Differential Expression of Type III Effector BteA Protein Due to IS481 Insertion in Bordetella pertussis

Background Bordetella pertussis is the primary etiologic agent of the disease pertussis. Universal immunization programs have contributed to a significant reduction in morbidity and mortality of pertussis; however, incidence of the disease, especially in adolescents and adults, has increased in several countries despite high vaccination coverage. During the last three decades, strains of Bordetella pertussis in circulation have shifted from the vaccine-type to the nonvaccine-type in many countries. A comparative proteomic analysis of the strains was performed to identify protein(s) involved in the type shift. Methodology/Principal Finding Proteomic analysis identified one differentially expressed protein in the B. pertussis strains: the type III cytotoxic effector protein BteA, which is responsible for host cell death in Bordetella bronchiseptica infections. Immunoblot analysis confirmed the prominent expression of BteA protein in the nonvaccine-type strains but not in the vaccine-type strains. Sequence analysis of the vaccine-type strains revealed an IS481 insertion in the 5′ untranslated region of bteA, −136 bp upstream of the bteA start codon. A high level of bteA transcripts from the IS481 promoter was detected in the vaccine-type strains, indicating that the transcript might be an untranslatable form. Furthermore, BteA mutant studies demonstrated that BteA expression in the vaccine-type strains is down-regulated by the IS481 insertion. Conclusion/Significance The cytotoxic effector BteA protein is expressed at higher levels in B. pertussis nonvaccine-type strains than in vaccine-type strains. This type-dependent expression is due to an insertion of IS481 in B. pertussis clinical strains, suggesting that augmented expression of BteA protein might play a key role in the type shift of B. pertussis.


Introduction
Bordetella pertussis is a human-specific pathogen that is the etiologic agent of whooping cough, an acute respiratory disease that is often particularly severe in infants [1]. Universal immunization programs have contributed to a significant reduction in morbidity and mortality of pertussis, especially in infants and children; however, the incidence of pertussis has increased in several countries despite high vaccination coverage [2][3][4][5]. Since the 1980s, a considerable genetic transition has been observed between B. pertussis vaccine strains and circulating clinical strains in many countries [6][7][8][9][10][11]. Genetic variations have been found in the loci encoding the major B. pertussis virulence factors: pertussis toxin S1 subunit (ptxA), pertactin (prn) and fimbriae 3 (fim3). Among circulating B. pertussis strains, vaccine-type alleles (ptxA2, prn1 and fim3A) have been replaced mainly with nonvaccine-type alleles (ptxA1, prn2 and fim3B). It has been speculated that adaptation of the bacterial population to vaccine-induced immunity has produced this genetic shift, and is one possible explanation for the resurgence of pertussis [12][13][14][15]. However, there have been few reports of the exact mechanism underlying this phenomenon.
B. pertussis expresses various virulence factors, including adhesins and toxins, which function to establish and maintain host infection. Several virulence factors such as filamentous haemagglutinin (FHA) and pertussis toxin (PT) are expressed under the control of the BvgAS two-component regulatory system [1,16,17]. The BvgAS system also positively regulates virulence factor secretion via the type III secretion system (T3SS) [18,19]. T3SS is highly conserved among a number of Gram-negative bacteria and functions as an injector of virulence molecules (i.e., effectors) into the host cell through a needle-like injection apparatus [20,21]. In B. pertussis, T3SS plays a role in subverting the protective innate and adaptive immunity of the host. Three T3SS-secreted proteins, BopN, BopD and Bsp22, have been identified so far [22]. In the animal pathogen Bordetella bronchiseptica, BopN is involved in the up-regulation of cytokine IL-10 [23], while Bsp22 polymerizes to form a flexible filamentous structure at the tip of the needle structure and associates with the pore component BopD [24]. The Bsp22 translocon is expressed in a significant proportion of B. pertussis clinical isolates but not in Tohama and Wellcome 28, the common laboratory-adapted vaccine strains [22].
Genomic differences between B. pertussis clinical strains and the vaccine strain Tohama have been investigated. The comparative genomics profiling revealed that the genome of B. pertussis Tohama differs from clinical isolates in four regions (RD11 to RD14) [25]. In contrast, progressive gene loss mediated by homologous recombination between IS481 insertion sequence elements has been observed among recently circulating strains of B. pertussis isolates [26,27]. IS481 is present in multiple copies on the B. pertussis chromosome, and it plays a critical role in B. pertussis evolution through genomic rearrangement.
Proteomic analysis has been widely applied to comparisons of protein expression among different strains, and information accumulated from genomic studies of Bordetella spp. facilitates comparative proteomic approaches to the investigation of B. pertussis clinical strains [6,28]. In the present study, a proteomic approach was employed to identify the protein(s) involved in the genetic shift from vaccine-type to nonvaccine-type in B. pertussis strains. The protein profile analyses identified one differentially expressed protein, the T3SS effector BteA (alias BopC) [29,30], between the strain types. BteA is a 68 kDa cytotoxic effector that has been identified in B. bronchiseptica but not in the B. pertussis human pathogen. Here we studied the differential expression of BteA protein in B. pertussis clinical strains and identified a specific IS481 insertion in the 59 untranslated region (59-UTR) of bteA in vaccine-type strains.

Identification of BteA in B. pertussis nonvaccine-type strain
A comparative proteomic analysis of two clinical strains was performed to investigate the shift of B. pertussis strains from vaccine-type to nonvaccine-type. Figure 1 shows 2-dimensional electrophoretic (2-DE) maps of total protein expressed in the nonvaccine-type clinical strain BP235 and the vaccine-type BP233. Among .600 protein spots detected on the 2-DE gel, one was notably absent in the 2-DE map of BP233. The protein spot was observed in other nonvaccine-type strains (BP157, BP159, BP162 and BP228), but not in other vaccine-type strains (BP155, BP156, BP232 and BP243). The protein represented by the spot was identified by LC-MS/MS analysis using tryptic digests. The MS/MS of the protein digests provided four peptide sequences (RPDEFAAR, FDALR, ITALNLR and TQTQLLALQR) that matched the B. pertussis hypothetical protein BP0500 (NCBI accession: NP_879352). Hypothetical protein BP0500 was identified as the T3SS effector BteA, since the sequence is highly conserved with 98% amino acid identity to the BteA (BopC) of B. bronchiseptica [29,30].
High expression of BteA protein in nonvaccine-type strains Immunoblots of B. pertussis clinical strains using anti-BteA antiserum detected high levels of a protein of ,68 kDa in all nonvaccine-type clinical strains (BP157, BP159, BP162, BP228 and BP235), whereas BteA expression was greatly reduced in the vaccine-type clinical strains (BP155, BP156, BP232, BP233 and BP243). Additional products of .200 kDa were also detected in the nonvaccine-type clinical strains. These high molecular mass signals appear to be the protein bands that have been reported as a multimeric complex of BteA in B. bronchiseptica [29,30] (see Figure  S1). T3SS function in the nonvaccine-type strains was confirmed 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). 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, corresponding 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 nonvaccinetype strain BP159 ( Figure 5B). IS481 contains an outward-facing 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 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-phosphorylated proteins (PY) of host cells [30], and its 130-amino acid Nterminal 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 strains but not in the vaccine-type strains, and that BteA protein expression is down-regulated by IS481a insertion in the vaccinetype 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][13][14][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 nonvaccinetype 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 cytotoxicity 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. IS481 belongs to the recently defined IS481 family [38], and 238 copies of IS481 are present in the B. pertussis Tohama genome [39]. In B. pertussis clinical strains, IS481 is also present in multiple copies on the chromosome and it plays a critical role in B. pertussis evolution [26,40]. Many IS elements have been shown to activate the expression of neighboring genes. IS481 contains an outwardfacing promoter that is located in close proximity to the left terminal inverted repeat, and this promoter is responsible for the transcription of katA in certain B. pertussis strains [34]. Here we identified an IS481a insertion in the bteA 59-UTR in B. pertussis vaccine-type clinical strains and detected a high level of bteA transcripts from the IS481a promoter (P2) compared with its own promoter (P1). However, the vaccine-type strains showed a low level of BteA protein expression, suggesting that insertion of IS481a represses P1 promoter activity, and that P2 transcript has a low translational efficiency from the additional nucleotide sequence (79 nucleotides) at its 59 end. Use of a cell-free coupled transcription-translation system revealed that the additional nucleotide sequence is involved in down-regulation of transcription and/or translation ( Figure S2). The 59-UTR of bacterial mRNAs can bear regulatory elements that are involved in downor up-regulation of translation [41]. The regulatory mechanisms in this region are controlled by RNA-binding proteins, small noncoding RNAs and structural rearrangements with the 59-UTR. In addition, a 59 stem-loop structure that sequesters the ribosomal binding site has been shown to be involved in translational regulation. Bioinformatic analysis uncovered a predicted stem-loop structure in the bteA 59-UTR of P2 transcript ( Figure S2).
In this study, the 59-UTRs of five B. pertussis vaccine-type clinical strains were sequenced; all had an insertion of an IS481a in the bteA 59-UTR, both transcribed in the same direction. In one of the vaccine-type strains, BP155, the major bteA mRNA was transcribed from P2 in the IS481a-promoter. These observations raise the possibilities that (i) the P2 transcript is translated into BteA under certain environmental conditions, and (ii) the P2 transcript is translated into another novel protein by translational frameshifting. BteA is known to be regulated by the BvgAS system and an extracytoplasmic function (ECF) sigma factor BtrS in B. bronchiseptica [18,29]. In B. pertussis, it has been suggested that expression of the T3SS translocon Bsp22 is blocked by posttranscriptional regulation [18]. However, the molecular details of the regulatory mechanism are still unclear. Further studies are needed to determine the down-regulation of BteA protein in B. pertussis vaccine-type clinical strains.
In conclusion, B. pertussis vaccine-type strains have been replaced with the nonvaccine-type strains in many countries, and the resurgence of pertussis has been observed in several nations. In Japanese B. pertussis clinical strains, the T3SS effector BteA is highly expressed in nonvaccine-type strains as compared with the vaccine-type strains. Our findings indicate that augmented expression of BteA protein in B. pertussis circulating strains could play a key role in the type shift. However, it is unclear whether BteA protein is implicated in the resurgence of pertussis. Further studies are needed to determine the expression of BteA protein in B. pertussis circulating strains on a global scale.

Materials and Methods
Bacterial strains and growth conditions B. pertussis clinical strains were selected from the laboratory collection of the National Institute of Infectious Diseases, Tokyo, Japan. The selection criteria included the time and geographic location of isolation, and their ptxA and prn alleles. A total of 10 clinical strains from 2002 to 2004 in Japan were included. Of the 10 clinical strains, 5 harbored ptxA1 and prn2 alleles (BP157, BP159, BP162, BP228 and BP235; nonvaccine-type strains), while the others carried ptxA2 and prn1 (BP155, BP156, BP232, BP233 and BP243; vaccine-type strains). All strains were cultured on Bordet-Gengou agar (BG agar, Difco) supplemented with 1% glycerol and 15% defibrinated horse blood or in modified Stainer-Scholte (SS) medium [42].

Protein identification
2D-PAGE gels were stained with silver nitrate without glutaraldehyde fixation [44], and protein spots of interest were excised. Proteins were reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide, and digested with sequencing grademodified trypsin (Promega, Madison, WI). Digested peptides were separated on a C18 capillary column (0.2 by 50 mm, Michrom Bioresources, CA) equipped with a Chorus 220 solvent delivery system and an HTC PAL auto-sampler system (CTC Analytics AG, Zwingen, Switzerland). Separated peptides were analyzed by the Finnigan LCQ TM Deca XP ion trap mass spectrometer (Thermo Fisher Scientific Inc., MA) with electrospray ionization (ESI) interface using the Nanosprayer FS (GL Sciences Inc., Japan). To identify peptides, data files were generated from the MS/MS scans by Bioworks 3.0 using the SEQUEST algorithm (threshold, 10 5 ; minimum group scan 2, Xc .1.0, Thermo Fisher Scientific) and searched against the complete amino acid database derived from the B. pertussis Tohama genome database. Antibody production against recombinant BteA, BtcA and ACT The BteA gene (NCBI accession: NP_879352) was amplified by PCR from B. pertussis Tohama DNA using BteA-F and BteA-R primers, and cloned into the XmnI/HindIII sites of pMal-c2X (New England Biolabs, Beverly, MA) to generate a maltose binding protein (MBP) fusion with BteA. Production of this fusion protein was induced in E. coli BL21 with 0.5 mM isopropyl-b-Dthiogalactopyranoside (IPTG) and subsequently purified using amylose resin (New England Biolabs) and Resource Q (Amersham Pharmacia Biotech, Uppsala, Sweden) columns. A two-step PCR was carried out to amplify recombinant BtcA (NCBI accession: NP_879351). The first PCR was performed using the BtcA-BteA-F3 and BtcA-BteA-R3 primers (Table S1), which amplified the region between positions 165122 and 167190 of the B. pertussis Tohama genome (GenBank accession: BX640412). In the second PCR, btcA was amplified from the first PCR product with the 5-BtcA and 3-BtcA primers (Table S1) and cloned into the NdeI/ HindIII sites of pCold II DNA (TAKARA Bio Inc.). His-tagged BtcA was induced in E. coli BL21 with 0.5 mM IPTG at 15uC and purified using the HisTrap FF Crude Kit (GE Healthcare UK Ltd.). A recombinant catalytic domain of B. pertussis adenylate cyclase toxin (ACT) was a gift from Mineo Watanabe.
Antibodies against MBP-BteA, BtcA and ACT were generated in mice at Nippon Biotest Laboratories, Inc. (Tokyo, Japan). The MBP-BteA antiserum was pre-absorbed with MBP2 protein (New England BioLabs) and the resulting antiserum was used.

Immunoblot analysis
B. pertussis clinical strains were inoculated in modified SS medium with a starting optical density of 0.2 at 600 nm, and further cultured with shaking at 36uC. Total protein was extracted with SDS-lysis buffer, and culture supernatant (CS) proteins were precipitated with 10% TCA. Protein samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad) and incubated with anti-BteA, anti-BtcA, anti-BopD [32], anti-ACT, anti-FHA, or anti-PT antiserum. Antigen-antibody complexes were visualized using horseradish peroxidase (HRP)conjugated secondary antibody (Bio-Rad, Hercules, CA) and ECL Western Blotting Detection Reagents (GE Healthcare).

DNA sequencing
The region between the btcA and bteA gene corresponding to positions 165122 to 168021 of B. pertussis Tohama (GenBank accession: BX640412) was amplified in vaccine-type and nonvaccine-type clinical strains with the appropriate primers and sequenced. Sequencing reactions were carried out with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and the products were sequenced on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems).

Transcriptional analyses
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and treated with RNase-free DNase (Promega) to degrade contaminating DNA. Reverse transcriptase-PCR (RT-PCR) was performed with bteA RT-R and btcA RT-R primers (Table S1) using the TAKARA One Step RNA PCR Kit (AMV, TAKARA Bio Inc.). PCR was performed with the following conditions: one cycle of 50uC for 30 min, 95uC for 2 min; 25 cycles of 95uC for 30 s, 58uC for 30 s, 72uC for 1 min; and a final incubation at 72uC for 10 min. Primer sets, bteA RT-F/bteA RT-R and btcA RT-F/btcA RT-R, were used for bteA and btcA amplification, respectively (Table S1). Products were analyzed by electrophoresis on a 1.5% agarose gel. Reverse transcriptase was omitted from the negative control reaction mixtures.
For quantitative RT-PCR (qRT-PCR), 5 mg of RNA was reverse transcribed into cDNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) with random hexamer primers. Relative levels of total bteA and recA transcripts were determined using TaqMan probes (bteA-and recA-probes, Table S1) and Premix Ex TaqTM (Perfect Real Time, TAKARA Bio Inc.) with the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The qRT-PCR conditions were 30 s at 95uC, followed by 40 cycles of 95uC for 15 s and 60uC for 1 min. The expression of recA was used as an internal control [45]. All samples were run in triplicate and bteA transcript (P1 + P2 transcripts) was normalized to the recA transcript for each sample. The bteA IS481a-promoter transcript (P2 transcript) was determined using a TaqMan probe (IS481-bteA probe). The qRT-PCR conditions were 30 s at 95uC, followed by 40 cycles of 95uC for 15 s and 55uC for 1 min. The ratio of P2 transcript to total bteA transcript (P2 transcript/P1 + P2 transcripts) was estimated from four independent experiments. The regions amplified by qRT-PCR are shown in Figure 6A.
BteA-deficient mutants: A 2.2-kbp DNA fragment containing a 59 portion of the bteA gene was amplified by PCR with the B1-bteA and B2-bteA primers (Table S1) using the B. pertussis Tohama genomic DNA as the template. The PCR product was cloned into the pDONR221 vector (Invitrogen) to obtain pDONR-bteA by means of adaptor PCR and site-specific recombination techniques with the Gateway Cloning System (Invitrogen). Inverse PCR was then carried out with R1-bteA and R2-bteA primers (Table S1) using circular pDONR-bteA as the template. The R1-bteA and R2-bteA primers contained a BamHI site. The resulting PCR product was digested with BamHI and self-ligated to obtain pDONR-DbteA, which contained a 178-bp deletion around the 59 region of bteA. pDONR-DbteA was mixed with pABB-CRS2 [46] to obtain pABB-DbteA using the Gateway Cloning System. pABB-DbteA was then introduced into E. coli SM10lpir and transconjugated into streptomycin (SM)-resistant B. pertussis BP155 (vaccinetype) and BP157 (nonvaccine-type) clinical strains. The resultant mutant strains were designated DbteA-BP155 and DbteA-BP157.

Statistical analysis
The Student's t-test was employed. A value of P,0.05 was considered statistically significant.

Nucleotide sequence accession number
The IS481a sequence was deposited in the DDBJ/EMBL/ GenBank nucleotide sequence databases under accession number AB473880. Figure S1 High secretion of BteA protein in Bordetella bronchiseptica. B. bronchiseptica (BB R05), B. pertussis BP155 (vaccine-type) and BP157 (nonvaccine-type) were cultured in modified SS medium for 24 h. Total protein extracted from the bacterial cells (Cell) and culture supernatants (CS) was separated by SDS-PAGE followed by silver staining (left panel). Immunoblots were incubated with anti-BteA, anti-BtcA or anti-BopD antiserum (right panel). For BteA detection, 0.5 mg of total protein (for Cell) and 5 ml of CS were loaded in the indicated lanes. The amount of total protein loaded was one-twentieth of that in Figure 2, and the loaded CS volume was one-fortieth of that in Figure 3. (TIF) Figure S2 In vitro transcription-translation analysis of a bteA 59-UTR deletion series. (A) bteA 59-UTR deletion genes were PCR-amplified using B. pertussis BP155 (vaccine-type) as the template. Proteins were synthesized using the WakoPURE System (Wako Pure Chemical Industries, Ltd.). The 59-UTR deletion genes harbored the T7 promoter at their 59 end. (B) Expression of BteA protein in an in vitro transcription-translation system (WakoPURE System). The synthesized product was analyzed with immunoblots using anti-BteA antiserum. NC, negative control. (C) A predicted stem-loop structure in the 59-UTR of bteA mRNA (P2 transcript). The RNA secondary structure was analyzed by CentroidFold (http://www.ncrna.org/centroidfold). The schematic shows a simplified map. TIR, translation initiation region. (TIF)