Cabrera-Licona Ariana, Solano-González Eduardo, Fonseca-Liñán Rocío, Bazán-Tejeda Ma Luisa, Bermúdez-Cruz Rosa Ma, Ortega-Pierres Guadalupe
Departamento de Genética y Biología Molecular. Centro de Investigación y Estudios Avanzados, IPN, México City, CA, 07360, Mexico.
Exp Parasitol. 2017 Aug;179:49-64. doi: 10.1016/j.exppara.2017.06.006. Epub 2017 Jun 28.
Giardia duodenalis is the protozoan parasite responsible for most cases of parasitic diarrhea worldwide. The pathogenic mechanisms of giardiasis have not yet been fully characterized. In this context parasite's excretory/secretory products have been related to the damage induced by the parasite on enterocytes. Among these is the Variable Surface Proteins (VSPs) family involved in antigenic variation and in the induction of protective response. In proteomic analyses carried out to identify the proteases with high molecular weight secreted by Giardia trophozoites during the initial phase of interaction with IEC-6 cell monolayers we identified the VSP9B10A protein. In silico bioinformatics analyses predicted a central region in residues 324-684 displaying the catalytic triad and the substrate binding pocket of cysteine proteases. The analysis of the effect of the VSP9B10A protein on epithelial cell monolayers using trophozoites that were transfected with a plasmid carrying the vsp9b10a gene sequence under the control of a constitutive promoter showed that transfected trophozoites expressing the VSP9B10A protein caused cytotoxic damages on IEC-6 and MDCK cell monolayers. This was characterized by loss of cell-cell contacts and cell detachment from the substrate while no damage was observed with trophozoites that did not express the VSP9B10A protein. The same cytotoxic effect was detected when IEC-6 cell monolayers were incubated only with supernatants from co-cultures of IEC-6 cell monolayers with VSP9B10A transfected trophozoites and this effect was not observed when transfected trophozoites were incubated with a monospecific polyclonal antibody anti-VSP9B10A previous to interaction with IEC-6 monolayers. These results demonstrate that the VSP9B10A protein secreted upon interaction with epithelial cells caused damage in these cells. Thus this protein might be considered as a conditional virulence factor candidate. To our knowledge this is the first report on the proteolytic activity from a Giardia VSP opening new research lines on these proteins.
十二指肠贾第虫是导致全球大多数寄生虫性腹泻病例的原生动物寄生虫。贾第虫病的致病机制尚未完全明确。在这种情况下，寄生虫的排泄/分泌产物与寄生虫对肠上皮细胞造成的损伤有关。其中包括参与抗原变异和诱导保护性反应的可变表面蛋白（VSPs）家族。在蛋白质组学分析中，为了鉴定贾第虫滋养体在与IEC-6细胞单层相互作用的初始阶段分泌的高分子量蛋白酶，我们鉴定出了VSP9B10A蛋白。计算机生物信息学分析预测，在残基324 - 684中有一个中央区域，显示出半胱氨酸蛋白酶的催化三联体和底物结合口袋。使用在组成型启动子控制下携带vsp9b10a基因序列的质粒转染的滋养体，分析VSP9B10A蛋白对上皮细胞单层的影响，结果表明，表达VSP9B10A蛋白的转染滋养体对IEC-6和MDCK细胞单层造成了细胞毒性损伤。其特征是细胞间接触丧失和细胞从基质上脱落，而未表达VSP9B10A蛋白的滋养体未观察到损伤。当IEC-6细胞单层仅与VSP9B10A转染滋养体的共培养上清液孵育时，也检测到了相同的细胞毒性作用，而在转染滋养体与IEC-6单层相互作用之前，用抗VSP9B10A单特异性多克隆抗体孵育时，则未观察到这种作用。这些结果表明，与上皮细胞相互作用时分泌的VSP9B10A蛋白对这些细胞造成了损伤。因此，这种蛋白可能被视为一种条件性毒力因子候选物。据我们所知，这是关于贾第虫VSP蛋白水解活性的首次报道，为这些蛋白开辟了新的研究方向。
