Outer membrane proteins of wild-type and intimin-deficient enteropathogenic Escherichia coli induce Hep-2 cell death through intrinsic and extrinsic pathways of apoptosis.
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.
Shankar
Balakrishna
B
Centre for Biotechnology, Anna University, Chennai, India.
Krishnan
Subramanian
S
Malladi
Vasantha
V
Balakrishnan
Arun
A
Williams
Peter H
PH
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
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