Hung Chiung-Yu, Yu Jieh-Juen, Seshan Kalpathi R, Reichard Utz, Cole Garry T
Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614-5086, USA.
Infect Immun. 2002 Jul;70(7):3443-56. doi: 10.1128/IAI.70.7.3443-3456.2002.
We report the isolation of a Coccidioides immitis gene (SOWgp) which encodes an immunodominant, spherule outer wall glycoprotein that is presented as a component of a parasitic phase-specific, membranous layer at the cell surface. The open reading frame of the gene from C. immitis isolate C735 translates a 422-amino-acid (aa) polypeptide that contains 6 copies of a 41- to 47-residue tandem repeat enriched in proline (20.4 mol%) and aspartate (19.7%). Two additional isolates of C. immitis produce SOWgps of different molecular sizes (328 and 375 aa) and show a corresponding difference in the number of tandem repeats (four and five, respectively). The accurate molecular sizes of these proline-rich antigens, as determined by surface-enhanced laser desorption/ionization mass spectrometry, are comparable to the predicted sizes from the translated protein sequences rather than the estimated sizes based on gel-electrophoretic separation. The results of Northern hybridization confirmed that SOWgp expression is parasitic phase specific, and immunoblot studies showed that elevated levels of production of this antigen occurred during early spherule development. The recombinant polypeptide (rSOWp) was shown to bind to mammalian extracellular matrix (ECM) proteins in an in vitro assay (laminin > fibronectin > collagen type IV), suggesting that the parasitic cell surface antigen may function as an adhesin. Deletion of the SOWgp gene by using a targeted gene replacement strategy resulted in partial loss of the ability of intact spherules to bind to ECM proteins and a significant reduction in virulence of the mutant strain. The wild-type gene was restored in the mutant by homologous recombination, and the revertant strain was shown to be as virulent as the parental isolate in our murine model of coccidioidomycosis. The parasitic cell surface glycoprotein encoded by the SOWgp gene appears to function as an adhesin and contributes to the virulence of C. immitis.
我们报告了一种粗球孢子菌基因（SOWgp）的分离情况，该基因编码一种免疫显性的球形体外壁糖蛋白，它作为细胞表面寄生虫阶段特异性膜层的一个组成部分呈现出来。来自粗球孢子菌分离株C735的该基因开放阅读框翻译出一个422个氨基酸（aa）的多肽，该多肽包含6个富含脯氨酸（20.4摩尔%）和天冬氨酸（19.7%）的41至47个残基的串联重复序列。另外两个粗球孢子菌分离株产生不同分子大小（328和375个aa）的SOWgps，并在串联重复序列数量上表现出相应差异（分别为4个和5个）。通过表面增强激光解吸/电离质谱法确定的这些富含脯氨酸抗原的准确分子大小，与根据翻译后的蛋白质序列预测的大小相当，而不是基于凝胶电泳分离估计的大小。Northern杂交结果证实SOWgp表达具有寄生虫阶段特异性，免疫印迹研究表明该抗原在球形体早期发育过程中产量升高。在体外试验中，重组多肽（rSOWp）显示能与哺乳动物细胞外基质（ECM）蛋白结合（层粘连蛋白>纤连蛋白>IV型胶原），这表明寄生虫细胞表面抗原可能起黏附素的作用。通过靶向基因置换策略缺失SOWgp基因导致完整球形体与ECM蛋白结合的能力部分丧失，且突变株的毒力显著降低。通过同源重组在突变体中恢复野生型基因，在我们的球孢子菌病小鼠模型中，回复株显示出与亲本分离株一样的毒力。由SOWgp基因编码的寄生虫细胞表面糖蛋白似乎起黏附素的作用，并对粗球孢子菌的毒力有贡献。
