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Expression of the Virulence Factor, BfpA, by Enteropathogenic Escherichia coli is Essential for Apoptosis Signalling but not for NF‐κB Activation in Host Cells
Author(s) -
Melo A. R.,
Lasunskaia E. B.,
De Almeida C. M. C.,
Schriefer A.,
Kipnis T. L.,
Dias da Silva W.
Publication year - 2005
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.2005.01626.x
Subject(s) - enteropathogenic escherichia coli , virulence factor , apoptosis , biology , microbiology and biotechnology , escherichia coli , electrophoretic mobility shift assay , chemistry , virulence , transcription factor , gene , biochemistry
Localized adherence (LA) of enteropathogenic Escherichia coli (EPEC) to epithelial cells results in attaching and effacing of the surface of these cells. LA depends on the gene bfpA , which codes for the BfpA protein. We found that EPEC‐E. coli adherence factor (EAF) (+) , expressing BfpA, significantly reduced HeLa cell viability in comparison with EPEC‐EAF (–) , as evaluated by the mitochondrial‐dependent succinate dehydrogenase conversion of 3′‐[4,5,‐dimethylthiazol‐2yl]2,5‐diphenyltetrazolium bromide (MTT) to its formazan. Apoptosis accounts for a substantial loss of the cell viability, because the cells incubated with EPEC‐EAF (+) or with cloned BfpA (data not shown), but not with EPEC‐EAF (–) , were positive for annexin‐V binding, demonstrated chromatin condensation and nuclei fragmentation and exhibited a high level of caspase‐3 activity. Because the blockade of bacterial cell‐surface‐associated BfpA by anti‐BfpA immunoglobulin (Ig)Y antibody suppressed apoptotic death induced by EPEC‐EAF (+) , BfpA may be the trigger for apoptosis. Both EPEC‐EAF (+) and EPEC‐EAF (–) , as well as recombinant BfpA (data not shown), activated nuclear factor (NF)‐κB in a similar manner as analysed by the electrophoretic mobility shift assay (EMSA). EMSA supershift analysis demonstrated the presence of p65/RelA in a DNA‐binding complex. In contrast to DNA binding, NF‐κB‐dependent reporter gene transactivation was stimulated more strongly by EPEC B171/EAF (+) , suggesting a role for this virulence factor in the regulation of transcriptional activity of NF‐κB. Because suppression of NF‐κB activation by BAY11‐7085, a NF‐κB inhibitor, neither induced apoptosis by itself nor blocked apoptosis induction by EPEC‐EAF (+) , it may be suggested that apoptosis is not regulated by the NF‐κB pathway in HeLa cells.