Differential Expression of BteA in B. Pertussis Figure 3. BteA secretion from B. pertussis nonvaccine-type and vaccine type strains. Strains BP235 (nonvaccine-type) and BP233 (vaccine- type) were cultured in modified SS medium, and the culture supernatants (CS) were collected at 12, 24 and 48 h. Protein samples prepared by precipitation with 10% trichloroacetic acid were separated by 12.5% SDS-PAGE followed by silver staining (left panel). BteA, FHA and PT secretions were analyzed by immunoblots using anti-BteA, anti-FHA or anti-PT antiserum (right panels). For BteA detection, the equivalent of 200 ml of CS was loaded in each lane. doi:10.1371/journal.pone.0017797.g003 promoter at one end that is responsible for transcription of the flanking catalase gene (katA) in B. pertussis [34]. However, the P2 start site is different from the katA transcription start site. The transcription start site of btcA, also determined by 59-RACE, was mapped to a T residue 31 bp upstream of the btcA translation start codon in both the vaccine-type and nonvaccine-type strains (Figure 5B). Primer extension analysis was also performed in an attempt to resolve the bteA transcription start sites. However, the start sites could not be ascertained, probably due to low amounts of bteA transcript in B. pertussis. IS481a-promoter transcript is the major bteA transcript in the vaccine-type strain Expression of the IS481a-promoter transcript (P2 transcript) in B. pertussis vaccine-type strain BP155 was analyzed by qRT-PCR with TaqMan probes (Figure 5A). The P2 transcript and total bteA (P1 + P2) transcripts were determined individually and the ratio of P2 transcript to total bteA transcript was calculated. Based on four independent experiments, the ratio (P2 transcript/P1 + P2 transcripts) was estimated to be 0.88 (61SD range, 0.70 to 1.09), indicating that the P2 transcript is the major bteA transcript in the vaccine-type strain (data not shown). BteA expression in B. pertussis BteA mutants To clarify the effect of the IS481 insertion on BteA expression, four BteA mutants (DbteA-BP155, DIS481-BP155, DbteA-BP157 and +IS481-BP157) were constructed from B. pertussis BP155 (vaccine-type) and BP157 (nonvaccine-type) by homologous recombination (Figure 6A). The DbteA-BP155 and DbteA-BP157 mutants had a 178-bp deletion in the 59 region of bteA. In the DIS481-BP155 mutant, a 2.2-kb insertion containing an intact bteA 59-UTR (derived from B. pertussis Tohama) replaced the native bteA 59-UTR + IS481a gene. In contrast, +IS481-BP157 mutant had a 3.2-kb insertion containing a bteA 59-UTR + IS481a (derived from B. pertussis BP155) instead of its own bteA 59-UTR. Consequently, DIS481-BP155 had an IS481a deletion from the bteA 59-UTR, whereas the +IS481-BP157 mutant had an IS481a insertion in the bteA 59-UTR. The btcA2bteA region of the mutants was verified by DNA sequence analysis. BteA expression in the bacterial cells and CS after 24 h in culture was analyzed by immunoblot with anti-BteA antiserum (Figure 6B). In DIS481-BP155 bacterial cells and CS, BteA polypeptide(s) corresponding to ,68 kDa and .200 kDa were detected at the same level as was observed in the BP157 wild-type strain. In contrast, the signals of BteA polypeptide(s) from +IS481- BP157 mutant were very low in both bacterial cells and CS. Similarly, BteA polypeptide(s) were not detected in either DbteA- BP155 or DbteA-BP157. These results clearly showed that BteA protein expression is down-regulated by the IS481 insertion in B. pertussis, and that the anti-BteA antiserum is highly specific to BteA. Discussion The BteA effector (alias BopC) is required for the induction of necrotic cell death during B. bronchiseptica infections, and is thought to play a pivotal role in T3SS-mediated cell death [29,30,35]. BteA is also involved in dephosphorylation of tyrosine-phosphor- ylated proteins (PY) of host cells [30], and its 130-amino acid N- terminal sequence is associated with target lipid rafts [31]. BteA is the only cytotoxic effector that has been identified in Bordetella spp. In B. pertussis, low-passage clinical strains have an ability to express a functionally active T3SS; however, BteA protein had not been detected in the clinical and common laboratory-adapted strains by MALDI-TOF mass spectrometry [22]. Here we demonstrate that BteA protein is highly expressed in B. pertussis nonvaccine-type PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e17797