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 accu- mulated 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. Results 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 Differential Expression of BteA in B. Pertussis 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 Figure 1. Comparative proteomic analysis of B. pertussis nonvaccine-type and vaccine-type strains. Total protein (10 mg) from the nonvaccine-type and vaccine-type clinical strains was separated by 2-D gel electrophoresis and silver stained. The left upper panel shows the protein profile of the nonvaccine-type BP235. The right upper panel shows the protein profile of the vaccine-type BP233. The red-boxed areas are enlarged (lower panels). The arrow in left lower panel indicates the spot that was identified as type III effector BteA by LC-MS/MS analysis. doi:10.1371/journal.pone.0017797.g001 PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17797