Bioinformatic Analysis of Chlamydia trachomatis Polymorphic Membrane Proteins PmpE, PmpF, PmpG and PmpH as Potential Vaccine Antigens

RESEARCH ARTICLE Bioinformatic Analysis of Chlamydia trachomatis Polymorphic Membrane Proteins PmpE, PmpF, PmpG and PmpH as Potential Vaccine Antigens Alexandra Nunes1, João P. Gomes1, Karuna P. Karunakaran2, Robert C. Brunham2* 1 Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal, 2 Vaccine Research Laboratory, University of British Columbia Centre for Disease Control, Vancouver, Canada * a11111 Abstract OPEN ACCESS Citation: Nunes A, Gomes JP, Karunakaran KP, Brunham RC (2015) Bioinformatic Analysis of Chlamydia trachomatis Polymorphic Membrane Proteins PmpE, PmpF, PmpG and PmpH as Potential Vaccine Antigens. PLoS ONE 10(7): e0131695. doi:10.1371/journal.pone.0131695 Academic Editor: David M. Ojcius, University of the Pacific, UNITED STATES Received: April 23, 2015 Accepted: June 5, 2015 Published: July 1, 2015 Copyright: © 2015 Nunes 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: AN is a recipient of a post-doctoral fellowship (SFRH/BPD/75295/2010) from Fundação para a Ciência e a Tecnologia (FCT). Chlamydia trachomatis is the most important infectious cause of infertility in women with important implications in public health and for which a vaccine is urgently needed. Recent immunoproteomic vaccine studies found that four polymorphic membrane proteins (PmpE, PmpF, PmpG and PmpH) are immunodominant, recognized by various MHC class II haplotypes and protective in mouse models. In the present study, we aimed to evaluate genetic and protein features of Pmps (focusing on the N-terminal 600 amino acids where MHC class II epitopes were mapped) in order to understand antigen variation that may emerge following vaccine induced immune selection. We used several bioinformatics platforms to study: i) Pmps’ phylogeny and genetic polymorphism; ii) the location and distribution of protein features (GGA(I, L)/FxxN motifs and cysteine residues) that may impact pathogen-host interactions and protein conformation; and iii) the existence of phase variation mechanisms that may impact Pmps’ expression. We used a well-characterized collection of 53 fullysequenced strains that represent the C. trachomatis serovars associated with the three disease groups: ocular (N=8), epithelial-genital (N=25) and lymphogranuloma venereum (LGV) (N=20). We observed that PmpF and PmpE are highly polymorphic between LGV and epithelial-genital strains, and also within populations of the latter. We also found heterogeneous representation among strains for GGA(I, L)/FxxN motifs and cysteine residues, suggesting possible alterations in adhesion properties, tissue specificity and immunogenicity. PmpG and, to a lesser extent, PmpH revealed low polymorphism and high conservation of protein features among the genital strains (including the LGV group). Uniquely among the four Pmps, pmpG has regulatory sequences suggestive of phase variation. In aggregate, the results suggest that PmpG may be the lead vaccine candidate because of sequence conservation but may need to be paired with another protective antigen (like PmpH) in order to prevent immune selection of phase variants. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0131695 July 1, 2015 1 / 16 Bioinformatic Analysis of Chlamydia trachomatis Pmp Antigens Introduction Chlamydia trachomatis is an obligate intracellular human bacterial pathogen, comprised of 15 to 18 major serovars. Serovars A-C are responsible for ocular infections that result in trachoma [1]. Serovars D-K cause sexually transmitted diseases such as cervicitis and pelvic inflammatory disease (PID), and globally are an important infectious cause of infertility and ectopic pregnancy in women [2]. Serovars L1-L3 also enter through the ano-urogenital tract, but may dissiminate via infection of macrophages to regional draining lymph nodes, causing lymphogranuloma venereum (LGV) [3]. C. trachomatis is the major bacterial cause of sexually transmitted infections (STIs), accounting for ~106 million of the 500 million new cases of STIs that occur each year worldwide [4]. In Europe, almost half of the estimated 47 million STI cases are due to C. trachomatis [4], while nearly 1.4 million infections are reported each year in the United States [5]. Although the estimated global economic burden is uncalculated, over $516 million are spent annually in direct medical costs on genital chlamydial infections in the United States, making C. trachomatis the most costly infection among the nonviral STIs [6–7]. Despite screening and treatment public health programs to control Chlamydia, the incidence of C. trachomatis infection has increased [4, 8–10]. Thus, there is an urgent need for an efficacious vaccine that prevents acquisition and transmission of infection and the development of pelvic inflammatory disease sequelae. Cumulative studies in animal models and human infections [8, 11–23] have shown that systemic and mucosal CD4 T cell-mediated immunity is necessary for protection against C. trachomatis infection. Among antigen candidates that have been studied [11, 15, 24], members of the polymorphic membrane protein family (PmpA-I) have shown promise as vaccine components as they are dominant antigenic targets for cellular immune responses [25–29]. Four of the nine Pmps (PmpE, PmpF, PmpG and PmpH) have been identified via immunoproteomics as dominant T-cell antigens with multiple MHC class II binding peptides for both C. trachomatis and C. muridarum and observed to be protective in the murine genital tract infection model [25, 28–30]. The fact that each Pmp generates different peptides recognized by different MHC class II haplotypes confers on them the capability of immunizing outbred populations [25]. In the murine model, a PmpG epitope was found to persist on splenic antigen presenting cells for at least 6 months [26]. Tetramer staining also demonstrated PmpG as one of the quantitatively dominant antigens recognized by murine CD4 T cells [17]. Pmps are Chlamydia-specific outer membrane proteins whose precise functions remain unknown, but which have been implicated in pathogenesis and host cell adherence. As typical type V autotransporters [31–32], Pmps are capable of translocating to the bacterial surface their N-terminal Sec-dependent leader sequence (passenger domain), containing multiple short repetitive motifs (GGA(I, L, V) and FxxN) [33]. The proteins may also undergo complex infection-dependent post-translational proteolytic processing [34–37]. These proteins mediate in vitro chlamydial attachment to human epithelial and endothelial cells [34, 38]. Previous bioinformatics (...truncated)