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

粘蛋白型 O-糖基化在克氏锥虫生物学中的作用

• 批准号: 7944915
• 负责人: CHRISTOPHER M. WEST
• 金额: $ 6.15万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7944915
• 关键词: Address; Alternative Complement Pathway; Amoeba genus; Antibodies; Architecture; Back; Binding; Biochemical; Biological Assay; Biology; Cardiac; Cell Nucleus; Cell physiology; Cell surface; Cells; Chagas Disease; Chimera organism; Codon Nucleotides; Complement; Complex; Cyclic AMP-Dependent Protein Kinases; Cytolysis; Cytoplasm; Defect; Dependence; Development; Dictyostelium; Drug Delivery Systems; Environment; Enzymes; Epitopes; Future; Galactosides; Galactosyltransferases; Gene Expression; Gene Targeting; Genes; Genetic; Genome; Glycoconjugates; Glycopeptides; Glycoproteins; Goals; Golgi Apparatus; Homologous Gene; Human; Hydroxylation; Hypoxia; Immunomodulators; Immunoprecipitation; In Vitro; Infection; Insect Vectors; Leishmania; Life Cycle Stages; Lytic; Maps; Mediating; Modification; Monitor; Mucins; Mutation; Nuclear; Orthologous Gene; Parasites; Pathogenesis; Pathway interactions; Patients; Peptide Hydrolases; Peripheral; Phagocytosis; Phenotype; Play; Polysaccharides; Positioning Attribute; Proline; Property; Protein Isoforms; Proteins; Regulation; Reporter; Reproduction spores; Role; Sialic Acids; Signal Transduction; Surface; Testing; Toxoplasma; Toxoplasma gondii; Transferase; Trisaccharides; Trypanosoma cruzi; UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; Virulence; Virulence Factors; Western Blotting; Work; abstracting; cell type; directed evolution; enzyme activity; gastrointestinal; gene replacement; glycosylation; glycosyltransferase; in vivo; invertebrate host; mutant; polypeptide; promoter; social; sugar

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.

描述(申请人提供)：克氏锥虫是一种单细胞原生动物寄生虫，可导致恰加斯病，其特征是慢性感染患者出现心脏或胃肠道并发症。目前还没有治愈的方法，迫切需要针对这种寄生虫的新药靶点。克氏锥虫呈现了一个复杂的生命周期，包括一个三聚氰胺昆虫媒介和包括人类在内的几种可能的脊椎动物宿主。寄生虫表面成分被认为在它们的连续宿主环境中发挥着重要的作用，以实现生存策略。间接实验证据表明，表面糖蛋白磷脂(GIPLs)和粘蛋白类型的糖蛋白保护寄生虫免受蛋白酶、补体、裂解抗体、吞噬作用的侵袭，并作为潜在的免疫调节剂发挥作用，甚至可能在感染过程中决定发病的命运。粘蛋白型糖蛋白的独特特征包括：1GlcNAc形成粘蛋白O-糖链和Thr残基之间的桥梁，2Gal残基以吡喃糖苷和/或呋喃糖苷的形式存在，以及13Gal或唾液酸残基，后者通过寄生虫细胞表面转唾液酸酶的作用从宿主糖共轭供体转移到寄生虫的表面Gal受体。我们的目标是确定粘蛋白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和成功。我们推测，高尔基体酶UDP-GlcNAc：多肽N-乙酰氨基葡萄糖基转移酶(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添加到苏氨酸和粘蛋白类型O-葡聚糖中的末端13Gal残基，是正确组装克氏锥虫表面结构所必需的。为了检验这一点，我们提出了两个具体目标。在目标1中，通过靶向基因置换获得编码pp-1GlcNActs的三个TcOGNT基因(1、2和L)中每一个的条件双敲除，这三个TcOGNT同源物将在相关的非致病锥虫塔氏利什曼原虫和克氏锥虫中单独或联合表达，并将TcOGNTs的表达映射到一系列的生活史阶段。这些转基因寄生虫将被用来评估糖基化、酶活性和细胞功能方面的缺陷，包括生存能力、定植无脊椎动物宿主的能力、分化能力、通过补体的替代途径抵抗溶解和/或在哺乳动物宿主细胞内黏附、感染和存活的能力。目的2对克氏毛滴虫高尔基体13GalT基因进行分析，检测其异源表达后预测的13GalT活性，并在克氏毛滴虫中通过靶向基因干扰和过度表达来研究其生化和细胞功能。本项目的重点是关于糖基转移酶的生化和细胞功能的基本问题，但未来的工作有望调查它们在毒力中的作用，以寻求治疗恰加斯病的新药物靶点。