Vector Biological Studies in Leishmaniasis

National Institute Of Allergy And Infectious Diseases

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Abstract

Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies, and directly demonstrated by laboratory crosses. In nature, opportunities for Leishmania interstrain mating are restricted to flies biting multiply infected hosts or through multiple bites of different hosts. In contrast, self-mating could occur in any infected sand fly. By crossing two recombinant lines derived from the same Leishmania major strain, each expressing a different drug-resistance marker, self-hybridization in L. major was confirmed in a natural sand fly vector, Phlebotomus duboscqi, and in frequencies comparable to interstrain crosses. We provide the first high resolution, whole-genome sequencing analysis of large numbers of selfing progeny. Genetic exchange consistent with classical meiosis was supported by the biallelic inheritance of the rare homozygous single nucleotide polymorphisms (SNPs) that arose by mutation during the generation of the parental clones. SNPs that were heterozygous in both parents, recombined to produce homozygous alleles in some hybrids. For trisomic chromosomes present in both parents, transmittal to the progeny was only altered by self-hybridization, involving a gain or loss of somy in frequencies predicted by a meiotic process. Thus, self-hybridization in Leishmania, with its potential to occur in any infected sand fly, may be an important source of karyotype variation, loss of heterozygosity, and functional diversity. Leishmania donovani species complex is known to have a vast diversity of clinical manifestations in humans, but underlying mechanisms for such diversity are yet unknown. Natural populations of hybrid parasites have been associated with the rise of atypical clinical outcomes in the Indian subcontinent (ISC). However, formal demonstration of genetic crossing in the major endemic sand fly species in the ISC remain unexplored. We investigated the ability of two distinct variants of L. donovani associated with strikingly different forms of the disease to undergo genetic exchange inside its natural vector, Phlebotomus argentipes. Clinical isolates of L. donovani either from a Sri Lankan cutaneous leishmaniasis (CL) patient or an Indian visceral leishmaniasis (VL) patient were genetically engineered to express different fluorescent proteins and drug-resistance markers and subsequently used as parental strains in experimental co-infection in Ph. argentipes sandflies. After 8 days of infection, sand flies were dissected and midgut promastigotes were transferred into double drug-selective media. Two double drug-resistant, dual fluorescent hybrid cell lines were recovered, which after cloning and whole genome sequencing, were shown to be full genomic hybrids. This study provides the first evidence of L. donovani hybridization within its natural vector Ph. argentipes. As mentioned, the generation of intra- and interspecific hybrids has been demonstrated in laboratory crosses during sand fly infections and, more recently, in culture after DNA damage induced by gamma-irradiation. Although no gametes or meiotic forms have been identified, allele inheritance patterns strongly suggest a meiotic process. We employed a functional genomics approach to identify genes required for Leishmania mating in vitro and in vivo. Using CRISPR/Cas9 editing tools to generate null mutants of core meiosis gene homologs (HOP1, SPO11, DMC1, HOP2 and MND1), as well as two genes related to the HAP2 fusogen (here named HAP2-1 and HAP2-2). We identified HOP1, a synaptonemal complex (SC) protein involved in chromosome pairing, and HAP2-2, as essential to Leishmania mating in vitro and in the sand fly, since deletion of either gene in one or both parents significantly reduced or completely abrogated mating competence. Complementation with the targeted genes could in each case partially rescue mating competence, and reporter constructs permitted detection of subpopulations of expressing cells and their localization to midgut microenvironments. This is the first demonstration in a trypanosomatid for the involvement of a meiotic protein homolog in genetic exchange, and the first description of a distinct fusogen mediating non-canonical, bilateral function in the hybridizing cells.