by using whole cell protein extracts for immunoblots of BtcA (the BteA chaperone) [29,31] and BopD (the T3SS translocon) [32]. BtcA and BopD polypeptides were detected in both strain types, but the BtcA signals produced by the nonvaccine-type strains were apparently lower than those of the vaccine-type strains (Figure 2). The reason for the different expression is not clear. In contrast, adenylate cyclase toxin (ACT), another Bordetella spp. virulence factor, was detected at similar levels in both strain types. In order to confirm BteA secretion by B. pertussis strains, BteA polypeptide in the culture supernatants (CS) was subjected to immunoblot analysis. BteA was detected in secreted proteins from the nonvaccine-type clinical strain BP159 at 12, 24 and 48 h, whereas the signal was very low in the vaccine-type clinical strain BP155 over the 48-h time period (Figure 3). Conversely, signals corresponding to PT-S1 subunit and FHA polypeptides were detected in the supernatants of both cultures throughout the sampling period, although silver staining revealed small differences in their protein profiles after 24 h in culture. Transcription of bteA bteA gene expression in B. pertussis strains was investigated with conventional RT-PCR and quantitative RT-PCR. bteA was transcribed in both the nonvaccine-type (BP157, BP159, BP162, BP228 and BP235) and vaccine-type (BP155, BP156, BP232, BP233 and BP243) clinical strains (Figure 4A). Similarly, btcA transcripts were detected in both strain groups. RT-PCR experiments lacking reverse transcriptase showed no specific product for bteA amplification, confirming negligible genomic DNA contamination in the RNA preparations. Quantitative RT- PCR (qRT-PCR) showed an average bteA transcript level of 0.146 (61SD range, 0.107 to 0.184) in nonvaccine-type strains and 0.095 (61SD range, 0.076 to 0.113) in vaccine-type clinical strains, a difference that was not statistically significant (P = 0.11) (Figure 4B). Differential Expression of BteA in B. Pertussis IS481 insertion in the bteA 59-UTR in vaccine-type strains Sequencing of the bteA 59-UTR of the five vaccine-type strains (BP155, BP156, BP232, BP233 and BP243), revealed a 1,043-bp insertion sequence (IS481) 2136 bp upstream of the bteA start codon (Figure 5A). IS481a, which is newly identified in B. pertussis, showed 99% nucleotide sequence identity with IS481 of B. pertussis Tohama. The CCTAAC sequence in the bteA 59-UTR is an insertion site of IS481a and is duplicated by the insertion, although the 6-bp consensus recognition sequence of IS481 has been reported as NCTAGN [33]. IS481 insertions were not found in the nonvaccine-type clinical strains, which had nucleotide sequences that were 99% identical to that of B. pertussis Tohama. In the bteA 59-UTR of the nonvaccine-type strains (BP157, BP159, BP162 BP228 and BP235), one single nucleotide polymorphism (ARG) was observed at 207 bp upstream of the bteA translation start site (Figure 5B). The bteA 59-UTR was PCR-amplified from chromosomal DNA of other B. pertussis strains to confirm insertion of IS481. Among 61 vaccine-type clinical strains, 60 (98%) produced amplicons of ,3.1 kb, a size indicative of an IS481 insertion in the bteA 59- UTR. One strain (BP121) had a product of ,2.1 kb, correspond- ing to the predicted size of the native 59-UTR (data not shown). Of the 23 nonvaccine-type strains examined, all generated ,2.1 kb amplicons, confirming the absence of the IS481 insertion. Determination of the bteA transcription start site 59-RACE mapping was used to identify the bteA transcription start site in vaccine-type strain BP155. Nucleotide sequences of the 59-RACE PCR products revealed two transcription start sites, P1 and P2, located 268 and 2147 bp from the bteA translation start codon (Figure 5A). The P1 start site (+1) was located within the bteA 59-UTR, whereas the P2 start site (279) was located within IS481a. Only the P1 start site was also found in the nonvaccine- type strain BP159 (Figure 5B). IS481 contains an outward-facing Figure 2. Expression of BteA, BtcA, BopD and ACT proteins in B. pertussis nonvaccine-type and vaccine-type strains. The nonvaccine- type clinical strains (BP157, BP159, BP162, BP228 and BP235) and vaccine-type clinical strains (BP155, BP156, BP232, BP233 and BP243) were cultured in modified SS medium for 18 h. Total protein extracted from bacterial cells was subjected to immunoblot analysis with anti-BteA, anti-BtcA, anti- BopD or anti-ACT antiserum. For BteA detection, 10 mg of total protein was loaded in each lane. doi:10.1371/journal.pone.0017797.g002 PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17797