Determining the molecular mechanism of anthelmintic resistance in hookworms

确定钩虫驱虫药耐药性的分子机制

基本信息

批准号：
8807346
负责人：
JOHN M HAWDON
金额：
$ 23.73万
依托单位：
GEORGE WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8807346
关键词：
Adult Albendazole Ancylostoma caninum Anemia Animal Model Anthelmintics Appearance Area Backcrossings Benzimidazoles Caenorhabditis elegans Canis familiaris Chemicals Child DNA Sequence Alteration Detection Developing Countries Development Elderly Ensure Exploratory/Developmental Grant Exposure to Family Frequencies Future Generations Genes Genetic Genetic Variation Haemonchus Health Helminths Hemorrhage Hookworm Infections Hookworms Human In Vitro Individual Intestines Investigation Iron deficiency anemia Knowledge Livestock Mediating Modeling Modification Molecular Monitor Morbidity - disease rate Mutagenesis Mutation Nature Nematoda Parasite resistance Parasites Parasitic nematode Partner in relationship Pharmaceutical Preparations Population Pregnant Women Procedures Protocols documentation Public Health Reporting Resistance Resistance development Role Schedule Soil Survivors Testing Time Transgenes Tubulin Variant Virulent assay development base benzimidazole egg feeding genetic approach genetic resistance genome sequencing hatching killings member mutant novel offspring pressure prevent programs resistance allele resistance mechanism resistance mutation resistant strain sample fixation scale up tool tool development

项目摘要

DESCRIPTION (provided by applicant): Hookworm infection remains one of the most important public health threats worldwide, with an estimated 800 million people infected. Heavy hookworm infection is the leading cause of anemia in the tropics, resulting in debilitating and sometimes fatal iron-deficiency anemia caused by blood loss to feeding adult worms in the intestine. Children, pregnant women, and the elderly are particularly susceptible to morbidity from hookworm infection. Control strategies are restricted to periodic de-worming of infected individuals with benzimidazole (BZ) anthelmintics. There is considerable concern that resistance to BZ drugs will develop with increased use in mass drug administration (MDA) programs to control hookworms. Molecular tests to monitor the emergence of resistance are necessary, but the genetic mutations that confer resistance to BZ are unknown in hookworms, and appear to be different than those that confer resistance in other parasitic nematodes. The lack of a BZ resistant strain of hookworm prevents identification of the resistance mutations. To determine the underlying genetic mutations that confer BZ resistance in hookworms, we propose using a novel in vitro mutagenesis and selection protocol to generate a strain of the canine hookworm Ancylostoma caninum that is phenotypically resistant to BZ. In Aim 1 we will mutagenize adult hookworms in vitro to generate genetic variation, and return them to a naïve host to reproduce. F2 offspring will be selected for resistance by exposure to BZ drugs in vitro. Survivors will be used to infect new hosts, and the F3 offspring selected with BZ. F3 survivors will be reciprocally backcrossed to the wild type parental strain, and BZ used to select resistant worms for propagation of the resistant strains. Phenotypic resistance will be confirmed by standard egg hatch and larval development assays. In Aim 2, we will identify mutations associated with resistance by whole genome sequencing and comparison to the susceptible wild type parental strain. Candidate resistance alleles will be confirmed by their ability to confer BZ resistance on the model nematode Caenorhabditis elegans in trans. Generation of a resistant strain of hookworm and the identification of the genetic mutations that cause resistance to BZ anthelmintics will permit the development of molecular based tools to monitor the frequency of resistance alleles in populations undergoing treatment. This will allow modification of treatment regiments in time to prevent widespread resistance from developing.

描述（由申请人提供）：钩虫感染仍然是全球最重要的公共卫生威胁之一，估计有 8 亿人感染。重度钩虫感染是热带地区贫血的主要原因，导致虚弱甚至致命的缺铁。喂养肠道内成虫而失血引起的贫血儿童、孕妇和老年人特别容易因钩虫感染而发病。控制策略仅限于定期驱虫。人们非常担心，随着控制钩虫的大规模药物管理（MDA）计划的增加，对苯并咪唑（BZ）驱虫药的耐药性将会产生，但监测耐药性出现的分子测试是必要的。钩虫中赋予 BZ 抗性的物质是未知的，并且似乎与其他寄生线虫中赋予 BZ 抗性的物质不同。钩虫缺乏 BZ 抗性菌株，妨碍了抗性的鉴定。为了确定赋予钩虫 BZ 抗性的潜在基因突变，我们建议使用一种新颖的体外诱变和选择方案来产生对 BZ 表型具有抗性的犬钩虫菌株。钩虫在体外产生遗传变异，并将其返回到幼稚宿主中进行繁殖，将通过在体外接触 BZ 药物来选择具有抗性的 F2 后代。感染新宿主，用 BZ 幸存者选择的 F3 后代将与野生型亲本菌株相互回交，并且用于选择抗性蠕虫以繁殖抗性菌株的表型抗性将通过标准卵孵化和确认。在目标 2 中，我们将通过全基因组测序鉴定与抗性相关的突变，并通过其与易感野生型亲本菌株的比较来确认其抗性等位基因的能力。赋予模型线虫秀丽隐杆线虫抗性钩虫抗性菌株的产生以及导致对 BZ 驱虫药抗性的基因突变的鉴定将允许开发基于分子的工具来监测经历群体中抗性等位基因的频率。这将有助于及时调整治疗方案，以防止出现广泛的耐药性。