Secreted <i>Giardia intestinalis</i> cysteine proteases disrupt intestinal epithelial cell junctional complexes and degrade chemokines

Giardiasis is a common diarrheal disease caused by the protozoan parasite <i>Giardia intestinalis</i>. Cysteine proteases (CPs) are acknowledged as virulence factors in <i>Giardia</i> but their specific role in the molecular pathogenesis of disease is not known. Herein, we aimed to characterize the three main secreted CPs (CP14019, CP16160 and CP16779), which were identified by mass spectrometry in the medium during interaction with intestinal epithelial cells (IECs) <i>in vitro</i>. First, the CPs were epitope-tagged and localized to the endoplasmic reticulum and cytoplasmic vesicle-like structures. Second, we showed that recombinant CPs, expressed in <i>Pichia pastoris</i>, are more active in acidic environment (pH 5.5-6) and we determined the kinetic parameters using fluorogenic substrates. Third, excretory-secretory proteins (ESPs) from <i>Giardia</i> trophozoites affect the localization of apical junctional complex (AJC) proteins and recombinant CPs cleave or re-localize the AJC proteins (claudin-1 and -4, occludin, JAM-1, β-catenin and E-cadherin) of IECs. Finally, we showed that the ESPs and recombinant CPs can degrade several chemokines, including CXCL1, CXCL2, CXCL3, IL-8, CCL2, and CCL20, which are up-regulated in IECs during <i>Giardia</i>-host cell interactions. This is the first study that characterizes the role of specific CPs secreted from <i>Giardia</i> and our results collectively indicate their roles in the disruption of the intestinal epithelial barrier and modulating immune responses during <i>Giardia</i> infections.

贾第虫病（Giardiasis）是一种由原生动物寄生虫肠道贾第鞭毛虫（Giardia intestinalis）引发的常见腹泻性疾病。半胱氨酸蛋白酶（Cysteine proteases, CPs）已被证实为贾第虫（Giardia）的毒力因子，但其在疾病分子致病机制中的具体功能尚未明确。本研究旨在对体外（in vitro）与肠上皮细胞（intestinal epithelial cells, IECs）共培养的培养基中经质谱鉴定得到的三种主要分泌型半胱氨酸蛋白酶（CP14019、CP16160与CP16779）进行功能表征。

首先，我们对上述半胱氨酸蛋白酶进行表位标签标记，发现其定位于内质网及细胞质囊泡样结构中。其次，我们在毕赤酵母（Pichia pastoris）中表达了重组半胱氨酸蛋白酶，证实其在酸性环境（pH 5.5~6）中活性更高，并利用荧光底物测定了其动力学参数。第三，贾第虫滋养体的排泄分泌蛋白（excretory-secretory proteins, ESPs）可影响肠上皮细胞顶端连接复合体（apical junctional complex, AJC）蛋白的定位；重组半胱氨酸蛋白酶可切割或重新定位肠上皮细胞的顶端连接复合体蛋白，包括紧密连接蛋白-1（claudin-1）、紧密连接蛋白-4（claudin-4）、闭合蛋白（occludin）、JAM-1、β-连环蛋白（β-catenin）及E-钙粘蛋白（E-cadherin）。最后，我们证实排泄分泌蛋白与重组半胱氨酸蛋白酶均可降解多种趋化因子，包括在贾第虫-宿主细胞互作过程中于肠上皮细胞内上调表达的CXCL1、CXCL2、CXCL3、IL-8、CCL2及CCL20。

本研究首次针对贾第虫分泌的特异性半胱氨酸蛋白酶的功能展开表征，研究结果整体表明，此类蛋白酶在贾第虫感染期间可通过破坏肠上皮屏障及调控免疫应答参与致病过程。

搜集汇总

背景与挑战

背景概述

该数据集聚焦于贾第虫分泌的半胱氨酸蛋白酶（CP14019、CP16160、CP16779）在贾第虫病发病机制中的作用，通过体外实验表征了这些蛋白酶的活性和功能。研究发现，这些蛋白酶在酸性环境中更活跃，能破坏肠道上皮细胞的连接复合物（如claudin-1和occludin），并降解多种趋化因子（如CXCL1和IL-8），从而影响肠道屏障完整性和免疫调节。这是首次针对贾第虫特定分泌蛋白酶的系统研究，揭示了其在疾病进展中的关键角色。