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Abstract：
Proteases are virulence factors with a recognized impact on the Leishmania spp. life cycle. This study considers a set of analyses measuring phenotypic factors of L. (V.) braziliensis clinical isolates as promastigotes growth curves, murine peritoneal macrophages infection, inflammatory mediators production, and serine proteases gene expression (subtilisin 13: S13, subtilisin 28: S28, oligopeptidase B: OPB) assessing these isolates\' fitness on in vitro conditions. Parasites had different behavior during the early growth phase from day zero to day three, and all isolates reached the stationary growth phase between days four and seven. Macrophages infection showed two tendencies, one of decreased infection rate and number of parasites per macrophage (Infection Index <1000) and another with a constant infection index (≥1400). TNF-α (≥10 pg/mL) detected in infections by 75% of isolates, IL-6 (≥80 pg/mL) by 30% of isolates and low levels of NO (≥0.01µM) in almost all infections. Gene expression showed higher values of S13 (≥2RQ) in the intracellular amastigotes of all the isolates evaluated. On the contrary, S28 expression was low (≤1RQ) in all isolates. OPB expression was different between promastigotes and intracellular amastigotes, being significantly higher (≥2RQ) in the latter form of 58% of the isolates. Predictive structural assays of S13 and OPB were performed to explore temperature influence on gene expression and the encoded proteases. Gene expression data is discussed based on in silico predictions of regulatory regions that show plasticity in the linearity index of secondary structures of S13 and OPB 3\'-untranslated regions of mRNA, dependent on temperature changes. While hairpin structures suggest an active region of mRNA for both genes above 26°C, pseudoknot structure found in S13 is an indication of a particular profile of this gene at mammalian host temperatures (37°C). Furthermore, the predicted 3D structures are in accordance with the influence of these temperatures on the catalytic site stability of both enzymes, favoring their action over peptide substrates. Data gathered here suggest that L. (V.) braziliensis serine proteases can be influenced by the temperature conditions affecting parasite fitness throughout its life cycle.
摘要：
蛋白酶是对利什曼原虫属有公认影响的毒力因子。生命周期。本研究考虑了一组测量L.(V.)巴西临床分离株作为前鞭毛生长曲线，小鼠腹膜巨噬细胞感染，炎症介质的产生，和丝氨酸蛋白酶基因表达（枯草杆菌蛋白酶13：S13，枯草杆菌蛋白酶28：S28，寡肽酶B：OPB）在体外条件下评估这些分离株的适应性。寄生虫在从零天到第三天的早期生长阶段有不同的行为，所有分离株在第4天到第7天之间达到静止生长期。巨噬细胞感染有两种倾向,一种是降低感染率和每个巨噬细胞的寄生虫数量（感染指数<1000），另一种是恒定感染指数（≥1400）。75％的分离株在感染中检测到TNF-α（≥10pg/mL），IL-6（≥80pg/mL）在几乎所有感染中都有30％的分离株和低水平的NO（≥0.01µM）。在所有评估的分离株的细胞内amastigotes中，基因表达均显示较高的S13值（≥2RQ）。相反,S28在所有分离株中表达较低(≤1RQ)。OPB在前鞭毛和细胞内的表达是不同的，在58%的分离株的后一种形式中显著更高(≥2RQ)。进行S13和OPB的预测性结构测定以探索温度对基因表达和编码的蛋白酶的影响。基因表达数据是根据对调节区的计算机预测进行讨论的，这些调节区在S13和OPB3'-mRNA非翻译区的二级结构的线性指数中显示出可塑性，取决于温度的变化。虽然发夹结构表明两个基因在26°C以上的mRNA活性区域，在S13中发现的假结结构是该基因在哺乳动物宿主温度(37°C)下的特定谱的指示。此外，预测的3D结构符合这些温度对两种酶催化位点稳定性的影响，优于肽底物。这里收集的数据表明L.(V.）巴西丝氨酸蛋白酶在整个生命周期中都会受到影响寄生虫适应性的温度条件的影响。
