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: 8284346
• 负责人: CHRISTOPHER M. WEST
• 金额: $ 5.44万
• 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 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8284346
• 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（由申请方提供）：克氏锥虫是一种单细胞原生动物寄生虫，可引起查加斯病，其特征是慢性感染患者的心脏或胃肠道并发症。目前还没有有效的治疗方法，迫切需要针对寄生虫的新药靶标。T.克鲁兹提出了一个复杂的生命周期，涉及锥蝽昆虫载体和几个可能的脊椎动物宿主，包括人类。寄生虫表面成分被怀疑在其连续的宿主环境中发挥重要作用，以实现生存策略。间接的实验证据表明，表面糖肌醇磷脂（GIPL）和粘蛋白型糖蛋白保护寄生虫蛋白酶，补体，溶解性抗体，吞噬作用，并作为潜在的免疫调节剂，甚至可以决定在感染过程中的发病的命运。粘蛋白型糖蛋白的独有特征包括在粘蛋白的0-聚糖和Thr残基之间形成桥的糖1GlcNAc、在吡喃糖苷和/或呋喃糖苷形式中发现的2Gal残基以及末端13 Gal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缀合物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型O-糖基化是否作为毒力因子起作用，并帮助寄生虫在其生命周期的不同环境中生存和成功。我们假设高尔基体酶UDP-GlcNAc：多肽N-乙酰葡糖胺基转移酶（pp-1GlcNAcT）和UDP-Gal：半乳糖苷13-半乳糖基转移酶（13 GalT）分别负责将粘蛋白型O-聚糖中的第一个1GlcNAc添加到Thr和末端13 Gal残基，它们对T的正确组装是必需的。cruzi表面结构。提出了两个具体目标来检验这一点。在目的1中，将通过靶向基因置换获得编码pp-1GlcNAcTs的三个TcOGNT基因（1、2和L）中的每一个的条件性双敲除，三个TcOGNT同源物将在相关的非致病性锥虫利什曼原虫tarentolae和T. cruzi，并且TcOGNT的表达将在连续的生命周期阶段上被映射。转基因寄生虫将用于评价糖基化缺陷、酶活性和细胞功能，包括活力、定殖无脊椎动物宿主、分化、抵抗补体替代途径裂解和/或粘附、感染和在哺乳动物宿主细胞内存活的能力。目的2：克隆T. cruzi的13 GalT活性，并通过靶向基因破坏和在T.克鲁兹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调查其在追求治疗恰加斯病的新药靶点的毒力中的作用。

项目成果

Venom alkaloids against Chagas disease parasite: search for effective therapies.

针对恰加斯病寄生虫的毒液生物碱：寻找有效的疗法。

• DOI: 10.1038/s41598-020-67324-8
• 发表时间: 2020
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Silva,RafaelCMCosta;Fox,EduardoGP;Gomes,FabioM;Feijó,DanielF;Ramos,Isabela;Koeller,CarolinaM;Costa,TatianaFR;Rodrigues,NathaliaS;Lima,AnaP;Atella,GeorgiaC;Miranda,Kildare;Schoijet,AlejandraC;Alonso,GuillermoD;deAl
• 通讯作者: deAl