Ramírez-Flores Carlos J, Cruz-Mirón Rosalba, Arroyo Rossana, Mondragón-Castelán Mónica E, Nopal-Guerrero Tais, González-Pozos Sirenia, Ríos-Castro Emmanuel, Mondragón-Flores Ricardo
Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN No. 2508, Col. Zacatenco, C.P. 07360, Ciudad de México, Mexico.
Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Ciudad de México, Mexico.
Parasitol Res. 2019 Jan;118(1):289-306. doi: 10.1007/s00436-018-6163-5. Epub 2018 Dec 1.
Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, Toxoplasma spreads throughout the body and migrates across biological barriers, such as the intestinal and blood-brain barriers, as well as the placenta in pregnant women. The mechanisms for parasite dissemination are still unknown; however, proteases could play a role as a virulence factor. The aim of this study was to detect and to characterize proteases in whole-cell extracts and in excretion/secretion products from tachyzoites of the RH strain isolated from infected mice. Both fractions were analyzed by gelatin and casein zymography and by azocasein degradation. The biochemical characterization of proteases included standardization of optimal conditions for their activation, such as pH, the presence of cofactors, and a reducing agent. In both fractions, we detected at least nine gelatin-degrading metalloproteases in the range of 50 to 290 kDa. The proteases present in the excretion/secretion products were found as soluble proteins and not associated with exosome-like vesicles or other secretory vesicles. Moreover, by using casein zymography, it was possible to detect three serine proteases. Exposure of MDCK cells to excretion/secretion products modified the organization of the cell monolayer, and this effect was reverted after washing thoroughly with PBS and inhibition by metalloprotease and serine protease inhibitors. Proteomic analysis of excretion/secretion products identified 19 proteases. These findings suggest that tachyzoites of a highly virulent strain of Toxoplasma use a battery of proteases to modify the epithelium, probably as a strategy to facilitate their tissue dissemination.
刚地弓形虫可感染温血动物的所有有核细胞。感染后，弓形虫会扩散至全身，并穿越生物屏障，如肠道和血脑屏障，以及孕妇的胎盘。寄生虫传播的机制尚不清楚；然而，蛋白酶可能作为一种毒力因子发挥作用。本研究的目的是检测并鉴定从感染小鼠分离出的RH株速殖子全细胞提取物及排泄/分泌产物中的蛋白酶。通过明胶和酪蛋白酶谱法以及偶氮酪蛋白降解法对这两种组分进行分析。蛋白酶的生化特性包括对其激活的最佳条件进行标准化，如pH值、辅因子的存在以及还原剂的使用。在这两种组分中，我们检测到至少9种分子量在50至290 kDa范围内的明胶降解金属蛋白酶。排泄/分泌产物中存在的蛋白酶为可溶性蛋白，与外泌体样囊泡或其他分泌囊泡无关。此外，通过酪蛋白酶谱法，有可能检测到3种丝氨酸蛋白酶。将MDCK细胞暴露于排泄/分泌产物会改变细胞单层的组织结构，在用PBS彻底洗涤并用金属蛋白酶和丝氨酸蛋白酶抑制剂抑制后，这种效应会逆转。对排泄/分泌产物的蛋白质组分析鉴定出19种蛋白酶。这些发现表明，高毒力弓形虫株的速殖子利用一系列蛋白酶来改变上皮细胞，这可能是促进其在组织中传播的一种策略。
