Giardipain-1, a protease secreted by Giardia duodenalis trophozoites, causes junctional, barrier and apoptotic damage in epithelial cell monolayers
Giardipain-1, a protease secreted by Giardia duodenalis trophozoites, causes junctional, barrier and apoptotic damage in epithelial cell monolayers
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DOI：
10.1016/j.ijpara.2018.01.006
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发表时间：
2018-07-01
影响因子：
4
通讯作者：
Gonzalez-Mariscal, Lorenza
中科院分区：
文献类型：
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作者：
Ortega-Pierres, Guadalupe;Arguello-Garcia, Raul;Gonzalez-Mariscal, Lorenza
贾第鞭毛虫滋养体与肠上皮细胞的粘附使贾第鞭毛虫病得以发生和维持。在这些相互作用过程中，上皮细胞可以通过寄生虫分泌的酶（包括半胱氨酸蛋白酶，其越来越多地被鉴定为寄生原生动物中的毒力因子）进行凋亡。本工作制备了抗G.发现在G.尿道滋养体当表达这种蛋白质的滋养体与IEC-6肠上皮细胞单层培养时，用mAbIG 3观察到这种蛋白质的动态释放。蛋白质组学分析鉴定该蛋白为成熟的组织蛋白酶B样（gCatB）酶，其蛋白水解活性，在酶谱中检测到，被CatB抑制剂E-64消除。该蛋白质由于其功能性木瓜蛋白酶样特征而被命名为贾地蛋白酶-1，并通过使用mAbIG 3的亲和色谱法纯化。暴露于纯化的、成熟的和分泌形式的贾地蛋白酶-1后，IEC-6上皮细胞单层显示膜起泡和细胞外表面的磷脂酰丝氨酸暴露，表明细胞凋亡过程。在Madin达比犬肾（MDCK）细胞单层中，giardipain-1导致出现孔隙样区域和沿着细胞-细胞连接的间隙，导致跨上皮电阻（TER）降低、caspase-3激活和聚ADP核糖聚合酶（PARP）断裂。在暴露过程中的早期，贾地蛋白酶-1共定位于细胞-细胞连接处，与闭合蛋白相关，并诱导紧密连接蛋白闭合蛋白和密蛋白-1的离域和降解。通过与CatB B抑制剂E-64预孵育来阻断贾地蛋白酶-1对上皮单层造成的损伤。此外，沉默滋养体中的giardipain-1基因降低了giardipain-1的蛋白水解活性，并减少了IEC-6单层的损伤。观察到的损伤似乎是特异性的贾地蛋白酶活性，因为几乎没有观察到损伤时，IEC-6单层与木瓜蛋白酶，一种非相关的半胱氨酸蛋白酶孵育。因此，这项研究表明，贾第虫蛋白酶-1触发，在上皮细胞中，细胞-细胞连接组件的降解和凋亡损伤，支持贾第虫蛋白酶-1作为贾第虫的毒力因子的概念。(C)2018年澳大利亚寄生虫学会。由爱思唯尔有限公司出版。保留所有权利。
The adhesion of Giardia duodenalis trophozoites to intestinal epithelial cells allows the onset and maintenance of giardiasis. During these interactions, epithelial cells can be committed to apoptosis by enzymes secreted by the parasites, including cysteine proteases that are increasingly identified as virulence factors in parasitic protozoa. In this work, a monoclonal antibody (mAb1G3) raised against G. duodenalis surface components was found to react with a 25 kDa protein expressed in the cell surface and flagella of G. duodenalis trophozoites. When trophozoites expressing this protein were cultured with IEC-6 intestinal epithelial cell monolayers, a dynamic release of this protein was observed with mAbIG3. Proteomic analysis identified the protein as a mature cathepsin B-like (gCatB) enzyme, whose proteolytic activity, detected in zymograms, was eliminated by CatB inhibitor E-64. This protein was named giardipain-1 due to its functional papain-like features and was purified by affinity chromatography using mAbIG3. Upon exposure to the purified, mature and secreted forms of giardipain-1, IEC-6 epithelial cell monolayers displayed membrane blebbing and phosphatidylserine exposure on the outer cell surface, indicating an apoptotic process. In Madin Darby Canine Kidney (MDCK) cell monolayers, giardipain-1 leads to the appearance of pore-like regions and of gaps along cell-cell junctions, to decreased transepithelial electrical resistance (TER), caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation. At early times during exposure, giardipain-1 co-localized at cell-cell junctions, associated with occludin and induced the delocalization and degradation of tight junction proteins occludin and claudin-1. The damage caused to epithelial monolayers by giardipain-1 was blocked by pre-incubation with the CatB B Inhibitor E-64. Furthermore, silencing the giardipain-1 gene in trophozoites lowered the proteolytic activity of giardipain-1 and reduced the damage in IEC-6 monolayers. The damage observed appears to be specific to giardipain activity since almost no damage was observed when IEC-6 monolayers were incubated with papain, a non-related cysteine protease. Hence this study suggests that giardipain-1 triggers, in epithelial cells, degradation of cell-cell junctional components and apoptotic damage, supporting the notion of giardiapain-1 as a virulence factor of Giardia. (C) 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.