Vector Biological Studies in Leishmaniasis

利什曼病媒介生物学研究

• 批准号: 10692011
• 负责人: David Sacks
• 金额: $ 77.51万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692011
• 关键词: Acetyltransferase; Alleles; Biological; Biology; Bite; CRISPR/Cas technology; Cell Line; Cells; Clinical; Competence; DNA Damage; Deaminase; Development; Disease; Dose; Frequencies; Generations; Genes; Genetic; Genetic Population Study; Genetic Recombination; Genotype; Germ Cells; Goals; Heterogeneity; Housekeeping Gene; Human; Huntingtin-Associated protein 1; Hybrids; Immune response; In Vitro; India; Infection; Inflammatory Response; Inheritance Patterns; Laboratories; Leishmania; Leishmania donovani; Leishmaniasis; Lutzomyia genus; Maps; Meiosis; Midgut; Outcome; Parasites; Partner in relationship; Pathogenesis; Ploidies; Population; Process; Proteins; Puromycin; Reporter; Reproduction; Resistance; Ribosomal RNA; Role; Saliva; Sand Flies; Stress; Synaptonemal Complex; Testing; Untranslated Regions; Vaccines; Visceral Leishmaniasis; Work; experimental study; fly; gamma irradiation; genome sequencing; in vivo; irradiation; microbiota; mutant; single-cell RNA sequencing; trait; transcriptome; transcriptomics; transmission process; vector; whole genome

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.

利什曼原虫是一种原生动物寄生虫，通过沙蝇媒介叮咬传播，在哺乳动物宿主中产生多种疾病。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。 尽管利什曼原虫的繁殖主要是克隆性的，但从群体遗传学研究中推断出能够产生杂交基因型的神秘性周期，并通过实验室杂交直接证明。实验上，交配能力很大程度上局限于白蛉中肠中发育的前鞭毛体。迄今为止，体外培养的前鞭毛体杂交的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，而这两种菌株在果蝇中的交配效率很高。我们发现，将前鞭毛体培养物暴露于DNA损伤应激下，热带乳杆菌菌株的体外杂交效率显着提高，并扩展到其他物种，包括杜氏乳杆菌、婴儿乳杆菌和巴西乳杆菌，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，这些杂种在每种情况下都是全基因组，大部分是四倍体杂种。我们还在利什曼原虫中首次应用了单细胞 RNA 测序，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，包括祖先配子融合剂 HAP2。 通过生成 HAP2 的报告基因构建体，我们可以选择可以在体外杂交或不能杂交的前鞭毛体。总体而言，这项工作揭示了利什曼原虫杂交涉及与可辨别的转录组特征相关的特定种群，并且应激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。 如前所述，种内和种间杂种的产生已在白蛉感染期间的实验室杂交中得到证实，并且最近在伽马辐射诱导的 DNA 损伤后的培养中得到了证实。尽管尚未鉴定出配子或减数分裂形式，但等位基因遗传模式强烈表明减数分裂过程。 我们利用具有高交配效率的热带乳杆菌亲本菌株MA37和L747生成CRISPR-Cas9感受态细胞系，并删除减数分裂相关基因HAP2-1、HAP2-2、SPO11、MND1、DMC1、HOP1和HOP2，以研究它们各自在遗传交换中的作用。通过用整个 CDS 替换侧翼为利什曼原虫管家基因 5 个和 3 个 UTR 的嘌呤霉素 N-乙酰转移酶 (PAC) 基因，我们能够为 L747 和 MA37 中的每个基因生成无效突变体。我们将其中一个菌株的无效突变体与含有整合到 SSU rRNA 基因座中的杀稻瘟菌素 S 脱氨酶 (BSD) 基因的对照系结合使用，并选择对 PAC 和 BSD 都有抗性的杂种。当热带乳杆菌 MA37 的无效突变体用于体外杂交时，只有 HAP2-2 的无效突变体显示杂交感受态细胞的最小频率显着降低（降低 5.8 倍，p = 0.0021）。 相比之下，L747 无效突变体在删除 HAP2-2、DMC1、HOP2、尤其是 HOP1 后表现出体外交配能力缺陷，在 3 个重复实验中均未恢复到杂交体。使用 Lutzomyia longipis 沙蝇的初步结果表明，L. tropica L747 HOP1 突变体也是体内杂交所必需的。 这些发现暗示了利什曼原虫杂交中减数分裂机制的几种蛋白质成分的参与，包括质配子、联会复合体的形成和重组。 正在进行进一步的实验，包括重新表达系的产生和测试，以进一步研究减数分裂相关基因在遗传交换中的作用。