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一系列疾病。这些不同的临床结果与寄生虫菌株和物种多样性直接相关。虽然利什曼原虫的繁殖主要是克隆的，但从群体遗传学研究中已经推断出能够产生杂交基因型的隐蔽的有性周期，并通过实验室杂交直接证明了这一点。在实验上，交配能力在很大程度上局限于沙蝇中肠中发育的前鞭毛体。到目前为止，体外杂交培养前鞭毛体的能力仅限于两个热带乳杆菌菌株L747和MA37之间的低效杂交，这两个品系在果蝇中进行高效交配。我们发现，将前鞭毛体培养置于DNA损伤胁迫下，可以显著提高热带乳杆菌品系的体外杂交效率，并延伸到其他物种，包括多诺瓦尼乳杆菌、婴儿乳杆菌和巴西乳杆菌，它们具有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，每一株杂种都是全基因组，大多数是四倍体杂种。我们还首次在利什曼原虫中进行了单细胞RNA测序，这突出了培养的前鞭毛体的转录组异质性，并揭示了辐射后出现的离散簇，在这些簇中可能涉及遗传交换的基因被表达，包括祖先配子FusoGen HAP2。通过为HAP2生成报告结构，我们可以选择可以在体外杂交或不杂交的前鞭毛体。总体而言，这项工作揭示了利什曼原虫杂交中涉及的特定群体与可辨别的转录特征相关，并且胁迫促进的体外杂交可以成为一种在不同物种和菌株之间产生大量杂交基因的变革性方法。 如上所述，在沙蝇感染期间的实验室杂交中以及最近在伽马辐射导致DNA损伤后的培养中都证明了种内和种间杂交的产生。虽然还没有鉴定出配子或减数分裂形式，但等位基因遗传模式强烈地表明了减数分裂过程。我们以交配效率高的热带乳杆菌亲本MA37和L747为材料，建立CRISPR-Cas9感受态细胞系，并删除与减数分裂相关的基因HAP2-1、HAP2-2、SPO11、MND1、DMC1、HOP1和HOP2，以研究它们在遗传交换中的各自作用。我们通过用完整的CDS替换位于利什曼原虫看家基因5个和3个UTRs两侧的嘌呤霉素N-乙酰转移酶(PAC)基因，从而为L747和MA37中的每个基因产生了零突变。我们利用其中一个菌株的零突变体与一个含有整合到SSU rRNA基因座的杀青素S脱氨酶基因的对照系相结合，进行了对PAC和BSD都具有抗性的杂交种的筛选。当热带乳杆菌MA37的零突变体用于体外杂交时，只有HAP2-2的零突变体显著降低了杂交能力细胞的最低频率(降低5.8%，p=0.0021)。相反，在HAP2-2、DMC1、HOP2，尤其是HOP1缺失后，L747缺失突变体的体外交配能力出现缺陷，在3次重复实验中均未获得杂交后代。用长斑潜蝇的初步结果表明，热带丝虫L747HOP1突变体也需要进行体内杂交。这些发现暗示了包括胞质配子、联会复合体形成和重组在内的减数分裂机制的几个蛋白质成分参与了利什曼原虫的杂交。进一步的实验，包括重新表达系的产生和测试，正在进行中，以进一步研究减数分裂相关基因在遗传交换中的作用。

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.