Conditioned medium from enterohemorrhagic Escherichia coli-infected T84 cells inhibits signal transducer and activator of transcription 1 activation by gamma interferon.
Gamma interferon (IFN-gamma) is a cytokine important to host defense which can signal through signal transducer and activator of transcription 1 (Stat1). Enterohemorrhagic Escherichia coli (EHEC) modulates host cell signal transduction to establish infection, and EHEC serotypes O113:H21 and O157:H7 both inhibit IFN-gamma-induced Stat1 tyrosine phosphorylation in vitro. The aim of this study was to delineate both bacterial and host cell factors involved in the inhibition of Stat1 tyrosine phosphorylation. Human T84 colonic epithelial cells were challenged with direct infection, viable EHEC separated from T84 cells by a filter, sodium orthovanadate, isolated flagellin, bacterial culture supernatants, and conditioned medium treated with proteinase K, trypsin, or heat inactivation. Epithelial cells were then stimulated with IFN-gamma and protein extracts were analyzed by immunoblotting. The data showed that IFN-gamma-inducible Stat1 tyrosine phosphorylation was inhibited when EHEC adhered to T84 cells, but not by bacterial culture supernatants or bacteria separated from the epithelial monolayer. Conditioned medium from T84 cells infected with EHEC O157:H7 suppressed Stat1 activation, and this was not reversed by treatment with proteinases or heat inactivation. Use of pharmacological inhibitors showed that time-dependent bacterial, but not epithelial, protein synthesis was involved. Stat1 inhibition was also independent of bacterial flagellin, host proteasome activity, and protein tyrosine phosphatases. Infection led to altered IFN-gamma receptor domain 1 subcellular distribution and decreased expression in cholesterol-enriched membrane microdomains. Thus, suppression of host cell IFN-gamma signaling by production of a contact-dependent, soluble EHEC factor may represent a novel mechanism for this pathogen to evade the host immune system.
Research Institute, Gastroenterology and Nutrition Room 8409, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8.
Narveen
N
Jandu
Peter J M
PJ
Ceponis
Seiichi
S
Kato
Jason D
JD
Riff
Derek M
DM
McKay
Philip M
PM
Sherman
D017077:Culture Media, Conditioned / Q000494:pharmacology*; D004347:Drug Interactions; D004847:Epithelial Cells / Q000378:metabolism; D004927:Escherichia coli Infections / Q000378:metabolism* / Q000503:physiopathology; D019453:Escherichia coli O157 / Q000502:physiology*; D007371:Interferon-gamma / Q000494:pharmacology*; D050794:STAT1 Transcription Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015534:Trans-Activators / Q000378:metabolism
D017077:Culture Media, Conditioned; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D007371:Interferon-gamma