Differential Expression of BteA in B. Pertussis Figure 6. Construction and characterization of B. pertussis BteA mutants. (A) Physical map of the btcA2bteA region of BteA mutants derived from B. pertussis BP155 (vaccine-type) and BP157 (nonvaccine-type). WT, wild-type; DbteA, a 178-bp deletion around the 59 region of bteA; DIS481, IS481a deletion from the bteA 59-UTR; +IS481, IS481a insertion in the bteA 59-UTR. (B) Expression of BteA protein in the BteA mutants. The mutants were cultured in modified SS medium for 24 h. Total protein from the bacterial cells (Cell) and culture supernatants (CS) was analyzed with immunoblot using anti-BteA antiserum. doi:10.1371/journal.pone.0017797.g006 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 augment- ed 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]. Two-dimensional gel electrophoresis (2D-PAGE) 2D-PAGE was performed based on the O’Farrell method [43] with minor modifications. B. pertussis clinical strains grown on BG agar plates were resuspended in casamino acid solution (1% casamino acid, 0.6% NaCl, pH 7.1). Bacterial cells were precipi- tated by centrifugation (12,000 6 g, 10 min) and resuspended in SDS-lysis buffer (62.5 mM Tris-HCl, 1% SDS, 10% glycerol, 5% 2-mercaptoethanol, pH 6.8) by sonication. Total protein was extracted by boiling for 3 min, followed by centrifugation. A portion (10 mg, approximately 2 ml) of the protein solution was mixed with 20 ml of sample buffer [8.5 M urea, 2% Nonidet P-40, 2% Ampholine (pH 3.5 to 10)], and applied to an isoelectric focusing tube gel (2.0 mm inside diameter by 12.0 cm) containing 4% polyacrylamide, 8.5 M urea, 2% Nonidet P-40, and 2% Ampholine (pH 5 to 7 and pH 3.5 to 10 in a ratio of 1:4). Proteins were focused at 10uC for 17 h (1 h at 200 V, 2 h at 400 V, and 14 h at 800 V) with 10 mM H3PO4 (anolyte) and 20 mM NaOH (catholyte). In the second dimension, the electrofocused tube gel was electrophoresed in 12% SDS-PAGE. The separated polypeptides were visualized by silver staining and analyzed with the PDQuest 2-D Analysis Software (Bio-Rad, Hercules, CA). The Lowry assay was used to measure protein concentrations in a trichloroacetic acid (TCA) pellet (resuspended in 1 N NaOH) using bovine serum albumin as a standard. 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 grade- modified 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 LCQTM 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, 105; 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. PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17797