title: Expression and regulation of the Escherichia coli O157:H7 effector proteins NleH1 and NleH2.
abstract: BACKGROUND
  E. coli O157 carries two genes encoding the effector proteins NleH1 and NleH2 which are 87% identical. Despite the similarity between the proteins, the promoter regions upstream of the genes encoding the effectors are more divergent suggesting that the actual expression of the genes may be differentially regulated. This was tested by creating reporter fusions and examining their expression in different genetic backgrounds, media and on contact with host cells. The function of the proteins was also tested following transfection into host cells.
  
  
  RESULTS
  Expression of both NleH1 and NleH2 was enhanced when cultured under conditions that stimulated expression of the Type Three Secretion System (T3SS) and was influenced by the regulators Ler and GrlA. Maximal expression of NleH1 required 531 bp of the upstream untranslated region but NleH2 required only 113 bp. Interestingly, contact with host cells strongly repressed expression of both NleH1 and NleH2. Following transfection, both proteins produced only minor effects on NF-κB activation when assessed using a NF-κB luciferase reporter assay, a result that is consistent with the recent report demonstrating the dependence on RPS3 for NleH1 modulation of NF-κB.
  
  
  CONCLUSIONS
  This study demonstrates the importance of considering gene regulation when studying bacterial effector proteins. Despite their sequence similarity, NleH1 and NleH2 are expressed differentially and may, therefore, be translocated at distinct times during an infection.
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Ashleigh
authors.initials: A
authors.lastname: Holmes
authors.forename: Cecilia S
authors.initials: CS
authors.lastname: Lindestam Arlehamn
authors.forename: Dai
authors.initials: D
authors.lastname: Wang
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Mitchell
authors.forename: Tom J
authors.initials: TJ
authors.lastname: Evans
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Roe
mesh_terms: D058947:Bacterial Secretion Systems / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D019453:Escherichia coli O157 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D058947:Bacterial Secretion Systems; D017931:DNA Primers; D029968:Escherichia coli Proteins; C513430:GrlA protein, E coli; C465556:Ler protein, E coli; C556114:NleH protein, E coli; D015534:Trans-Activators; D008156:Luciferases