Vector Biological Studies in Leishmaniasis

National Institute Of Allergy And Infectious Diseases

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Abstract

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.