Determining the molecular mechanism of anthelmintic resistance in hookworms

确定钩虫抗蠕虫药的分子机制

• 批准号: 9089987
• 负责人: JOHN M HAWDON
• 金额: $ 19.77万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9089987
• 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; whole genome; 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; whole genome

 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 na¿ve 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（由适用提供）：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。重钩虫感染是热带贫血的主要原因，导致因失血而导致肠道中的成年蠕虫失血引起的衰弱，有时是致命的铁缺陷性贫血。儿童，孕妇和较早的儿童尤其容易受到钩虫感染的发病率。控制策略仅限于苯甲酰唑（BZ）驱虫药的周期性驱动。人们非常担心的是，对BZ药物的抵抗力将随着大规模药物管理（MDA）的控制计划而发展，以控制钩虫。要监测抗性的出现的分子测试是必要的，但是在钩虫中，抗BZ的会议抗性的遗传突变与在其他寄生虫中赋予耐药性的基因突变是未知的。缺乏钩虫的抗BZ菌株阻止了抗性突变的识别。 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 phenotyically resistant to BZ.在AIM 1中，我们将在体外诱变成年钩虫以产生遗传变异，并将其返回到NATO宿主中繁殖。 F2后代将通过体外暴露于BZ药物来选择抗性。幸存者将用于感染新宿主，并选择使用BZ选择的F3后代。 F3幸存者将与野生型亲代菌株相互反向交叉，BZ用于选择抗性蠕虫以传播抗性菌株。表型抗性将通过标准卵舱口和幼虫发育测定法证实。在AIM 2中，我们将确定与整个基因组测序与耐药性相关的突变，并与易感野生型亲本菌株进行比较。候选抗性等位基因将通过其在Trans中赋予秀丽隐杆线虫模型的抗BZ耐药性来确认。产生钩虫的抗性菌株以及对BZ驱虫药引起耐药性的遗传突变的鉴定将允许开发基于分子的工具，以监测接受治疗的人群中抗性等位基因的频率。这将允许及时修改治疗机构，以防止宽度抗性发展。