Nsal E. coli against ROSMaterials and Methods Bacterial Strains, Cells Lines, and Culture ConditionsThe non-pathogenic murine E. coli strain NC101 was isolated as described previously[24]. E. coli strain O157:H7 was a kind gift from Dr. Ann Matthysse at UNC, Chapel Hill. E. coli were grown in Luria-Burtani (LB) broth at 37 with shaking at 250 rpm. The J774 murine macrophage and L929 fibroblast cell lines were originally obtained from ATCC (Manassas, VA) and cultured in RPMI containing 10 fetal bovine serum (FBS), 100U/mL penicillin, 1000 g/mL streptomycin, and 10mM glutamine in 37 humidified incubators with 5 CO2. Conditioned media from L929 cells was used as a source of macrophage colony stimulating factor (M-CSF) for the production of bone marrow-derived macrophages (BMDMs) and was made as described previously[25]. The mutant E. coli NC101 strain STI-571 biological activity lacking ibpA and ibpB (NC101ibpAB) that was used in this study had been generated previously using the -red PX-478 site recombinase method[23,26]. We used identical methods to create a mutant E. coli O157:H7 strain that lacks ibpA and ibpB (O157: H7ibpAB). However, since the pCP20 plasmid encoding Flp recombinase failed to induce recombination at the FRT sites in E. coli O157:H7, we used strains of NC101ibpAB and O157: H7ibpAB that still contained the kanamycin resistance gene. Mutant E. coli NC101 lacking oxyS (NC101oxyS) was also generated using the -red recombinase method. Primers 5’GCATAGCAACGAACGATTATCCCTATCAAGCATTCTGACTGTGTAGGCTGGAGCTGCTTC and 5′ ACCGTTACTATCAGGCTCTCTTGCTGTGGGCCTGTAGAATCATATGAATATCCTCCTTAGTTCC were used to amplify the kanamycin resistance cassette from pKD4. Transformation and site-specific recombination of the PCR product into the oxyS locus on the E. coli NC101 chromosome followed by excision of the kanamycin resistance gene using pCP20 was performed as previously described[23,26]. Recombinant bacterial cell lines were generated in accordance with procedures outlined by the Environmental Health and Safety Department at University of North Carolina at Chapel Hill.Mouse Strains and Production of Bone Marrow-Derived MacrophagesWild-type, gp91phox-/-, and Inos-/- mice (all on the C57/B6 genetic background) were originally obtained from Jackson Laboratories and maintained in specific-pathogen-free conditions in Department of Lab and Animal Medicine facilities at UNC, Chapel Hill. All animal protocols were approved by the UNC-Chapel Hill Institutional Animal Care and Use Committee. Bone marrow derived macrophages (BMDMs) were obtained similar to methods described previously[27]. Briefly, bone marrow was harvested from femurs and tibias of mice by flushing marrow cavities with sterile RPMI through a 26G needle and red cells lysed with 0.8 ammonium chloride for 5 minutes. After washing twice with RPMI containing 10 FBS, 2.5 x 107 cells/ plate were added to 25cm petri dishes in 50mL RPMI/10 FBS/100U/mL penicillin/ 1000 g/ mL streptomycin/25ng/mL Fungizone/10 conditioned L929 media. Three days later, 10mL of RPMI/10 FBS/100U/mL penicillin/ 1000 g/mL streptomycin/25ng/mL Fungizone/10 conditioned L929 media was added to each plate. On day 6, adherent cells (BMDMs) were removed with TrypLE-Express (Invitrogen), counted, and plated into experimental wells.Gentamicin Protection AssaysIntra-macrophage bacterial survival assays were performed as described previously[14,23]. Briefly, approximately 10 mid-log phase bacteria/cell were added to 5?.5 x 105 BMDMs/well in 12-well plates in a tot.Nsal E. coli against ROSMaterials and Methods Bacterial Strains, Cells Lines, and Culture ConditionsThe non-pathogenic murine E. coli strain NC101 was isolated as described previously[24]. E. coli strain O157:H7 was a kind gift from Dr. Ann Matthysse at UNC, Chapel Hill. E. coli were grown in Luria-Burtani (LB) broth at 37 with shaking at 250 rpm. The J774 murine macrophage and L929 fibroblast cell lines were originally obtained from ATCC (Manassas, VA) and cultured in RPMI containing 10 fetal bovine serum (FBS), 100U/mL penicillin, 1000 g/mL streptomycin, and 10mM glutamine in 37 humidified incubators with 5 CO2. Conditioned media from L929 cells was used as a source of macrophage colony stimulating factor (M-CSF) for the production of bone marrow-derived macrophages (BMDMs) and was made as described previously[25]. The mutant E. coli NC101 strain lacking ibpA and ibpB (NC101ibpAB) that was used in this study had been generated previously using the -red recombinase method[23,26]. We used identical methods to create a mutant E. coli O157:H7 strain that lacks ibpA and ibpB (O157: H7ibpAB). However, since the pCP20 plasmid encoding Flp recombinase failed to induce recombination at the FRT sites in E. coli O157:H7, we used strains of NC101ibpAB and O157: H7ibpAB that still contained the kanamycin resistance gene. Mutant E. coli NC101 lacking oxyS (NC101oxyS) was also generated using the -red recombinase method. Primers 5’GCATAGCAACGAACGATTATCCCTATCAAGCATTCTGACTGTGTAGGCTGGAGCTGCTTC and 5′ ACCGTTACTATCAGGCTCTCTTGCTGTGGGCCTGTAGAATCATATGAATATCCTCCTTAGTTCC were used to amplify the kanamycin resistance cassette from pKD4. Transformation and site-specific recombination of the PCR product into the oxyS locus on the E. coli NC101 chromosome followed by excision of the kanamycin resistance gene using pCP20 was performed as previously described[23,26]. Recombinant bacterial cell lines were generated in accordance with procedures outlined by the Environmental Health and Safety Department at University of North Carolina at Chapel Hill.Mouse Strains and Production of Bone Marrow-Derived MacrophagesWild-type, gp91phox-/-, and Inos-/- mice (all on the C57/B6 genetic background) were originally obtained from Jackson Laboratories and maintained in specific-pathogen-free conditions in Department of Lab and Animal Medicine facilities at UNC, Chapel Hill. All animal protocols were approved by the UNC-Chapel Hill Institutional Animal Care and Use Committee. Bone marrow derived macrophages (BMDMs) were obtained similar to methods described previously[27]. Briefly, bone marrow was harvested from femurs and tibias of mice by flushing marrow cavities with sterile RPMI through a 26G needle and red cells lysed with 0.8 ammonium chloride for 5 minutes. After washing twice with RPMI containing 10 FBS, 2.5 x 107 cells/ plate were added to 25cm petri dishes in 50mL RPMI/10 FBS/100U/mL penicillin/ 1000 g/ mL streptomycin/25ng/mL Fungizone/10 conditioned L929 media. Three days later, 10mL of RPMI/10 FBS/100U/mL penicillin/ 1000 g/mL streptomycin/25ng/mL Fungizone/10 conditioned L929 media was added to each plate. On day 6, adherent cells (BMDMs) were removed with TrypLE-Express (Invitrogen), counted, and plated into experimental wells.Gentamicin Protection AssaysIntra-macrophage bacterial survival assays were performed as described previously[14,23]. Briefly, approximately 10 mid-log phase bacteria/cell were added to 5?.5 x 105 BMDMs/well in 12-well plates in a tot.
