Vector Biological Studies in Leishmaniasis

利什曼病媒介生物学研究

• 批准号: 8946248
• 负责人: David Sacks
• 金额: $ 46.77万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8946248
• 关键词: Antibiotics; Biological; Bite; Chromosome Mapping; Chromosomes; Communities; Cutaneous; Development; Differentiation and Growth; Disease; Dose; Drug resistance; Event; Future; Generations; Genes; Genetic; Genetic Polymorphism; Genome; Genotype; Goals; Growth and Development function; Hybrids; Immune response; Infection; Inflammatory Response; Inherited; Invertebrates; Kinetoplast DNA; Leishmania; Leishmaniasis; Loss of Heterozygosity; Lutzomyia genus; Methods; Midgut; Nutrient; Parasites; Parents; Partner in relationship; Pathogenesis; Pathology; Penicillins; Phenotype; Ploidies; Population; Recombinant DNA; Role; Saliva; Sand Flies; Skin; Staging; Streptomycin; Stress; Vaccines; Viscera; Visceral; deprivation; dietary supplements; fly; genetic linkage analysis; in vivo; metacyclogenesis; reconstitution; trait; vector

We have previously provided conclusive evidence that the invertebrate stages of Leishmania are fully capable of a sexual cycle during their growth and development in the sand fly vector. Our prior studies involved only mating between parental lines of the same species, L. major. In 2014, we investigated the possibility of genetic exchange between different Leishmania species. A cutaneous strain of L. major and a visceral strain of L. infantum, each bearing a different drug resistant marker, were used to co-infect Lutzomyia longipalpis sand flies. Eleven double drug resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome indicated that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental inheritance of maxicircle kDNA. Hybrids with DNA contents of 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies represent the first experimental confirmation of cross-species mating in Leishmania, opening the way towards genetic linkage analysis of important traits, and providing strong evidence that genetic exchange is responsible for the generation of the mixed species genotypes observed in natural populations. We have identified the normal midgut microbiota as an essential factor controlling metacyclogenesis - the development of Leishmania promastigotes into their infective stage. L. major infected P. duboscqi sand flies treated with penicillin/streptomycin were completely inhibited in their ability to support the normal growth and differentiation of promastigotes into the metacyclic stage. Metacyclogenesis in antibiotic treated flies could be rescued by reconstituting the sand fly midguts with defined bacterial species isolated from the midguts. The bacterial diversity in normal and antibiotic treated flies was characterized by both culture dependent and independent methods by sequencing of the 16s rDNA. The possible roles of the normal bacterial communities in providing nutritional supplements essential to parasite differentiation, or to promoting nutrient deprivation or other stress conditions in the midgut required to trigger metacyclogenesis, will be investigated in future studies.

我们以前提供了确凿的证据，利什曼原虫的无脊椎动物阶段是完全能够在他们的生长和发展过程中的性周期的白蛉载体。 我们以前的研究只涉及同一物种的亲本系之间的交配，L。少校 2014年，我们研究了不同利什曼原虫物种之间遗传交换的可能性。 皮肤型L. major和L.婴儿，每个携带不同的药物抗性标记，用于共感染Lutzomyia longipalpis沙蝇。产生了11个双耐药后代克隆，每个克隆都是独立交配事件的产物，并进行了基因型和表型分析。多个等位基因标记的基因组分析表明，每个后代克隆遗传至少一个完整的染色体从每个父母，在一些位点的杂合性丢失，和单亲遗传的大环kDNA。 观察到DNA含量为2n、3 n和4 n的杂种。体内研究揭示了杂种在皮肤中产生病理或在内脏中传播和生长的能力的明显差异，表明控制这些性状的基因的多态性和差异遗传。这些研究代表了利什曼原虫跨物种交配的首次实验确认，为重要性状的遗传连锁分析开辟了道路，并提供了强有力的证据表明，遗传交换是负责自然种群中观察到的混合物种基因型的产生。 我们已经确定了正常的中肠微生物群作为一个重要因素控制后循环-发展利什曼原虫前鞭毛体进入其感染阶段。 L.用青霉素/链霉素处理的主要感染的P. duboscqi沙蝇在其支持前鞭毛体正常生长和分化到元周期阶段的能力方面被完全抑制。 抗生素处理的苍蝇中的代谢循环可以通过用从中肠分离的确定的细菌物种重建沙蝇中肠来拯救。 正常和抗生素处理的苍蝇的细菌多样性的特征在于培养依赖性和独立的方法，通过测序的16 S rDNA。正常细菌群落在提供寄生虫分化所必需的营养补充剂，或促进营养剥夺或中肠中触发代谢循环所需的其他应激条件方面的可能作用，将在未来的研究中进行调查。