MtzR和Ciar Hp分离株通过不同于rdxA失活(MtzR)或23S rRNA突变(CIAR)的机制获得耐药性，这两种机制在美国主流人群中被发现具有较低的耐药性。

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.