Characterization of Calflagin, a Flagellar Calcium-Binding Protein from Trypanosoma congolense

BackgroundIdentification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests.MethodsImmuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay.ResultsThe antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves, associated with host IgG and IgM, were calculated to be 0.623 and 0.709 respectively, indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies.ConclusionsWhile calflagin is conserved among different species of African trypanosomes, our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections.

背景：鉴定物种特异性的锥虫分子，对于开展基于实验室和现场的流行病学及疾病诊断研究具有重要意义。尽管刚果锥虫（Trypanosoma congolense）是非洲牛类最主要的锥虫病原体，但目前尚未在该寄生虫的感染性血流期（BSF）中发现物种特异性分子，这一局限阻碍了诊断试剂的开发。

方法：本研究采用免疫质谱法，鉴定出一种可被刚果锥虫特异性单克隆抗体（mAb）Tc6/42.6.4识别的蛋白质。将该鉴定得到的分子在大肠杆菌（E. coli）中表达为重组蛋白，并通过多种免疫试验验证其与该单克隆抗体的结合能力。为探究导致刚果锥虫该分子免疫反应特异性差异的结构基础，我们对该蛋白质的三维结构进行建模，并分别与克氏锥虫（T. cruzi）和布氏锥虫（T. brucei）同源蛋白的晶体结构及核磁共振（NMR）结构进行比对。此外，本研究采用酶联免疫吸附试验（ELISA），检测感染锥虫的非洲牛体内产生的抗体，以评估刚果锥虫钙鞭毛蛋白（calflagin）用于血清学诊断试验的潜力。

结果：刚果锥虫特异性单克隆抗体Tc6/42.6.4所识别的抗原，被鉴定为鞭毛钙结合蛋白——钙鞭毛蛋白（calflagin）。重组后的该分子可与刚果锥虫特异性单克隆抗体结合，证实其即为同源抗原。免疫荧光试验结果显示，钙离子（Ca²+）可调控钙鞭毛蛋白在锥虫体内的定位。结构建模及与其他锥虫科原生动物钙鞭毛蛋白同源物的比对结果显示，刚果锥虫钙鞭毛蛋白表面存在4个非保守区域；由于表面化学性质及结构形貌的差异，这些区域或可形成物种特异性表位。以重组钙鞭毛蛋白为抗原的ELISA试验结果表明，多数感染锥虫的牛体内均存在针对该蛋白的抗体应答。针对宿主免疫球蛋白G（IgG）与免疫球蛋白M（IgM）的受试者工作特征（ROC）曲线下面积分别为0.623与0.709，提示锥虫感染与抗钙鞭毛蛋白抗体的存在呈正相关。

结论：尽管钙鞭毛蛋白在非洲锥虫的不同物种间具有保守性，但本研究结果表明，刚果锥虫钙鞭毛蛋白拥有独特的表位，可将其与其他锥虫物种的同源蛋白区分开来。单克隆抗体Tc6/42.6.4可作为实验室工具，用于刚果锥虫的鉴定。刚果锥虫钙鞭毛蛋白具备作为血清学诊断抗原的潜力，其在牛感染诊断的抗原检测试验中的应用价值值得进一步探索。