CO2 Responsive Regulon in Bordetella Figure 6. Differential recognition of antigens of a Bvg2 mutant grown in atmospheric or 5% CO2 conditions. C57BL/6 mice were inoculated with 56105 CFU B. bronchiseptica strain RB50 and sera were collected 28 days post-inoculation. Strains RB50 and RB54 were grown in atmospheric or in 5% CO2 concentrations, and were probed with serum against RB50. 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.g006 B. pertussis and B. parapertussis Modulate Virulence Factor Expression in Response to 5% CO2 Since up-regulation of virulence factors in response to growth in 5% CO2 conditions is common to multiple B. bronchiseptica strains, we hypothesized that B. pertussis and B. parapertussis may also regulate virulence factor expression in response to growth in 5% CO2 conditions. Genes shown to be CO2 responsive (cyaA, fhaB, fimA) or non-responsive to CO2 (bvgS) in B. bronchiseptica (Fig. 3), were chosen to be analyzed by qPCR in B. pertussis and B. parapertussis. B. pertussis and B. parapertussis had increased expression of fhaB and cyaA, but not bvgS in response to CO2 (Fig. 8A, B). B. pertussis, unlike B. parapertussis, also had increased expression of fimA, indicating that the 5% CO2 responsive regulon may be different among the three classical Bordetella species. To further investigate this effect, Western blots with lysates of B. pertussis and B. parapertussis grown in ambient air or 5% CO2 were probed with either B. pertussis-induced sera or B. parapertussis-induced sera (Fig. 8C, D). B. pertussis strains 536, 18323 and a recent clinical isolate CHOC 0012 grown in ambient air showed a different antigenic profile from the lysates prepared from strains grown in 5% CO2, with bands from roughly 55 to 250 kDa which were more numerous and intense in 5% CO2 for strains 536 and CHOC 0012 (Fig. 8C). Notably, B. pertussis strains 536 and CHOC 0012 appeared to increase similar antigens in response to growth in 5% CO2 conditions, while strain 18323 grown in 5% CO2 decreased production of several antigens (Figure 8C). B. parapertussis grown in ambient air produced a more intense band between 130 and 250 kDa, while growth in 5% CO2 produced a more intense band between 36 and 55 kDa (Fig. 8D). Overall, B. parapertussis did not appear to differentially regulate many antigens in response to growth in 5% CO2 conditions (Figure 8D). These data indicate that several antigens in B. pertussis, but few in B. parapertussis isolate 12822, are differentially regulated in response to growth in 5% CO2 compared to growth in ambient air and that there is strain variation in CO2 responsiveness in the bordetellae. Discussion BvgAS was originally considered an ON/OFF switch, modu- lating Bordetella species between two distinct states, avirulent (Bvg2) and virulent (Bvg+). The discovery of an intermediate phase has led to a view of BvgAS gene regulation as a rheostat visualized as varying along a one dimensional gradient [23,32,34]. In this view of the two-component system few signals, temperature and some chemical cues, are known to affect virulence factor regulation PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47635