title: Outer membrane proteins of wild-type and intimin-deficient enteropathogenic Escherichia coli induce Hep-2 cell death through intrinsic and extrinsic pathways of apoptosis.
abstract: Enteropathogenic Escherichia coli cause protracted diarrhoea and malnutrition in infants by cytoskeletal depolymerisation and effacement of enterocyte microvilli. In this study, outer membrane proteins of wild-type enteropathogenic E. coli and an intimin-deficient mutant are shown to induce apoptosis by up-regulation of tumour necrosis factor alpha and activation of c-jun N-terminal kinase. Fluorescence-activated cell sorter analysis revealed apoptosis of cells treated with outer membrane proteins of wild-type and intimin-deficient strains. Proteinase K treatment of outer membrane proteins reduced apoptosis significantly, as did neutralising tumour necrosis factor alpha with specific antibodies. Elevated tumour necrosis factor receptor 1-associated death domain and caspase-3 expression were also observed on treatment with both types of outer membrane proteins. Furthermore, apoptosis was associated with suppression of Bcl-2 protein expression, up-regulation of Bax mRNA levels and increased cytochrome c release from mitochondria. Elevated phospho-c-jun N-terminal kinase, c-jun mRNA and activator protein-1 expression were observed, and phosphorylation of activator protein-1 was also observed by DNA-binding assays. Inhibition of c-jun N-terminal kinase, but not inhibition of p38 mitogen-activated protein kinase, resulted in reduction of tumour necrosis factor alpha mRNA levels and caspase-3 protein levels, and a reduction in apoptosis as observed by fluorescence-activated cell sorter analysis. From the host point of view, this study suggests a possible interplay between the death receptor and mitochondrial pathways when cell-free bacterial outer membrane preparations are used to trigger apoptosis.
authors.affiliation: Centre for Biotechnology, Anna University, Chennai, India.
authors.forename: Balakrishna
authors.initials: B
authors.lastname: Shankar
authors.forename: Subramanian
authors.initials: S
authors.lastname: Krishnan
authors.forename: Vasantha
authors.initials: V
authors.lastname: Malladi
authors.forename: Arun
authors.initials: A
authors.lastname: Balakrishnan
authors.forename: Peter H
authors.initials: PH
authors.lastname: Williams
mesh_terms: D017209:Apoptosis; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D053148:Caspase 3 / Q000096:biosynthesis; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D054308:Enteropathogenic Escherichia coli / Q000276:immunology*; D004847:Epithelial Cells / Q000382:microbiology*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D015854:Up-Regulation; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis
chemical_list: D001425:Bacterial Outer Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D048031:JNK Mitogen-Activated Protein Kinases; D053148:Caspase 3