Vector Biological Studies in Leishmaniasis

National Institute Of Allergy And Infectious Diseases

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Abstract

Leishmania have two main cellular morphologies: amastigotes, found principally within macrophages of the mammalian host; and promastigotes, found as extracellular, motile forms within the digestive tract of the sand fly vector. During the development of transmissible infections in the vector, there are at least five major promastigote morphotypes that have been identified based on the length and width of the cell body and flagellum, designated as: procyclics, nectomonads, leptomonads, haptomonads and metacyclics. These forms are further distinguished by whether or not they are observed in division, and by the timing and positioning of their appearance within the midgut. Importantly, the non-dividing, infectious metacyclic promastigotes have long been thought to be the most mature stage, positioned in the anterior midgut for transmission by bite, and uniquely pre-adapted for survival in the mammalian host. Given their distinctive morphologies, temporal appearance, and spatial localization, it has been generally assumed that these different forms represent true developmental stages, adapted to distinct microenvironments, and characterized by specific molecules and transcriptional programs. Because of the difficulty in obtaining homogeneous populations from infected sand fly midguts, prior bulk RNA-sequencing analyses failed to provide the true transcriptomic complexity of the various promastigote stages colonizing the midgut throughout their development. Using a low bias, single cell RNA sequencing (scRNAseq) approach carried out on parasites obtained at different times post-infection, we generated the first single cells atlas of Leishmania developmental stages in sand flies. Our single-cell study of L. major development in a natural vector, Phlebotomus duboscqi, unveiled the transcriptomic programs and major markers of 8 subpopulations, including each of the promastigote stages defined by their morphology, as well as transitional stages of some of these forms, and a novel subpopulation of metacyclics promastigotes that were found in division. Based on pseudotime trajectory analysis, our results challenge conventional models that describe a linear progression of promastigote development culminating in the differentiation of non-dividing metacyclics as the only infective forms transmitted to the mammalian host. We demonstrate that leptomonads are the precursors of two separate lineages, haptomonads and dividing early metacyclics, with the latter serving as the precursors of non-dividing, late metacyclics. Most importantly, we have applied scRNA-seq to the transmitted inoculum to show that flies harboring mature infections can transmit multiple stages, with haptomonads and late metacyclics the predominant stages among egested promastigotes. Finally, using endogenously tagged, stage-specific markers to select for discreet populations of the transmitted cells, we could demonstrate the previously unrecognized contribution of haptomonads to host infection. L. donovani is the classical agent of visceral leishmaniasis (VL) in the Indian subcontinent. More recently, however, it has been associated with an atypical form of cutaneous leishmaniasis (CL) in Sri Lanka. Based on the phylogenetic evidence, the atypical L. donovani cutaneous isolates found in Sri Lanka might in some cases be the result of the introgression of genes acquired from crosses involving classical Old World cutaneous species, either L. tropica or L. major. To better address the possible strain and species heterogeneity of the parasites producing CL in Sri Lanka, whole genome sequencing was carried out on 27 clinical isolates obtained from CL patients in three administrative districts. Multiple genetic comparisons made using 32 different Old World Leishmania strains revealed that 8 patient isolates clustered with L. tropica, and 19 with Sri Lankan L. donovani. The patients with CL caused by L. tropica had never travelled overseas. Thus, it is almost certain that these are autochthonous cases of CL due to L. tropica. Our findings indicate L. donovani is not the only cause of CL in Sri Lanka, and potentially identify a haplotype that gave rise to interspecies dermotropic L. donovani hybrids within the island.