Differential Expression of Type III Effector BteA Protein Due to IS481 Insertion in Bordetella pertussis Hyun-Ja Han1¤, Asaomi Kuwae2, Akio Abe2, Yoshichika Arakawa 1 , Kazunari Kamachi1* 1 Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, Japan, 2 Laboratory of Bacterial Infection, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan Abstract 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 59 untranslated region of bteA, 2136 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. Citation: Han H-J, Kuwae A, Abe A, Arakawa Y, Kamachi K (2011) Differential Expression of Type III Effector BteA Protein Due to IS481 Insertion in Bordetella pertussis. PLoS ONE 6(3): e17797. doi:10.1371/journal.pone.0017797 Editor: Olivier Neyrolles, Institut de Pharmacologie et de Biologie Structurale, France Received September 29, 2010; Accepted February 11, 2011; Published March 10, 2011 Copyright: 2011 Han et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants for Research on Emerging and Re-emerging Infectious Diseases (09158691 and 09158699) from the Ministry of Health, Labor and Welfare of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kamachi@nih.go.jp ¤ Current address: National Fisheries Research and Development Institute, Busan, Republic of Korea 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 reduc- tion 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 [2–5]. Since the 1980s, a considerable genetic transition has been observed between B. pertussis vaccine strains and circulating clinical strains in many countries [6–11]. 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 [12–15]. 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 PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17797