Expression of the virulence factor, BfpA, by enteropathogenic Escherichia coli is essential for apoptosis signalling but not for NF-kappaB activation in host cells.
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)-kappaB 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-kappaB-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-kappaB. Because suppression of NF-kappaB activation by BAY11-7085, a NF-kappaB 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-kappaB pathway in HeLa cells.
Melo
A R
AR
Laboratório de Biologia do Reconhecer, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil.
Lasunskaia
E B
EB
de Almeida
C M C
CM
Schriefer
A
A
Kipnis
T L
TL
Dias da Silva
W
W
D017209:Apoptosis; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism
C104901:BfpA protein, E coli; D029968:Escherichia coli Proteins; D016328:NF-kappa B; D037521:Virulence Factors; D036781:Fimbriae Proteins