Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
Sci Rep. 2020 Feb 20;10(1):3120. doi: 10.1038/s41598-020-60077-4.
Bacterial EVs have been related to inter-kingdom communication between probiotic/pathogenic bacteria and their hosts. Our aim was to investigate the transcytosis process of B. subtilis EVs using an in vitro intestinal epithelial cell model. In this study, using Confocal Laser Scanning Microscopy, we report that uptake and internalization of CFSE-labeled B. subtilis EVs (115 nm ± 27 nm) by Caco-2 cells are time-dependent. To study the transcytosis process we used a transwell system and EVs were quantified in the lower chamber by Fluorescence and Nanoparticle Tracking Analysis measurements. Intact EVs are transported across a polarized cell monolayer at 60-120 min and increased after 240 min with an estimated average uptake efficiency of 30% and this process is dose-dependent. EVs movement into intestinal epithelial cells was mainly through Z axis and scarcely on X and Y axis. This work demonstrates that EVs could be transported across the gastrointestinal epithelium. We speculate this mechanism could be the first step allowing EVs to reach the bloodstream for further delivery up to extraintestinal tissues and organs. The expression and further encapsulation of bioactive molecules into natural nanoparticles produced by probiotic bacteria could have practical implications in food, nutraceuticals and clinical therapies.
细菌细胞外囊泡已被证明在益生菌/病原菌与其宿主之间存在跨物种通讯。本研究旨在利用体外肠上皮细胞模型研究枯草芽孢杆菌细胞外囊泡的转胞吞作用。在这项研究中，我们使用共聚焦激光扫描显微镜报告了 CFSE 标记的枯草芽孢杆菌细胞外囊泡（115nm±27nm）被 Caco-2 细胞摄取和内化是时间依赖性的。为了研究转胞吞过程，我们使用了 Transwell 系统，并通过荧光和纳米颗粒跟踪分析测量来定量下室中的细胞外囊泡。完整的细胞外囊泡在 60-120 分钟内跨极化细胞单层运输，并在 240 分钟后增加，估计平均摄取效率为 30%，且该过程呈剂量依赖性。细胞外囊泡进入肠上皮细胞的主要途径是 Z 轴，而 X 和 Y 轴则很少。这项工作表明细胞外囊泡可以穿过胃肠道上皮细胞。我们推测，这种机制可能是允许细胞外囊泡进入血液，进一步递送至肠外组织和器官的第一步。益生菌产生的天然纳米颗粒表达和进一步封装生物活性分子在食品、营养保健品和临床治疗方面具有实际意义。
