Vector Biological Studies in Leishmaniasis

利什曼病媒介生物学研究

• 批准号: 10014015
• 负责人: David Sacks
• 金额: $ 91.18万
• 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/10014015
• 关键词: Alleles; Backcrossings; Bacteria; Biological; Biology; Bite; CRISPR/Cas technology; Cell Cycle; Cell fusion; Chromosome Segregation; Competence; Data; Development; Disease; Dose; Drug resistance; Early Diagnosis; Early treatment; Epidemiology; Epigenetic Process; Etiology; Event; Gene Expression; Generations; Genes; Genetic; Genetic Crossing Over; Genetic Recombination; Genome; Genotype; Germ Cells; Goals; Growth; Heterozygote; Histones; Human; Hybrids; Immune; Immune response; Immunologic Factors; India; Individual; Infection; Inflammatory Response; Inheritance Patterns; Insecta; Laboratories; Leishmania; Leishmania donovani; Leishmaniasis; Loss of Heterozygosity; Maps; Meiosis; Messenger RNA; Mitotic; Mutation; Organism; Parasites; Parents; Partner in relationship; Pathogenesis; Pathway interactions; Patients; Pattern; Phlebotomus; Population; Protocols documentation; Recovery; Resolution; Saliva; Sand Flies; Self-Fertilizations; Site; Sterility; Techniques; Time; Up-Regulation; Vaccines; Virulence; Visceral Leishmaniasis; base; cohort; fly; genetic analysis; genome editing; genome sequencing; genome-wide; human subject; member; microbiota; mutant; positional cloning; prevent; sex; trait; transcription factor; transmission process; vector; volunteer; whole genome

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. We used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97-99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes. We also provide direct evidence for self-mating in Leishmania and present the first high resolution genotype analysis of self-mating progeny using whole genome sequencing data. By singly introducing two different drug resistance markers into the same L. major strain, we could select for double drug resistant, self-mating progeny in co-infected sand flies. Genetic exchange consistent with meiotic sex was supported by: 1) the biallelic inheritance of the rare homozygous SNPs that arose by mutation during the generation of the parental clones, and 2) segregation of chromosomes in a Mendelian proportion resulting in homozygous conversion in one of the few blocks of heterozygosity that we could observe in this lineage. What might be the non-recombinatorial benefits of sex between two largely identical genotypes? Our findings document the loss of virulence that was associated with clonal growth of the parasite, and the reacquisition of virulence resulting from self-mating that in many cases produced progeny with greater virulence than either parent. The recovery of virulence was positively correlated with mRNA levels of histones and other cell cycle related genes at the metacyclic promastigote stage, suggesting that the ability of the parasite to establish successful infection in the mammalian host is dependent on upregulation of components of the replication machinery already in the non-dividing, infectious parasite stage in the sand fly. The data suggest that epigenetic reprogramming through self-fertilization in the sand fly is a mechanism to reset the expression of genes required for growth and survival in the mammalian host. Studies of sand fly / Leishmania interactions have been limited by the absence of genome editing techniques applied to these insects. We adapted the CRISPR/Cas9 technology to Phlebotomus papatasi sand flies, a natural vector for L. major, targeting the sand fly Immune Deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in P. papatasi and were able to generate transmissible null mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. In addition to an expected sensitivity to bacteria, rel mutant sand flies infected with L. major presented higher parasite loads and greater numbers of infective stage promastigotes. Together, our data show 1) the successful adaptation of the CRISPR/Cas9 technology to sand flies, and 2) the sand fly immune response impacts the vector competence for Leishmania parasites. Visceral leishmaniasis (VL), which is endemic in the northeast Indian state of Bihar, is thought to have an anthroponotic transmission cycle as no mammalian host other than humans has ever been shown to harbor the etiologic agent, L. donovani. However, which infected humans can act as important reservoirs for transmission to the vector, Phlebotomus argentipes, remains poorly studied. In particular, whether or not infected but healthy, asymptomatic individuals can transmit to the vector remains a critical question since these individuals represent the vast majority infections. Understanding the dynamics and epidemiology of anthroponotic transmission holds clear importance for the development of control strategies. For xenodiagnostic studies aimed at defining the ability of specific human-subject groups across the infection spectrum to transmit viable L. donovani organisms to sand flies, an on-site, self-sustaining sand fly colony, has been established in Bihar, India, and is the first and only self-sustaining laboratory-based colony of sand flies in the Indian sub-continent. Completion of the xenodiagnostic studies on a cohort of 76 active cases of VL has revealed that patients in the early stage of disease transmit poorly, or not at all, to the vector, and none of the VL patients transmitted following treatment. More critically, none of the 186 asymptomatic volunteers was able to transmit infection to even a single sand fly. This is crucial data to indicate that early diagnosis and treatment of patients with active disease will prevent infected individuals from contributing to the transmission cycle of human VL on the Indian sub-continent.

在利什曼原虫的自然分离株中已经多次描述了杂交基因型，并且从与利什曼原虫不同菌株或物种共感染的沙蝇中恢复的实验性杂交品种已正式证明该属的成员具有遗传交换机制。由于在载体中的寄生虫发育过程中没有直接观察到配子阶段和细胞融合事件，因此我们依靠经典的遗传分析来确定利什曼原虫是否具有真正的性周期。利用全基因组测序技术对大乳杆菌与婴儿乳杆菌不同品系内及品系间产生的实验杂种染色体遗传模式进行了研究。我们还产生了第一个热带L.的实验杂交种并对其进行了测序。我们发现，在每种情况下，亲本染色体和等位基因的贡献在97-99%的情况下符合减数分裂预期的遗传模式。杂种相当于F1后代，在大多数基因组中是杂合的，因为在亲本之间是纯合的和不同的标记。罕见的，非孟德尔模式的染色体遗传被观察到，包括染色体的增加或减少，以及杂合性的丧失，可能出现在减数分裂或有丝分裂期间的后代克隆在苍蝇或培养。种间杂交种表现为不育，种内杂交种则能产生回交和异交后代。通过对一个主要利什曼原虫F1杂交的5个回交和异交后代克隆，以及一个热带利什曼原虫自然杂交的17个回交后代克隆的分析，揭示了利什曼原虫全基因组重组模式，表明经典杂交发生在减数分裂时期，并使我们得以构建利什曼原虫的第一个物理和遗传图谱。总之，这些发现为利什曼原虫减数分裂样性重组提供了强有力的证据，为重要基因的前向遗传分析和定位克隆提供了明确的机会。