title: Generation of a MDCK cell line with constitutive expression of the Enteropathogenic E. coli effector protein Map as an in vitro model of pathogenesis.
abstract: 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.
authors.affiliation: a Centre for Molecular Medicine; Jawaharlal Nehru University ; New Delhi, India.
authors.forename: Anand Prakash
authors.initials: AP
authors.lastname: Singh
authors.affiliation: a Centre for Molecular Medicine; Jawaharlal Nehru University ; New Delhi, India.
authors.forename: Saima
authors.initials: S
authors.lastname: Aijaz
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D003001:Cloning, Molecular; D004285:Dogs; D054308:Enteropathogenic Escherichia coli / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005818:Genetic Engineering; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008954:Models, Biological; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism; D014162:Transfection; D014774:Virulence / Q000235:genetics
chemical_list: D000907:Antibodies, Bacterial; D029968:Escherichia coli Proteins; C512346:Map protein, E coli; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: MDCK; enteropathogenic E coli; mitochondrial associated protein; tight junctions