CO2 Responsive Regulon in Bordetella Figure 5. Differential transcription of 5% CO2 responsive genes independent of bvgS expression. qRT-PCR analysis was performed on RB50 grown in 5% CO2 (light grey bars), RB54 in ambient air (dark gray bars), and RB54 in 5% CO2 (black bars) compared to RB50 grown in ambient air (white bars). Fold-change expression (FCE) in all strains was expressed as mean 6 standard deviation for cyaA (A), fhaB (B), bopD (C), bopB (D), cheZ (E), and flgB (F). Data shown are averages obtained from quadruplicate cultures. * indicates a p-value less than 0.05. doi:10.1371/journal.pone.0047635.g005 (Fig. 6). Strain RB54 grown in 5% CO2 also produced antigens .250 kDa and ,60 kDa in greater amounts (Fig. 6). RB54 grown in ambient air produced an antigen between 95 and 130 kDa in greater amounts (Fig. 6, arrowhead). These data demonstrate that antigen production is differentially regulated in response to 5% CO2 even when a functional BvgS is absent. Growth in 5% CO2 Affects Cytotoxicity and Adherence of B. bronchiseptica Strains Since genes, cyaA and the TTSS genes, associated with the cytotoxicity of B. bronchiseptica were increased when strains RB50 and 761 were grown in 5% CO2 conditions (Fig. 3), we assessed the relative cytotoxicity to J774 murine macrophages of strains grown in atmospheric or 5% CO2 conditions. Similar to previous findings [52], RB50 killed .90% of cells at an MOI of 10 or 100; however, RB50 only killed ,65% at an MOI of 1 (Fig. 7A). RB50 grown in 5% CO2 killed .90% at all MOIs, indicating that RB50 grown in 5% CO2 killed more macrophages at a lower MOI than RB50 grown in atmospheric conditions (Fig. 7A). Strain 761 had detectable (,30%) killing only at high MOIs (10 and 100), while 761 grown in 5% CO2 was cytotoxic at an MOI of 1 (,45%) and comparable to RB50 at higher MOIs (Fig. 7A). These data show that growth in 5% CO2 increased killing of murine macrophages by both strains. ACT and TTSS have been previously shown to account for all cytotoxicity of macrophages when RB50 is grown in ambient air [37,52]. Since 5% CO2 increased expression of several genes, we examined whether the increased cytotoxicity is due to increases in these known factors or a new cytotoxic mechanism. Cells were exposed for 4 hours at MOIs of 1, 10 or 100 with wild-type RB50, RB50DcyaA, RB50DbscN (encoding the ATPase of the TTSS) or a mutant lacking bscN and cyaA (RB50DcyaADbscN). Growth in 5% CO2 increased cytotoxicity of RB50Dcya to macrophages at MOIs of 1, 10 and 100, while growth 5% CO2 caused increased cytotoxicity of RB50DbscN only at an MOI of 100, likely indicating the differential roles of the TTSS and ACT in cytotoxicity (Figure 7B). RB50DcyaADbscN caused very low levels of cytotox- icity as observed previously [37], and growth in 5% CO2 did not increase cytotoxicity(Fig. 7B). These data suggest that there are no other cytotoxic mechanisms and that increased ACT and TTSS function accounts for the increased cytotoxicity when strains are grown in 5% CO2 conditions. Since many genes encoding adhesins were increased in transcription in response to growth in 5% CO2 conditions, we hypothesized that strains grown under these conditions, in comparison to growth in ambient air, would be more adherent to epithelial cells. L2 cells were incubated with RB50 or 761 pre- grown in either atmospheric or 5% CO2 conditions. Both strains pre-grown in 5% CO2 conditions adhered to lung epithelial cells more efficiently than bacteria grown in normal atmospheric conditions (Fig. 7C). PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e47635