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Title: Pfit Is a Structurally Novel Crohn's Disease-Associated Superantigen}
Article:
Abstract:
T cell responses to enteric bacteria are important in inflammatory bowel disease. I2, encoded by the pfiT gene of Pseudomonas fluorescens, is a T-cell superantigen associated with human Crohn's disease. Here we report the crystal structure of pfiT at 1. 7Å resolution and provide a functional analysis of the interaction of pfiT and its homolog, PA2885, with human class II MHC. Both pfiT and PA2885 bound to mammalian cells and stimulated the proliferation of human lymphocytes. This binding was greatly inhibited by anti-class II MHC HLA-DR antibodies, and to a lesser extent, by anti HLA-DQ and DP antibodies, indicating that the binding was class II MHC-specific. GST-pfiT efficiently precipitated both endogenous and in vitro purified recombinant HLA-DR1 molecules, indicating that pfiT directly interacted with HLA-DR1. Competition studies revealed that pfiT and the superantigen Mycoplasma arthritidis mitogen( MAM) competed for binding to HLA-DR, indicating that their binding sites overlap. Structural analyses established that pfiT belongs to the TetR-family of DNA-binding transcription regulators. The distinct structure of pfiT indicates that it represents a new family of T cell superantigens.
Introduction:
T-cell receptors( TCRs) typically recognize antigens in the form of peptides or peptide-lipids bound to major histocompatibility complex( MHC) molecules. TCRs and class II MHC proteins may directly interact with viral or bacterial proteins known as superantigens( SAgs) to activate T cells. Strong primary T cell responses are elicited by microbial toxin superantigens via their interaction with TCR Vβ[1],[2],[3],[4],[5] elements, and in some cases to TCR Vα chains[6],[7],[8]. A novel enteric T cell superantigen, I2, and its full-length gene product pfiT, are encoded by a microbial gene associated with Crohn's disease( CD)[9],[10],[11]. Several lines of evidence has implicated I2 and pfiT in the pathogenesis of CD[10],[12],[13],[14],[15],[16],[17],[18]. The I2 sequence was selectively detected in active CD lesional colonic tissue, but not in healthy controls or inactive CD ileum. Serum antibody studies revealed that IgA seroreactivity to a recombinant I2 protein was CD specific. Such IgA antibodies specific for I2 were detected in 50–60% of patients( both children and adults) with CD, 10% of patients with ulcerative colitis, and 4% of healthy controls. Patients expressing I2 were distinguished by greater disease severity and progression, including a greater frequency of strictures, internal perforations, and small bowel surgery( 72% in antibody-positive patients, versus 23% in antibody-negative patients)[19]. Through genetic analysis and genomic cloning, a full-length I2 gene( pfiT) was identified as a genomic element of Pseudomonas fluorescens, a normal environmental bacterium detectable in the human gastrointestinal tract[11]. Commensal bacteria have emerged as an important factor in the pathogenesis of CD[20], although P. fluorescens has had limited testing as a potential pathobiont in IBD[21]. Several lines of evidence suggest that the I2 protein pfiT is a novel T-cell SAg[9],[11]. I2 protein induced proliferation and IL-10 responses by normal CD4+ T cells[9]. The I2 response was dependent on MHC class II-mediated recognition, but the antigen did not require antigen processing, a feature typical of SAgs, and antibody blockade of I-Ab specifically blocked the T-cell proliferative response[9]. Selective activation was observed for the murine TCR-Vβ5 subpopulation, and pfiT also exhibited T-cell SAg bioactivity[11]. PA2885, the apparent Pseudomonas aeruginosa homolog of pfiT( 78% sequence identity) also stimulated murine CD4+ T cell proliferation[11]. Accordingly, colonization by the I2 and pfiT-expressing microorganism, P. fluorescens, in IBD susceptible hosts, was speculated to provide a superantigenic stimulus pertinent to the pathogenesis of Crohn's disease. In this study, we describe structural and functional studies of the CD-associated superantigen pfiT. We show that pfiT can stimulate the activation of both mouse splenocytes and human peripheral blood mononuclear cells( PBMCs). We show that pfiT specifically and directly interacts with class II MHC HLA-DR, and to a lesser extent HLA-DP or HLA-DQ, and show that pfiT shares an overlapping binding site on HLA-DR1 with other superantigens. Crystal structure of pfiT at high resolution revealed that pfiT belongs to the bacterial transcription factor family of tetracycline repressor( TetR). The distinct structure of pfiT relative to other SAgs suggests that it represents a novel family of SAg.
Discussion:
Superantigens( SAgs) activate large fractions of T cells expressing particular T-cell receptor( TCR) β chains, and are the pathogenic factors of many human diseases such as toxic shock syndrome and autoimmune diseases[4]. Although much is known about the structures and functions of the pyrogenic SAgs encoded by staphylococcal and streptococcal bacteria, far less is known about the non-pyrogenic SAgs, such as the Crohn's disease( CD)-associated protein I2( pfiT). I2 and its full-length protein pfiT encoded by P. fluorescens are novel enteric SAgs; they are important pathogenic factors and serological markers of CD, an inflammatory bowel disease. The discovery of pathogenic factor and serological marker I2( pfiT) suggest that these proteins play a role in the pathogenesis of Crohn's disease. However, how pfiT performs its biological function and its role in Crohn's disease are not known. In this study, we determined the structure and identified an immunologic function of pfiT. Based upon the high resolution crystal structure of pfiT, we found that pfiT belongs to the TetR transcription regulator protein family. The TetR family members are key players in multidrug resistance, virulence, and pathogenicity processes caused by bacterial pathogens. They act as repressors by binding to consecutive DNA major grooves through the N-terminal DBDs[31],[32],[33],[34],[35],[36]. TetR proteins control the expression of genes involved in multidrug resistance, enzymes implicated in catabolic pathways, biosynthesis of antibiotics, osmotic stress, and pathogenicity of gram-negative and gram-positive bacteria[29],[30]. TetR proteins function as a dimer to bind their DNA elements[31],[32],[33]. Our results demonstrated that pfiT exists as a dimer in solution; and crystal structure also confirmed that pfiT is a dimer at structural level. Structure-based alignment of sequences of pfiT with those of TetR members with known structures of the protein-DNA complexes indicated that most of residues important for TetR members to bind their DNA elements are conserved in the pfiT sequence( Fig. 7F & 7H). Similar to QacR and TetR, pfiT also displays a positively charged surface at the putative DNA binding site( Fig. 7G). Although it is currently unknown whether pfiT binds DNA, these structural evidences suggest that pfiT might also function as a transcription regulator, like other TetR members. However, further investigation will be required to determine whether pfiT acts as both as a SAg and as a DNA-binding protein. Pseudomonas is a large genus of Gram-negative aerobic γ proteobacteria, belonging to the family Pseudomonadaceae containing 191 species[38]. The genomes of many of these species have been sequenced[39],[40],[41],[42],[43]. BLAST search[44] indicated that many putative TetR-family members of the Pseudomonas bacteria share high sequence homology with pfiT( Fig. S1A). In addition, putative TetR members of a number of other bacterial species also display significant sequence homology with pfiT( Fig. S1B). It is currently unknown whether these other TetR members possess SAg activity. Nonetheless, our study suggests a new function for this group of proteins, at least for pfiT and PA2885. We further investigated the function of the CD-associated enteric SAg pfiT. Our data revealed that pfiT can stimulate the activation of not only murine splenocytes, as previously reported[9],[11], but also human peripheral blood T cells. We demonstrated that the class II MHC molecule is the human receptor for the SAg pfiT. Addition of antibodies against all three class II HLA alleles greatly reduced the binding of pfiT to human PBMC. Addition of anti-HLA-DR antibody alone also greatly reduced pfiT binding to human PBMC, whereas anti-DQ and anti-DP antibodies only moderately reduced the binding. The results indicated that HLA-DR, and to a lesser extent, HLA-DQ and HLA-DP, is responsible for pfiT binding. Moreover, using purified recombinant proteins, we demonstrated that pfiT and HLA-DR interacted directly. Competition binding data suggested that the pfiT-binding site on HLA-DR overlaps with that for MAM, a phylogenetically and structurally distinct SAg. Both pfiT and MAM are more selective for HLA-DR[45]. Using a genetic analysis, MAM was found to be immunologically active( T cell activation) in the context of murine H-2E( the murine homologue of HLA-DR), but also certain HLA-DQ and homologue H-2A alleles[45]. Using an antibody-blocking analysis, pfiT also showed immunologic activation with H-2Ab, but not H-2E or other HLA-D molecules([9],[11], and present study). What might be the basis for this overlapping but distinct MHC-dependence of pfiT and MAM? Whereas staphylococcal and streptococcal SAgs share similar three dimensional structures, SAgs from other sources display various structural folds, ranging from completely β-sheets to completely α-helices[4],[24],[46]. Our structural study indicated that the structures of both pfiT and MAM are completely helical, although pfiT displays a different folding pattern[24]. Our competition binding measurements indicated that MAM can compete binding of pfiT to its human receptor HLA-DR. We speculate that the distinct folding patterns might yield differences in molecular contacts that could account for the divergent MHC fine specificities of these two SAgs. Alternatively, these differences may reflect the outcome of genetic versus antibody analysis. These questions await integrated assessment of these two SAgs using the same immunologic assay context, and direct structural analysis of binding with HLA-DR and H-2A. In summary, the structural and functional studies of the pfiT-MHC interactions provide detailed insights into how a novel enteric SAg is recognized by its human receptor. Immune activity to I2, the peptide antigen encoded by pfiT, is a distinguishing biomarker for disease state and clinical phenotype of Crohn's disease[47],[48]. There is an intensifying focus on the interplay of environment and microbiota in Crohn's disease, both as a source of antigens in adaptive immune activation, and as a source of microbial products targeting innate immune and epithelial function[20],[49],[50]. This structural and functional study provides new insights into the SAg properties of pfiT, and accordingly, how P. fluorescens, a ubiquitous environmental microbial commensal, has emerged as an immunologic feature of Crohn's disease.
Human inflammatory bowel disease ( IBD ) is a family of chronic inflammatory disorders of the gastrointestinal tract which affect genetically susceptible individuals . IBD is a lifelong disease involving mostly young people , often severely . Crohn's disease ( CD ) and ulcerative colitis are the two major forms of IBD . Although the exact cause of these diseases remains unknown , both genetic and environmental factors together play significant roles in the disease pathogenesis . Several lines of evidence implicate commensal bacteria as an important pathogenic element in clinical disease , particularly in CD . We recently identified a novel microbial gene , I2 , encoded by Pseudomonas fluorescens , a gram-negative commensal , which may be involved in the pathogenesis of CD . Both molecular and immunological approaches were used to identify the human receptor for the microbial antigen encoded by I2 , to characterize the ligand-receptor interactions , and to determine the three-dimensional structure of the microbial gene product . In particular , we show that the pfiT is a T cell superantigen , which may help to explain how microbial flora can trigger immune activation in IBD , and may provide the groundwork for novel therapies to treat CD .