Differential Expression of BteA in B. Pertussis Figure 4. RT-PCR analysis of bteA transcript in B. pertussis nonvaccine-type and vaccine-type strains. (A) RT-PCR with primers specific for bteA and btcA. cDNA made from total RNA of nonvaccine-type (BP157, BP159, BP162, BP228 and BP235) and vaccine-type (BP155, BP156, BP232, BP233 and BP243) clinical strains was used as templates for PCR. Genomic DNA (G) from B. pertussis strain Tohama was used as a positive control. A mock reaction for bteA (2RT) consisted of reactions lacking reverse transcriptase. (B) Quantitative RT-PCR analysis of bteA transcript levels in the nonvaccine-type and vaccine-type clinical strains listed in (A). The recA transcript was used as a reference. Each point represents one strain and vertical bars indicate standard deviations. doi:10.1371/journal.pone.0017797.g004 strains but not in the vaccine-type strains, and that BteA protein expression is down-regulated by IS481a insertion in the vaccine- type strains. We provide the first evidence that BteA protein expression is type-dependent due to the IS481a insertion in B. pertussis clinical strains. In Japan, B. pertussis circulating strains began to change from vaccine-type to nonvaccine-type in the mid-1990s [8], and the reported incidence of adult cases of pertussis has dramatically increased since 2002 [36]. The genetic divergence in B. pertussis circulating strains has also been observed in many other countries. A possible explanation for the genetic divergence is that the type shift is a result of vaccine-driven evolution [12–15]. More recently, Mooi et al. [37] reported that expansion of B. pertussis strains with increased PT production has contributed to the resurgence of pertussis in the Netherlands. Here we showed prominent expression of the T3SS effector protein BteA in the nonvaccine- type strains, and that PT and ACT (important virulence factors of B. pertussis) are expressed at the same level in both the nonvaccine and vaccine-type strains. Besides vaccine-driven evolution, our findings could provide another possible explanation for the type shift from vaccine-type to nonvaccine-type, i.e., the augmented expression of BteA protein in B. pertussis nonvaccine-type strains may be involved in the type shift. B. bronchiseptica BteA has in vitro cytotoxic activity against cultured mammalian cells [18,22,29,30]. In this study, we determined the cytotoxicity of B. pertussis BteA mutants by measuring the release of lactate dehydrogenase (LDH) from L2 rat lung epithelial cells, J774 mouse macrophage-like cells, or HeLa cells. However, even BteA-expressing strains (DIS481- BP155 and wild-type BP157) showed low cytotoxicity (,10%), and consequently no statistically significant differences in cytotox- icity were observed among the wild-type and mutant strains. B. pertussis is known to have a lower in vitro cytotoxicity than B. bronchiseptica [18,22], which is consistent with the extremely low secretion of BteA in B. pertussis as compared to B. bronchiseptica (Figure S1). Therefore, a more sensitive and quantitative assay is required to determine the BteA-dependent cytotoxicity of B. pertussis. PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17797