Rodrigues Adriana C, Garcia Herakles A, Ortiz Paola A, Cortez Alane P, Martinkovic Franjo, Paiva Fernando, Batista Jael S, Minervino Antonio H, Campaner Marta, Pral Elizabeth M, Alfieri Silvia C, Teixeira Marta M G
Departamento de Parasitologia, Universidade de São Paulo, São Paulo, SP, Brazil.
Parasitol Int. 2010 Sep;59(3):318-25. doi: 10.1016/j.parint.2010.03.002. Epub 2010 Mar 15.
Although Trypanosomatheileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of approximately 1.7kb located in 2 chromosomal bands of 600-720kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes.
尽管泰勒锥虫及相关锥虫是牛科动物中分布最广的锥虫，但对这些物种中的蛋白水解酶却知之甚少。我们对来自牛、水牛和鹿的分离株中编码组织蛋白酶L样（CATL）半胱氨酸蛋白酶的基因进行了表征，这些基因与其他锥虫物种的同源物有很大差异。对来自22株泰勒锥虫的78个CATL催化结构域序列进行分析，发现6种基因型紧密聚集在泰勒锥虫进化枝中。这些锥虫中的CATL基因以约1.7kb的串联阵列形式组织，位于600 - 720kb的两条染色体带中。一种针对CATL序列的诊断性PCR检测方法能检测到来自牛、水牛、鹿以及虻类载体的所有基因型的泰勒锥虫。通过明胶凝胶中的蛋白水解活性和Z - Phe - Arg - AMC底物的水解证明了泰勒锥虫半胱氨酸蛋白酶的表达。这项工作的结果与先前使用核糖体和剪接前导基因的数据一致，表明CATL基因序列可用于泰勒锥虫的诊断、群体基因分型和进化研究。
