Augustine Swinburne A J, Kleshchenko Yuliya Y, Nde Pius N, Pratap Siddharth, Ager Edward A, Burns James M, Lima Maria F, Villalta Fernando
Division of Microbial Pathogenesis and Immune Response, Department of Biomedical Sciences, School of Medicine, Meharry Medical College, 1005 Dr. D. B. Todd Jr. Blvd., Nashville, TN 37208, USA.
Infect Immun. 2006 Jul;74(7):3922-9. doi: 10.1128/IAI.00045-06.
In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of Trypanosoma cruzi, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of T. cruzi. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the T. cruzi genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked T. cruzi infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.
在本研究中，我们报告了克氏锥虫（恰加斯病的病原体）首个细胞表面酪蛋白激酶II（CKII）底物（Tc-1）的克隆及特性分析。基因序列分析显示，一个1653 bp的开放阅读框编码550个氨基酸残基。Northern印迹分析表明，在侵袭性锥鞭毛体中表达的转录本为4.5 kb，而在克氏锥虫的非侵袭性上鞭毛体形式中不表达。Southern印迹分析表明，Tc-1是单拷贝基因。在氨基酸水平上，Tc-1与克氏锥虫基因组计划最近报道的两种假定蛋白具有95%和99%的同一性。对翻译后的氨基酸序列分析表明，Tc-1基因具有一个假定的跨膜结构域，带有多个胞质和胞外CKII磷酸化位点。外源性人CKII能够使重组Tc-1和完整锥鞭毛体的Tc-1上的丝氨酸残基磷酸化。这种磷酸化被CKII抑制剂肝素和4,5,6,7-四溴-2-氮杂苯并咪唑抑制。用抗重组Tc-1免疫球蛋白G探测可溶性锥鞭毛体、上鞭毛体和无鞭毛体的免疫印迹显示，一种62 kDa的蛋白仅在感染性锥鞭毛体中表达。锥鞭毛体标记表面蛋白的免疫沉淀表明，62 kDa的蛋白是一种表面蛋白，并且我们通过直接免疫荧光发现该蛋白均匀分布在锥鞭毛体表面。针对Tc-1的抗体有效阻断了锥鞭毛体对宿主细胞的侵袭，从而降低了寄生虫载量。用CKII抑制剂对锥鞭毛体或成肌细胞进行预孵育可阻断克氏锥虫感染。因此，我们首次描述了一种原生动物寄生虫的细胞表面CKII底物，该底物可被人CKII磷酸化并参与细胞感染。
