Vector Biological Studies in Leishmaniasis

National Institute Of Allergy And Infectious Diseases

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Abstract

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. 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. Experimentally, mating competence has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low efficiency crosses between two L. tropica strains, L747 and MA37, that mate with high efficiency in flies. We showed that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains, and extends to other species, including L. donovani, L. infantum, and L. braziliensis, a capacity to generate intra- and interspecific hybrids. Whole genome sequencing and total DNA content analyses indicated that the hybrids were in each case full genome, mostly tetraploid hybrids. We also carried out the first application of single-cell RNA sequencing in Leishmania, which highlighted the transcriptome heterogeneity of the cultured promastigotes and revealed discrete clusters that emerged post-irradiation in which genes potentially involved in genetic exchange were expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for promastigotes that could either hybridize or not in vitro. Overall, this work revealed that there are specific populations involved in Leishmania hybridization associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains. 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 used L. tropica parental strains MA37 and L747 that have a high mating efficiency to generate CRISPR-Cas9 competent cell lines and to delete the meiosis-related genes HAP2-1, HAP2-2, SPO11, MND1, DMC1, HOP1 and HOP2, to investigate their respective roles in genetic exchange. We were able to generate null mutants for each of these genes in both L747 and MA37 by substituting the whole CDS for the Puromycin N-acetyltransferase (PAC) gene flanked by 5 and 3 UTRs of a Leishmania housekeeping gene. We used the null mutants from one of the strains in combination with a control line containing the Blasticidin S deaminase (BSD) gene integrated into the SSU rRNA locus and selected for hybrids resistant to both PAC and BSD. When null mutants of L. tropica MA37 were used for in vitro crosses, only the null mutant for HAP2-2 showed a significant decrease in the minimum frequency of hybridization-competent cells (5.8-fold lower, p = 0.0021). By contrast, L747 null mutants showed a deficit in in vitro mating capacity following deletion of HAP2-2, DMC1, HOP2, and especially HOP1, for which no hybrids were recovered in any of the 3 replicate experiments. Preliminary results using Lutzomyia longipalpis sand flies indicated that the L. tropica L747 HOP1 mutant was also required for hybridization in vivo. These findings implicate the involvement of several protein components of the meiotic machinery, including plasmogamy, synaptonemal complex formation, and recombination, in Leishmania hybridization. Further experiments, including the generation and testing of re-expressor lines, are being performed to further investigate the role of meiosis-related genes in genetic exchange.