Vector Biological Studies in Leishmaniasis

利什曼原虫是一种原生动物寄生虫，通过沙蝇媒介的叮咬传播，在其哺乳动物宿主中产生广泛的疾病。这些不同的临床结果与寄生虫品系和物种多样性直接相关。虽然利什曼原虫的繁殖主要是无性繁殖，但从群体遗传研究中推断出能够产生杂交基因型的隐性周期，并通过实验室杂交直接证明。实验表明，交配能力在很大程度上仅限于在沙蝇中肠发育的原毛菌。到目前为止，培养的原毛体的体外杂交能力仅限于两种热带乳杆菌菌株L747和MA37之间的低效率杂交，而这两种菌株在苍蝇中交配效率很高。我们发现，暴露于DNA损伤胁迫下的promastigote培养物显著提高了L. tropica菌株的体外杂交效率，并扩展到其他物种，包括L. donovani， L. infantum和L. braziliensis，产生种内和种间杂交的能力。全基因组测序和总DNA含量分析表明，杂种均为全基因组，多为四倍体杂种。我们还首次在利什曼原虫中应用了单细胞RNA测序，突出了培养的原毛菌的转录组异质性，并揭示了辐照后出现的离散簇，其中可能参与遗传交换的基因表达，包括祖先配子的融合原HAP2。通过生成HAP2的报告基因结构，我们可以选择能够在体外杂交或不能杂交的原原体。总的来说，这项工作揭示了利什曼原虫杂交的特定种群与可识别的转录组特征相关，并且胁迫促进的体外杂交可以成为在不同物种和菌株之间产生大量杂交基因型的变革性方法。

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.