Ortiz P A, Maia da Silva F, Cortez A P, Lima L, Campaner M, Pral E M F, Alfieri S C, Teixeira M M G
Department of Parasitology, University of São Paulo (USP), São Paulo, SP, 05508-900, Brazil.
Acta Trop. 2009 Dec;112(3):249-59. doi: 10.1016/j.actatropica.2009.07.036. Epub 2009 Aug 14.
We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T. rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T. rangeli (rangelipain) closest to T. cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T. rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T. rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T. rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T. rangeli lineages associated with sympatric species of Rhodnius.
我们对来自中美洲和南美洲人类、野生哺乳动物以及红猎蝽属物种的克氏锥虫分离株中编码组织蛋白酶L样（CatL样）半胱氨酸蛋白酶的基因进行了测序。克氏锥虫成熟CatL样酶编码序列与其他锥虫、利什曼原虫属和波豆虫属同源基因的系统发育树显示，克氏锥虫（兰氏锥虫蛋白酶）的序列与克氏锥虫（克氏锥虫蛋白酶）最为接近。基于CatL样序列的动基体目系统发育树与基于小亚基核糖体RNA（SSU rRNA）和甘油醛-3-磷酸脱氢酶（gGAPDH）基因推导的系统发育树完全一致。对代表克氏锥虫整体系统发育多样性和地理分布范围的17个分离株的CatL样催化结构域序列进行分析，支持了先前使用核糖体和剪接前导基因定义的所有谱系（A - D）。克氏锥虫分离株的蛋白水解活性比较显示，明胶凝胶中半胱氨酸蛋白酶的条带图谱存在异质性，即使在同一谱系的分离株之间也存在差异。事实证明，CatL样序列是通过聚合酶链反应（PCR）对克氏锥虫进行诊断和基因分型的极佳靶点。来自CatL样编码基因的数据与先前关于动粒DNA标记、核糖体和剪接前导基因的研究结果一致，从而证实了克氏锥虫的克隆进化、独立传播周期以及与同域红猎蝽物种相关的克氏锥虫谱系的分化。
