Malladi Vasantha, Shankar Balakrishna, Williams Peter H, Balakrishnan Arun
Centre for Biotechnology, Anna University, 600 025, Chennai, India.
Microbes Infect. 2004 Jan;6(1):38-50. doi: 10.1016/j.micinf.2003.09.022.
Enteropathogenic Escherichia coli (EPEC) is a Gram-negative bacterial pathogen that adheres to human intestinal epithelial cells, resulting in watery, persistent diarrhoea. Despite the advances made in understanding EPEC-host cell interactions, the molecular mechanisms underlying watery diarrhoea have not been understood fully. Loss of transepithelial resistance and increased monolayer permeability by disruption of tight junctions has been implicated in this process. Apart from disruption of tight junctions, an important factor known to regulate monolayer permeability is E-cadherin and its interaction with beta-catenin, both of which constitute the adherens junctions. Our previous studies using HEp-2 cells demonstrated the morphological and cytoskeletal changes caused by cell-free outer membrane preparations (OMPs) of EPEC. In this study, we have shown that EPEC and its OMP induce significant changes in the adherens junctions of Caco-2 monolayers. We also observed significant phosphorylation of protein kinase Calpha (PKCalpha) in cells treated with either whole EPEC or its OMP. Immunoprecipitation of cell lysates with anti-E-cadherin and probing with phospho-PKCalpha monoclonal antibodies and anti-beta-catenins revealed that in these cells, phosphorylated PKCalpha is associated with cadherins, leading to the dissociation of the cadherin/beta-catenin complex. Immunofluorescence showed beta-catenins dissociated from the membrane-bound cadherins and redistributed into the cytoplasm. Expression of dominant negative PKCalpha reversed these effects caused by either whole EPEC or its OMP and also reduced the associated increase in monolayer permeability. It is possible that this mechanism may complement the earlier known pathways for loss of barrier function involving myosin light chain kinase activation and also may play a role in causing host cell death by apoptosis.
肠致病性大肠杆菌（EPEC）是一种革兰氏阴性细菌病原体，它粘附于人类肠道上皮细胞，导致水样持续性腹泻。尽管在理解EPEC与宿主细胞相互作用方面取得了进展，但水样腹泻的分子机制尚未完全明确。跨上皮电阻的丧失以及紧密连接破坏导致的单层通透性增加与这一过程有关。除了紧密连接的破坏外，已知调节单层通透性的一个重要因素是E-钙粘蛋白及其与β-连环蛋白的相互作用，二者共同构成粘着连接。我们之前使用HEp-2细胞的研究证明了EPEC的无细胞外膜制剂（OMPs）引起的形态学和细胞骨架变化。在本研究中，我们表明EPEC及其OMP会诱导Caco-2单层细胞粘着连接发生显著变化。我们还观察到，用完整的EPEC或其OMP处理的细胞中蛋白激酶Cα（PKCα）发生了显著磷酸化。用抗E-钙粘蛋白对细胞裂解物进行免疫沉淀，并用磷酸化PKCα单克隆抗体和抗β-连环蛋白进行检测，结果显示，在这些细胞中，磷酸化的PKCα与钙粘蛋白相关，导致钙粘蛋白/β-连环蛋白复合物解离。免疫荧光显示β-连环蛋白从膜结合的钙粘蛋白上解离并重新分布到细胞质中。显性负性PKCα的表达逆转了由完整的EPEC或其OMP引起的这些效应，也降低了相关的单层通透性增加。这种机制可能补充了早期已知的涉及肌球蛋白轻链激酶激活的屏障功能丧失途径，也可能在通过凋亡导致宿主细胞死亡中发挥作用。
