Molecular characterization, expression, and in vivo analysis of LmexCht1: the chitinase of the human pathogen, Leishmania mexicana.
Molecular characterization, expression, and in vivo analysis of LmexCht1: the chitinase of the human pathogen, Leishmania mexicana.
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DOI：
10.1074/jbc.m412299200
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发表时间：
2005-02-04
期刊：
影响因子：
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通讯作者：
Dwyer DM
中科院分区：
文献类型：
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作者：
Joshi MB;Rogers ME;Shakarian AM;Yamage M;Al-Harthi SA;Bates PA;Dwyer DM
几丁质酶在多种寄生生物的生命周期、发育和传播中具有重要作用。利用分子生物学方法，我们确定和表征了一个单拷贝LmexCht 1-几丁质酶基因的结构，从原始的锥虫病原体的人类，墨西哥利什曼原虫。LmexCht 1编码约50 kDa的蛋白，具有几丁质酶-18蛋白家族成员特有的高度保守的底物结合和催化结构域。此外，我们表明，LmexCht 1 mRNA组成型表达的昆虫载体（即前鞭毛体）和哺乳动物（即无鞭毛体）的生命周期发育形式的这种原生动物寄生虫。然而，有趣的是，发现无鞭毛体在其体外生长期间分泌/释放比前鞭毛体高~ >2-4倍水平的几丁质酶活性。此外，同源附加体表达系统的设计和用于表达表位标记的LmexCht 1嵌合构建体在这些寄生虫。通过RT-PCR、Western印迹和间接免疫荧光分析，在这些转染子中验证LmexCht 1嵌合体的表达。此外，偶联免疫沉淀/酶活性实验的结果表明，LmexCht 1嵌合蛋白由这些转染的L. mexicana寄生虫，并具有功能性几丁质酶活性。还评估了这种转染子在体外人巨噬细胞和两种不同品系小鼠中的感染性。这些实验的结果表明，与对照寄生虫相比，LmexCht 1转染子在人巨噬细胞中的存活率明显更好，并且在小鼠中产生了明显更大的病变。总而言之，我们的结果表明LmexCht 1嵌合体为这些哺乳动物宿主中的寄生虫提供了明确的生存优势。因此，LmexCht 1可能代表这种重要人类病原体在哺乳动物阶段的新毒力决定因子
Chitinases have been implicated to be of importance in the life cycle development and transmission of a variety of parasitic organisms. Using a molecular approach, we identified and characterized the structure of a single copy LmexCht1-chitinase gene from the primitive trypanosomatid pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an ~50 kDa protein, with well-conserved substrate-binding and catalytic domains characteristic of members of the Chitinase-18 protein family. Further, we showed that LmexCht1 mRNA is constitutively expressed by both the insect vector (i.e. promastigote) and mammalian (i.e. amastigote) life cycle developmental forms of this protozoan parasite. Interestingly, however, amastigotes were found to secrete/release ~ >2-4 fold higher levels of chitinase activity during their growth in vitro than promastigotes. Moreover, a homologous episomal-expression system was devised and used to express an epitope–tagged LmexCht1 chimeric construct in these parasites. Expression of the LmexCht1 chimera was verified in these transfectants by RT-PCR, Western blots and indirect immunofluorescence analyses. Further, results of coupled-immunoprecipitation/ enzyme activity experiments demonstrated that the LmexCht1 chimeric protein was secreted/released by these transfected L. mexicana parasites and that it possessed functional chitinase enzyme activity. Such transfectants were also evaluated for their infectivity both in human macrophages in vitro and in two different strains of mice. Results of those experiments demonstrated that the LmexCht1 transfectants survived significantly better in human macrophages and also produced significantly larger lesions in mice than control parasites. Taken together, our results indicate that the LmexCht1-chimera afforded a definitive survival advantage to the parasite within these mammalian hosts. Thus, the LmexCht1 could potentially represent a new virulence determinant in the mammalian phase of this important human pathogen