CO2 Responsive Regulon in Bordetella Figure 2. Differential recognition of antigens in B. bronchiseptica strains grown in atmospheric or 5% CO2 conditions. C57BL/6 mice were inoculated with 56105 CFU B. bronchiseptica strain RB50, and serum was collected 28 days later. Strains RB50 and 761 were grown in atmospheric or in elevated CO2 concentrations, and were probed with serum against RB50. Increased (arrows) or decreased (arrowheads) production of antigens in response to 5% CO2 conditions is denoted. The ratio of band intensity between antigens produced in ambient air and 5% CO2 conditions is indicated in the margins: 1 = equal amounts produced in either conditions, ,1 = more produced in ambient air, .1 = more produced in 5% CO2 conditions. doi:10.1371/journal.pone.0047635.g002 Cytotoxicity Assay Cytotoxicity assays were carried out as previously described [45]. J774A.1 cells, a murine macrophage cell line, obtained from the ATCC were cultured in DMEM with 10% FBS. Cells were grown to approximately 80% confluency, and bacteria were added at a MOI of 100, 10 or 1. After a 5 minute centrifugation at 2506g, the mixture was incubated at 37uC for the indicated times. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release using the Cytotox96 (Promega, Madison, WI) kit according to the manufacturer’s protocol. Results were analyzed using analysis of variance with a Tukey simultaneous test, and a P value of ,0.05 was considered significant. Adherence Assay Adherence assays were modified from a previously described protocol [47]. Rat epithelial cell line L2, obtained from the ATCC, was cultured in DMEM/Ham’s F12 50–50 mixture with 10% FBS. Cells were grown to approximately 80% confluency, and bacteria were added at an MOI of 100. After a 5 minute centrifugation at 2506g, the mixture was incubated for 30 minutes. Cell culture supernatant was removed and cells were washed 4 times with PBS to remove unbound bacteria. Epithelial cells were then trypsinized and resuspended in 1 mL of tissue culture media. The mixture of cells and bacteria were diluted in PBS and plated on BG agar to determine CFU. Results were analyzed using analysis of variance with a Tukey simultaneous test, and a P value of ,0.05 was considered significant. Results A B. bronchiseptica Isolate Regulates ACT Expression in Response to 5% CO2 Conditions B. bronchiseptica isolates are generally b-hemolytic when grown in Bvg+ conditions due to the production of ACT, which causes lysis of red blood cells. It was recently discovered that some B. bronchiseptica isolates do not have the genes required to produce a functional ACT and therefore are not hemolytic on blood agar plates [39]. However, through screening of 73 isolates based on hemolysis on blood agar plates and PCR amplification of the genes encoding ACT, 4 B. bronchiseptica isolates were found to be non-hemolytic, but still retained the genes for production of ACT (Fig. 1, data not shown). When one isolate displaying this phenotype, B. bronchiseptica strain 761, was grown in a tissue culture incubator where the CO2 concentration is increased to 5%, it was hemolytic (Fig. 1, compare panels B and D). RB50 was hemolytic even in ambient air (,0.03% CO2) (Fig. 1, compare panels A and C), but there appeared to be more hemolysis when it was grown in 5% CO2 conditions, suggesting both strains produce more ACT in response to growth in 5% CO2 conditions. Growth rate and pH were not significantly PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47635