Singh Anand Prakash, Aijaz Saima
a Centre for Molecular Medicine; Jawaharlal Nehru University ; New Delhi, India.
Bioengineered. 2015;6(6):335-41. doi: 10.1080/21655979.2015.1096456. Epub 2015 Oct 2.
Enteropathogenic E. coli (EPEC) cause diarrhea and are the major cause of mortality in developing countries. EPEC use a type III secretion system to deliver effector proteins into the host epithelial cells. To understand the functions of these effectors, majority of studies on EPEC pathogenesis have relied on infections of animals or cell lines with wild type strains of EPEC or mutant strains deficient in one or more effectors. While these studies have provided valuable data, it can be difficult to assess functions of an individual effector in the presence of other EPEC effectors. Recent studies have reported the use of transient transfections with plasmids encoding various EPEC effectors into different cell lines. However, variable transfection efficiencies and expression levels of the effector proteins coupled with their expression for relatively short periods of time pose a problem if the long term effects of these effectors need to be examined. We have generated a MDCK cell line with constitutive expression of the EPEC effector Map (Mitochondrial associated protein) for efficient stable expression of EGFP-tagged Map. We observed that the constitutive expression of Map increased the permeability of charged and non-charged molecules. We also generated polyclonal antibodies against Map and checked for their specificity in MDCK-Map expressing cells. Map has been reported to contribute to the onset of diarrhea but the underlying mechanism is yet to be identified. The MDCK-Map cell line and the anti-Map antibodies generated by us can be used for in vitro studies to examine the role of Map in EPEC pathogenesis.
肠致病性大肠杆菌（EPEC）可引起腹泻，是发展中国家死亡的主要原因。EPEC利用III型分泌系统将效应蛋白递送至宿主上皮细胞。为了解这些效应蛋白的功能，大多数关于EPEC发病机制的研究依赖于用EPEC野生型菌株或缺乏一种或多种效应蛋白的突变菌株感染动物或细胞系。虽然这些研究提供了有价值的数据，但在存在其他EPEC效应蛋白的情况下，很难评估单个效应蛋白的功能。最近的研究报道了将编码各种EPEC效应蛋白的质粒瞬时转染到不同细胞系中的方法。然而，如果需要研究这些效应蛋白的长期影响，可变的转染效率和效应蛋白的表达水平，以及它们相对较短时间的表达会带来问题。我们构建了一个组成型表达EPEC效应蛋白Map（线粒体相关蛋白）的MDCK细胞系，用于高效稳定表达EGFP标记的Map。我们观察到Map的组成型表达增加了带电和不带电分子的通透性。我们还制备了针对Map的多克隆抗体，并检测了它们在表达MDCK-Map的细胞中的特异性。据报道，Map会导致腹泻，但潜在机制尚未确定。我们构建的MDCK-Map细胞系和制备的抗Map抗体可用于体外研究，以检验Map在EPEC发病机制中的作用。
