Infection by bacterial pathogens expressing type III secretion decreases luciferase activity: ramifications for reporter gene studies.
Pathogenic microbes influence gene regulation in eukaryotic hosts. Reporter gene studies can define the roles of promoter regulatory sequences. The effect of pathogenic bacteria on reporter genes has not been examined. The aim of this study was to identify which reporter genes are reliable in studies concerning host gene regulation by bacterial pathogens expressing type III secretory systems. Human intestinal epithelial cells, T84, Caco-2 and HT-29, were transfected with plasmids containing luciferase (luc), chloramphenicol acetyltransferase (CAT) or beta-galactosidase (beta-gal) as reporter genes driven by the inducible interleukin-8 (IL-8) or constitutively active simian virus 40 (SV40) promoter. Cells were infected with enteropathogenic E. coli or Salmonella typhimurium, and the reporter activity was assessed. Luc activity significantly decreased following infection, regardless of the promoter. The activity of recombinant luc was nearly ablated by incubation with either EPEC or Salmonella in a cell-free system. Activity was partially preserved by protease inhibitors, and immunoblot analysis showed a decreased amount and molecular weight of recombinant luc, suggesting protein degradation. Neither beta-gal nor CAT activity was altered by infection. Disruption of type III secretion prevented the loss of luc activity. We conclude that CAT or beta-gal, but not luc, can be used as reliable reporter genes to assess the impact of pathogenic microbes, especially those expressing type III secretion on host cell gene regulation.
Savkovic
S D
SD
University of Illinois, West Side VA Medical Center, Chicago, USA.
Koutsouris
A
A
Wu
G
G
Hecht
G
G
D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology; D004926:Escherichia coli / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000382:microbiology; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000502:physiology*; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase