Gamboa D, Van Eys G, Victoir K, Torres K, Adaui V, Arevalo J, Dujardin J-C
Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, A.P. 4314, Lima 100, Peru.
Parasitology. 2007 Nov;134(Pt 12):1689-98. doi: 10.1017/S003118200700306X. Epub 2007 Sep 26.
Gene expression is known to vary significantly during the Leishmania life-cycle. Its monitoring might allow identification of molecular changes associated with the infective stages (metacyclics and amastigotes) and contribute to the understanding of the complex host-parasite relationships. So far, very few studies have been done on Leishmania (Viannia) braziliensis, one of the most pathogenic species. Such studies require, first of all, reference molecular markers. In the present work, we applied differential display analysis (DD analysis) in order to identify transcripts that might be (i) candidate markers of metacyclics and intracellular amastigotes of L. (V.) braziliensis or (ii) potential controls, i.e. constitutively expressed. In total, 48 DNA fragments gave reliable sequencing data, 29 of them being potential markers of infective stages and 12 potential controls. Eight sequences could be identified with reported genes. Validation of the results of DD analysis was done for 4 genes (2 differentially expressed and 2 controls) by quantitative real-time PCR. The infective insect stage-specific protein (meta 1) was more expressed in metacyclic-enriched preparations. The oligopeptidase b showed a higher expression in amastigotes. Two genes, glucose-6-phosphate dehydrogenase and a serine/threonine protein kinase, were found to be similarly expressed in the different biological samples.
已知基因表达在利什曼原虫的生命周期中会有显著变化。对其进行监测可能有助于识别与感染阶段（后循环体和无鞭毛体）相关的分子变化，并有助于理解复杂的宿主-寄生虫关系。到目前为止，针对最具致病性的物种之一巴西利什曼原虫（Viannia亚属）的研究非常少。此类研究首先需要参考分子标记。在本研究中，我们应用差异显示分析（DD分析）来鉴定可能是（i）巴西利什曼原虫（Viannia亚属）后循环体和细胞内无鞭毛体的候选标记物，或（ii）潜在对照，即组成性表达的转录本。总共48个DNA片段给出了可靠的测序数据，其中29个是感染阶段的潜在标记物，12个是潜在对照。8个序列可与已报道的基因进行比对。通过定量实时PCR对4个基因（2个差异表达基因和2个对照基因）的DD分析结果进行了验证。感染性昆虫阶段特异性蛋白（meta 1）在富含后循环体的制剂中表达更高。寡肽酶b在无鞭毛体中表达较高。发现葡萄糖-6-磷酸脱氢酶和一种丝氨酸/苏氨酸蛋白激酶这两个基因在不同生物样品中的表达相似。
