Alvarez Carina-Shianya, Badia Josefa, Bosch Manel, Giménez Rosa, Baldomà Laura
Secció de Bioquímica i Biologia Molecular, Departament de Bioquímica i Fisiologia, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de BarcelonaBarcelona, Spain; Institut de Biomedicina de la Universitat de Barcelona, Institut de Recerca Sant Joan De DéuBarcelona, Spain.
Unitat de Microscòpia Òptica Avançada, Centres Científics i Tecnològics, Universitat de Barcelona Barcelona, Spain.
Front Microbiol. 2016 Dec 15;7:1981. doi: 10.3389/fmicb.2016.01981. eCollection 2016.
The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other strains of human intestinal origin that contain the gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional regulation of claudin-2. Thus, in addition to OMVs and TcpC, other active factors released by these microbiota strains contribute to the reinforcement of the epithelial barrier.
胃肠道上皮层形成了一道物理和生化屏障，维持着宿主与肠道微生物群之间的分隔。这一屏障的完整性对于维持体内的稳态至关重要，其功能障碍与多种疾病相关，尤其是炎症性肠病。肠道微生物，特别是益生菌，通过降低肠道通透性和加强紧密连接来调节屏障的完整性。益生菌Nissle 1917（EcN）是人类肠道的良好定植菌，在人类溃疡性结肠炎缓解方面具有已证实的治疗效果。EcN通过紧密连接蛋白ZO-1、ZO-2和claudin-14的上调和重新分布来正向调节肠道上皮屏障。claudin-14的上调归因于分泌蛋白TcpC。ZO-1和ZO-2的调节是否由EcN分泌因子介导仍不清楚。本研究的目的是探讨EcN释放的外膜囊泡（OMV）是否能加强上皮屏障。本研究包括其他含该基因的人类肠道来源菌株，如ECOR63。从野生型菌株和衍生突变体收集的无细胞上清液被分离成分离的OMV和可溶性分泌因子。通过测量T-84和Caco-2极化单层中的跨上皮电阻和几种紧密连接蛋白的表达，评估这些细胞外组分对上皮屏障的影响。我们的结果表明，EcN和ECOR63的增强活性并不完全依赖于TcpC。这些菌株分泌的OMV和可溶性因子均促进ZO-1和claudin-14的上调以及claudin-2的下调。OMV介导的效应不依赖于TcpC。可溶性分泌的TcpC有助于ZO-1和claudin-14的上调，但该蛋白对claudin-2的转录调节没有影响。因此，除了OMV和TcpC外，这些微生物菌株释放的其他活性因子也有助于加强上皮屏障。
