Expression of calpain-like proteins and effects of calpain inhibitors on the growth rate of Angomonas deanei wild type and aposymbiotic strains.
Expression of calpain-like proteins and effects of calpain inhibitors on the growth rate of Angomonas deanei wild type and aposymbiotic strains.
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DOI：
10.1186/s12866-015-0519-0
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发表时间：
2015-09-29
期刊：
影响因子：
4.2
通讯作者：
Branquinha MH
中科院分区：
文献类型：
--
作者：
de Oliveira SS;Garcia-Gomes Ados S;d'Avila-Levy CM;dos Santos AL;Branquinha MH
迪恩囊单胞菌是一种寄生于昆虫的锥虫，它具有细菌内共生体，为宿主提供氨基酸和其他营养物质。原生动物的抗生素处理诱导的细菌损失导致对氨基酸的需求增加的脱共生菌株，并导致胞外肽酶的产生增加。在这项工作中，一个更详细的检查A。deanei进行了测定内共生体损失对宿主钙蛋白酶样蛋白（CALP）的影响，然后测试不同的钙蛋白酶抑制剂对寄生虫增殖的影响。Western blotting结果显示，抗果蝇钙蛋白酶（抗Dm-calpain）、抗龙虾钙蛋白酶（抗CDPIIb）和抗布氏锥虫钙蛋白酶（抗CAP 5.5）的抗体对CALP有不同的反应性，提示CALP可能受到内共生体的影响。在A. deanei野生型和非共生菌株中，80 kDa的蛋白质与抗Dm-钙蛋白酶抗体交叉反应;然而，未发现与抗CAP 5.5和抗CDPIIb抗体的交叉反应。在A. deanei基因组中的D. melanogaster calpain，T.布鲁氏菌CAP5.5和龙虾CDPIIb钙蛋白酶揭示了具有至少一个钙蛋白酶保守结构域的命中物的存在，并且还具有与每种抗体的识别一致的理论分子量。内共生体基因组中没有发现明显的命中，表明钙蛋白酶分子可能不存在于共生体中。用抗钙蛋白酶抗体处理的细胞的流式细胞术分析显示，更大量的反应性表位位于细胞内。可逆的钙蛋白酶抑制剂MDL 28170在减少两种菌株的生长方面显示出比非竞争性钙蛋白酶抑制剂PD 150606高得多的功效，而不可逆的钙蛋白酶抑制剂V仅略微减少增殖。总之，这些结果表明，不同的钙蛋白酶样分子表达的A。deanei，与一个可能的调制的表达影响的内共生体。此外，用MDL 28170处理影响两种菌株的生长速率，如先前在人类致病性物种亚马逊利什曼原虫和克氏锥虫中所确定的，A. deanei共享免疫学和生物化学关系。
Angomonas deanei is a trypanosomatid parasite of insects that has a bacterial endosymbiont, which supplies amino acids and other nutrients to its host. Bacterium loss induced by antibiotic treatment of the protozoan leads to an aposymbiotic strain with increased need for amino acids and results in increased production of extracellular peptidases. In this work, a more detailed examination of A. deanei was conducted to determine the effects of endosymbiont loss on the host calpain-like proteins (CALPs), followed by testing of different calpain inhibitors on parasite proliferation. Western blotting showed the presence of different protein bands reactive to antibodies against calpain from Drosophila melanogaster (anti-Dm-calpain), lobster calpain (anti-CDPIIb) and cytoskeleton-associated calpain from Trypanosoma brucei (anti-CAP5.5), suggesting a possible modulation of CALPs influenced by the endosymbiont. In the cell-free culture supernatant of A. deanei wild type and aposymbiotic strains, a protein of 80 kDa cross-reacted with the anti-Dm-calpain antibody; however, no cross-reactivity was found with anti-CAP5.5 and anti-CDPIIb antibodies. A search in A. deanei genome for homologues of D. melanogaster calpain, T. brucei CAP5.5 and lobster CDPIIb calpain revealed the presence of hits with at least one calpain conserved domain and also with theoretical molecular mass consistent with the recognition by each antibody. No significant hit was observed in the endosymbiont genome, indicating that calpain molecules might be absent from the symbiont. Flow cytometry analysis of cells treated with the anti-calpain antibodies showed that a larger amount of reactive epitopes was located intracellularly. The reversible calpain inhibitor MDL28170 displayed a much higher efficacy in diminishing the growth of both strains compared to the non-competitive calpain inhibitor PD150606, while the irreversible calpain inhibitor V only marginally diminished the proliferation. Altogether, these results indicate that distinct calpain-like molecules are expressed by A. deanei, with a possible modulation in the expression influenced by the endosymbiont. In addition, treatment with MDL28170 affects the growth rate of both strains, as previously determined in the human pathogenic species Leishmania amazonensis and Trypanosoma cruzi, with whom A. deanei shares immunological and biochemical relationships.