Role of mucin-type O-glycosylation in Trypanosoma cruzi biology

描述（由申请人提供）：克氏锥虫是一种单细胞原生动物寄生虫，可引起恰加斯病，其特征是慢性感染患者出现心脏或胃肠道并发症。目前还没有有效的治疗方法，迫切需要针对这种寄生虫的新药物靶点。克氏锥蝽具有复杂的生命周期，涉及锥蝽昆虫媒介和包括人类在内的几种可能的脊椎动物宿主。寄生虫表面成分被怀疑在其连续宿主环境中实现生存策略中发挥着重要作用。间接实验证据表明，表面糖肌醇磷脂（GIPL）和粘蛋白型糖蛋白可以保护寄生虫免受蛋白酶、补体、溶解抗体、吞噬作用的影响，并作为潜在的免疫调节剂发挥作用，甚至可以决定感染过程中发病机制的命运。粘蛋白型糖蛋白的独特特征包括糖 1GlcNAc 在 O-聚糖和粘蛋白的 Thr 残基之间形成桥梁，以吡喃糖苷和/或呋喃糖苷形式发现的 2Gal 残基，以及末端 13Gal 或唾液酸残基，后者通过寄生虫细胞表面反式的作用从宿主糖缀合物供体转移到寄生虫的表面 Gal 受体。唾液酸酶。我们的目标是确定粘蛋白型 O-糖基化是否作为毒力因子发挥作用，并帮助寄生虫在其生命周期的不同环境中生存并取得成功。我们假设高尔基体酶 UDP-GlcNAc: 多肽 N-乙酰氨基葡萄糖转移酶 (pp-1GlcNAcT) 和 UDP-Gal: 半乳糖苷 13-半乳糖基转移酶 (13GalT) 分别负责将第一个 1GlcNAc 添加到 Thr 和粘蛋白型 O-聚糖中的末端 13Gal 残基，对于正确组装T. cruzi 表面结构。提出了两个具体目标来测试这一点。在目标 1 中，将通过靶向基因替换获得编码 pp-1GlcNAcT 的三个 TcOGNT 基因（1、2 和 L）中每一个的条件双敲除，这三个 TcOGNT 同源物将在塔伦托利什曼原虫（一种相关的非致病性锥虫和克氏锥虫）中单独或组合表达，并且将在连续的生命周期阶段绘制 TcOGNT 的表达图谱。转基因寄生虫将用于评估糖基化、酶活性和细胞功能的缺陷，包括活力、定植无脊椎动物宿主、分化、抵抗补体替代途径裂解的能力，和/或在哺乳动物宿主细胞内粘附、感染和存活的能力。在目标 2 中，将通过测试其在盘基网柄菌中异源表达后预测的 13GalT 活性来分析 T. cruzi 的假定高尔基体 13GalT 基因，并通过靶向基因破坏和在 T. cruzi 中过度表达来研究其生化和细胞功能。该项目的重点是关于假定的糖基转移酶的生化和细胞功能的基本问题，但未来的工作预计将研究它们在毒力中的作用，以寻找治疗恰加斯病的新药物靶点。

DESCRIPTION (provided by applicant): Trypanosoma cruzi is a single-cell protozoan parasite that causes Chagas disease, characterized by cardiac or gastrointestinal complications in chronically infected patients. There is no curative treatment yet available and new-drug targets against the parasite are urgently needed. T. cruzi presents a complex life- cycle involving a Triatomine insect vector and several possible vertebrate hosts including humans. Parasite surface components are suspected to play essential roles to fulfill survival strategies in their sequential host environments. Indirect experimental evidences suggest that surface glycoinositolphospholipids (GIPLs) and mucin-type glycoproteins protect the parasites from proteases, complement, lytic antibodies, phagocytosis, and function as potential immunomodulators that could even determine the fate of pathogenesis during infection. Exclusive features of mucin-type glycoproteins include the sugar 1GlcNAc forming the bridge between O-glycans and Thr residues of the mucins, 2Gal residues found both in the pyranosidic and/or furanosidic forms, and terminal 13Gal or sialic-acid residues, the later being transferred from host glycoconjugate donors to the parasite's surface Gal acceptors by the action of parasite cell surface trans- sialidases. Our goal is to determine if mucin-type O-glycosylation works as virulence factor and help the parasite to survive and succeed in its different environments during its life-cycle. We hypothesize that the Golgi enzymes UDP-GlcNAc:polypeptide N-acetylglucosaminyl transferase (pp-1GlcNAcT) and UDP- Gal:galactoside 13-galactosyltransferase (13GalT), respectively responsible for the addition of the first 1GlcNAc to Thr and the terminal 13Gal residues in the mucin-type O-glycans, are essential for the correct assembly of T. cruzi surface architecture. Two specific aims are proposed to test this. In Aim 1, conditional double knock-outs for each of the three TcOGNT genes (1, 2 and L) that encode pp-1GlcNAcTs will be obtained by targeted gene replacement, the three TcOGNT homologs will be expressed alone or in combination in Leishmania tarentolae, a related non-pathogenic trypanosomatid, and T. cruzi, and expression of the TcOGNTs will be mapped over serial life cycle stages. The genetically modified parasites will be used to evaluate defects in glycosylation, enzyme activities and on cell function, including viability, capacity to colonize invertebrate hosts, differentiate, resist to lysis by the alternative pathway of complement, and/or to adhere, infect and survive inside mammalian host cells. In Aim 2, the putative Golgi 13GalT gene of T. cruzi will be analyzed, by testing its predicted 13GalT activity after heterologous expression in Dictyostelium, and investigating its biochemical and cellular function by targeted gene disruption and over-expression in T. cruzi. This project is focused on basic questions about the biochemical and cellular functions of the putative glycosyltransferases, but future work is expected to investigate their roles in virulence in pursuit of new drug targets for treatment of Chagas disease.