Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), México DF, México.
Front Immunol. 2020 Nov 17;11:564953. doi: 10.3389/fimmu.2020.564953. eCollection 2020.
A hallmark of enteroaggregative (EAEC) infection is the formation of an intestinal biofilm, which comprises a mucus layer with immersed bacteria. Pic is an autotransporter secreted by EAEC, and other pathotypes, and has been involved in two apparently contradictory phenotypes, as a mucus secretagogue and as a mucinase. Here, we investigated this Pic dual activity, mucus secretagogue capability and mucinolytic activity, in human goblet cells that secrete MUC2 and MUC5AC. Pic induced mucus hypersecretion directly in the goblet cells, without other intestinal cell types involved. At the same time, Pic exhibited strong proteolytic activity on the secreted mucins. These activities were independent since a mutation in the serine protease motif (PicS258I) abolished mucin degradation while maintaining the mucus secretagogue activity intact. Furthermore, deoxycholic acid (DCA)-induced mucins were proteolytically degraded when goblet cells were co-incubated with DCA/Pic, while co-incubation with DCA/PicS258I induced a synergistic effect on mucus hypersecretion. Pic was more efficient degrading MUC5AC than MUC2, but no degradation was detected with Pic inactivated at the active site by mutation or pharmacological inhibition. Remarkably, Pic cleaved MUC2 and MUC5AC in the C-terminal domain, leaving N-terminal subproducts, impacting the feature of gel-forming mucins and allowing mucus layer penetration by EAEC. Astonishingly, Pic stimulated rapid mucin secretion in goblet-like cells by activating the intracellular calcium pathway resulting from the PLC signal transduction pathway, leading to the production of DAG and releasing IP, a second messenger of calcium signaling. Therefore, the dual activity of Pic, as a mucus secretagogue and a mucinase, is relevant in the context of carbon source generation and mucus layer penetration, allowing EAEC to live within the layer of mucus but also access epithelial cells.
肠聚集性大肠杆菌（EAEC）感染的一个标志是形成肠道生物膜，它由包含浸入细菌的粘液层组成。Pic 是由 EAEC 和其他病原体分泌的一种自转运体，它与两种明显矛盾的表型有关，即粘液分泌激动剂和粘蛋白酶。在这里，我们研究了 Pic 的这种双重活性，即粘液分泌激动剂能力和粘蛋白水解活性，在分泌 MUC2 和 MUC5AC 的人杯状细胞中。Pic 直接在杯状细胞中诱导粘液过度分泌，而不涉及其他肠道细胞类型。与此同时，Pic 对分泌的粘蛋白表现出很强的蛋白水解活性。这些活性是独立的，因为丝氨酸蛋白酶基序（PicS258I）中的突变消除了粘蛋白降解，同时保持了粘液分泌激动剂活性的完整。此外，当杯状细胞与 DCA/Pic 共孵育时，DCA 诱导的粘蛋白被蛋白水解降解，而 DCA/PicS258I 的共孵育对粘液过度分泌产生协同作用。Pic 对 MUC5AC 的降解效率高于 MUC2，但在用突变或药理学抑制失活活性位点的 Pic 处理时，未检测到 MUC2 的降解。值得注意的是，Pic 在 C 端结构域切割 MUC2 和 MUC5AC，留下 N 端亚产物，影响凝胶形成粘蛋白的特征，并允许 EAEC 通过粘液层穿透。令人惊讶的是，Pic 通过激活 PLC 信号转导途径导致 DAG 的产生和 IP 的释放，从而刺激细胞内钙途径，导致快速分泌粘蛋白，IP 是钙信号的第二信使，在杯状细胞中刺激快速粘蛋白分泌。因此，Pic 的双重活性，作为粘液分泌激动剂和粘蛋白酶，与碳源生成和粘液层穿透有关，使 EAEC 能够在粘液层内生存，但也能够进入上皮细胞。
