Vector Biological Studies in Leishmaniasis

利什曼原虫是原生动物寄生虫，通过白蛉媒介的叮咬传播，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株和物种多样性直接相关。 虽然利什曼原虫繁殖主要是克隆的，一个神秘的性周期能够产生杂交基因型已推断出人口遗传学研究，并直接证明了实验室杂交。在实验上，交配能力主要局限于在沙蝇中肠发育的前鞭毛体。到目前为止，在体外杂交培养前鞭毛体的能力仅限于两种乳杆菌之间的低效杂交。Tropica菌株L747和MA 37在果蝇中具有高效率的交配。我们发现，前鞭毛体培养物暴露于DNA损伤应激产生了显着提高的效率在体外杂交的L。tropica菌株，并延伸到其他物种，包括L.多诺瓦尼湖infantum和L. braziliensis，产生种内和种间杂种的能力。全基因组测序和总DNA含量分析表明，在每种情况下的杂种是全基因组，大多数四倍体杂种。我们还进行了单细胞RNA测序在利什曼原虫中的首次应用，该应用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，其中表达了可能参与遗传交换的基因，包括祖先配子融合子HAP 2。 通过产生HAP 2的报告构建体，我们可以选择在体外可以杂交或不杂交的前鞭毛体。总的来说，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组特征相关，并且应力促进体外杂交可以是一种变革性的方法，以在不同的物种和菌株之间产生大量的杂交基因型。 如前所述，种内和种间杂种的产生已经在实验室杂交中在白蛉感染期间得到证实，最近，在伽马辐照诱导的DNA损伤后的培养中得到证实。虽然没有配子或减数分裂形式已被确定，等位基因遗传模式强烈建议减数分裂过程。 我们使用L。本研究使用具有高交配效率的热带假丝酵母亲本菌株MA 37和L747来产生CRISPR-Cas9感受态细胞系并缺失减数分裂相关基因HAP 2 -1、HAP 2 -2、SPO 11、MND 1、DMC 1、HOP 1和HOP 2，以研究它们各自在遗传交换中的作用。我们能够在L747和MA 37中产生这些基因中的每一个的无效突变体，通过将整个CDS替换为侧接利什曼原虫管家基因的5个和3个UTR的嘌呤霉素N-乙酰转移酶（PAC）基因。我们使用的无效突变体的菌株之一，与含有杀稻瘟菌素S脱氨酶（BSD）基因整合到SSU rRNA基因座的控制线，并选择耐PAC和BSD的杂交种。当L. tropica MA 37用于体外杂交，只有HAP 2 -2的无效突变体显示出杂交感受态细胞的最小频率的显著降低（降低5.8倍，p = 0.0021）。 相比之下，L747无效突变体在缺失HAP 2 -2、DMC 1、HOP 2，特别是HOP 1后显示出体外交配能力的缺陷，在3个重复实验的任何一个中都没有回收到杂交体。利用长须沙蝇的初步研究结果表明，长须沙蝇的种群数量与沙蝇的种群数量呈负相关。tropica L747 HOP 1突变体也需要用于体内杂交。 这些发现暗示参与减数分裂机制，包括胞浆融合，联会复合体的形成和重组，在利什曼原虫杂交的几种蛋白质成分。 正在进行进一步的实验，包括再表达株系的产生和测试，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.