Figure 7. Cytotoxicity and adherence of strains grown in 5% CO2 conditions. J774 murine macrophage cells were stimulated with RB50DcyaA media alone or (m), media RB50DbscN (X) containing (N), or RB50 RB50DcyaADbscN (&) or 761 (m) (A), (¤) RB50 (B) (&), in atmospheric (solid lines) or 5% CO2 conditions (dashed lines) at MOIs of 1, 10 or 100 for 4 hours, and LDH release was assayed. (C) Rat epithelial cells were incubated with RB50 or 761 at a MOI of 100 for 30 minutes. Adherence is expressed as the proportion of adherent bacteria to the amount in the original inoculum. The error bars represent standard deviations. * p-values less than 0.05 as compared to the same strain grown in atmospheric conditions. doi:10.1371/journal.pone.0047635.g007 CO2 Responsive Regulon in Bordetella through the BvgAS system. However, the respiratory tract contains many microenvironments, and within each environment there is likely to be great variation. For example, CO2 levels are thought to vary between air and epithelial cells of the respiratory tract, although these are separated by a fraction of a millimeter of mucous. These sites also change dramatically in the course of the various stages of an infection, and there is likely to be a selective advantage to any strain that can sense these differences and modulate virulence factor expression in response. Here we show that the classical bordetellae share the ability to sense and respond to physiological changes in CO2 concentrations likely to be encountered in the host. In mammalian tissues and blood, CO2 concentrations are higher than inhaled ambient air concentrations of CO2, which are approximately 0.03%. The observed changes in expression of various virulence factors (Fig. 3), and altered phenotypes (adherence and cytotoxicity, Fig. 7) provide additional evidence that the ability to respond to changes in CO2 concentrations allow Bordetella species to adjust to different microenvironments within the host respiratory tract. Recently, it has been shown that there is a zone of oxygenation between the anaerobic luminal environment and the host epithelium in the gastrointestinal tract, which can be sensed by Shigella flexneri [2]. The presence of oxygen alters the expression of TTSS effectors that are important for invasion of host cells, and this ‘aerobic zone’ may enhance secretion of these effectors thereby increasing invasion of host cells [2]. Similarly, the respiratory tract of mammals contains multiple sites where gradients of CO2 or oxygen likely influence virulence factor expression and how respiratory pathogens interact with host cells. B. bronchiseptica has been isolated from multiple sites within the respiratory tract (e.g. nasopharnyx, trachea, lungs) and the ability to detect these differences could allow this pathogen to respond by expressing the array of factors optimal for success under each condition [2]. As bacteria disseminate from the nasal cavity to the trachea, lung and potentially even invade tissues, CO2 concentra- tions may increase, serving as a signal for increased transcription of factors such as adhesinsand toxins that subvert the immune response, which is more robust in these regions [17,52–54]. B. bronchiseptica strains sense and differentially regulate virulence factor gene expression in response to 5% CO2 (Fig. 3), and differential regulation was observed in multiple strains and species of Bordetella demonstrating that sensing and responding to carbon dioxide levels is an ability shared among the classical bordetellae. Additionally, the transcription of several Bvg+-phase genes increased in response to 5% CO2, suggestive of BvgAS involve- ment in the response. Interestingly, regulation of some virulence factor genes (bopD, bopB) by BvgAS was epistatic to 5% CO2 regulation. However, not all virulence gene expression (cyaA, fhaB) was dependent on bvgS (Fig. 5A, B), demonstrating an independent mechanism for virulence factor gene regulation in response to 5% CO2. Standard Bvg+ conditions, without additional CO2, are sufficient for production of ACT (Fig. 1) in RB50, suggesting that additional mechanisms may contribute to increases in production, but are not required. Of 73 B. bronchiseptica strains screened, 4 strains were identified here, 761, 308, 448 and JC100, in which Bvg+ phase conditions are not sufficient for measurable production of ACT. In these strains both Bvg+ phase conditions and 5% CO2 are required for detectable production of ACT. The requirement for 5% CO2 for the production of virulence factors may reflect evolutionary adaption of B. bronchiseptica strains, and suggests that the mechanism of CO2 sensing may confer a selective advantage. Intriguingly, there also appears to be variation in responsiveness of both B. bronchiseptica and B. pertussis strains suggesting that although PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47635