Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases

The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease. We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8(+) cells both in a model of HLA-B27 transgenic mice and in patients. This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system. After preselection, immunodominant peptides were identified by Ag-specific flow cytometry. Using this approach we have identified for the first time nine peptides derived from different C. trachomatis proteins that are stimulatory for CD8(+) T cells. Eight of these nine murine-derived peptides were recognized by cytotoxic T cells. The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis. Eleven peptides were found to be stimulatory for patient-derived CD8(+) T cells, of which eight overlapped those found in mice. Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8(+) T cells in patients with Chlamydia-induced arthritis. This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.     

Endogenous Processing and Presentation of T-cell Epitopes from Chlamydia trachomatis with Relevance in HLA-B27-associated Reactive Arthritis

Chlamydia trachomatis triggers reactive arthritis, a spondyloarthropathy linked to the human major histocompatibility complex molecule HLA-B27, through an unknown mechanism that might involve molecular mimicry between chlamydial and self-derived HLA-B27 ligands. Chlamydia-specific CD8⁺ T-cells are found in reactive arthritis patients, but the immunogenic epitopes are unknown. A previous screening of the chlamydial genome for putative HLA-B27 ligands predicted multiple peptides that were recognized in vitro by CD8⁺ T-lymphocytes from patients. Here stable transfectants expressing bacterial fusion proteins in human cells were generated to investigate the endogenous processing and presentation by HLA-B27 of two such epitopes through comparative immunoproteomics of HLA-B27-bound peptide repertoires. A predicted T-cell epitope, from the CT610 gene product, was presented by HLA-B27. This is, to our knowledge, the first endogenously processed epitope involved in HLA-B27-restricted responses against C. trachomatis in reactive arthritis. A second predicted epitope, from the CT634 gene product, was not detected. Instead a non-predicted nonamer from the same protein was identified. Both bacterial peptides showed very high homology with human sequences containing the HLA-B27 binding motif. Thus, expression and intracellular processing of chlamydial proteins into human cells allowed us to identify two bacterial HLA-B27 ligands, including the first endogenous T-cell epitope from C. trachomatis involved in spondyloarthropathy. That human proteins contain sequences mimicking chlamydial T-cell epitopes suggests a basis for an autoimmune component of Chlamydia-induced HLA-B27-associated disease.Chlamydia trachomatis is an obligate intracellular parasite that infects the urogenital epithelium. It is a very common pathogen and one frequently inducing reactive arthritis (ReA)¹ (1). Multiple strategies, including down-regulation of major histocompatibility complex (MHC) class I and class II expression (2–4) and persistence, have been developed by the bacteria to evade the immune system. Yet both CD4⁺ and CD8⁺ T-cell responses are activated upon infection (5). In particular, HLA-B27-restricted CD8⁺ T-lymphocytes are found in patients with Chlamydia-induced ReA (6, 7). The role of these cells in the pathogenesis and evolution of ReA to chronic disease is probably mediated by IFN-γ. Secretion of this cytokine is critical for the protective immunity against Chlamydia (8) because it inhibits the bacterial growth (9). However, this is often insufficient to promote complete clearance of C. trachomatis, and actually IFN-γ-induced depletion of the tryptophan pool induces the differentiation of the metabolically active reticular bodies to persistent forms (10), which sustain chronic infection and ReA. The high prevalence of HLA-B27 among patients with Chlamydia-induced ReA (11), especially in its chronic form, suggests a pathogenetic mechanism based on interactive effects of the bacteria and HLA-B27 that seems unrelated to the capacity of C. trachomatis to infect or replicate into HLA-B27-positive cells (12). One such mechanism could be T-cell-mediated autoimmunity elicited by molecular/antigenic mimicry between chlamydial and self-derived HLA-B27 ligands. Antigenic mimicry between chlamydial and homologous α-myosin-derived peptides is crucial to inducing autoimmune myocarditis in mice (13). Breakdown of cytotoxic T-lymphocyte (CTL) tolerance to HLA-B27 was observed in transgenic rats upon exposure to C. trachomatis (14). Cross-reactivity between HLA-B27-restricted Chlamydia-specific CTL with self-derived HLA-B27 epitopes has not been reported. However, a biochemical basis for it was provided by the finding of an endogenously processed and presented peptide from the DNA primase of C. trachomatis with high homology to a self-derived HLA-B27 ligand (15, 16).Because of the likely involvement of HLA-B27 in the pathogenesis of chronically evolving ReA, the role of CD8⁺ T-cell responses in the protective immunity against C. trachomatis and the presence of HLA-B27-restricted T-cells in patients with Chlamydia-induced ReA, the identification of relevant chlamydial epitopes becomes crucial to establish the pathogenetic mechanism of this disease. Unfortunately a direct analysis of chlamydial HLA-B27 ligands expressed on infected cells is exceedingly difficult because of their extremely low amounts, which challenge even the most sensitive techniques of MS. In the case of Chlamydia, the situation is further complicated by the down-regulation of MHC class I expression shortly after infection (3, 4). To our knowledge, only one MHC class I ligand was recently identified, in the mouse system, from Chlamydia muridarum-infected cells using state-of-the-art MS techniques (17). Due in part to this difficulty, alternative approaches, such as expression cloning and synthetic peptide epitope mapping (18, 19) or MHC class I tetramer arrays (20), have been used to identify MHC class I-restricted chlamydial T-cell epitopes in mice. In a previous study (6) predictive algorithms were used to screen the whole genome of C. trachomatis for nonamer peptide sequences containing the HLA-B*2705 binding motif and a high probability of being generated by proteasomal cleavage. This led to identifying multiple sequences that, when used as synthetic peptides in vitro, stimulated CD8⁺ T-cells from patients with Chlamydia-induced ReA. Such cells could also be detected in the synovial fluid of these patients using HLA-B27 tetramers complexed to some of these peptides (7).Although these strategies identify chlamydial sequences that are recognized by CD8⁺ T-cells they do not prove that these peptides are the endogenously processed epitopes that activated the natural T-cell responses to the bacteria in vivo. Because of the intrinsic cross-reactivity of T-cells (21, 22), it is conceivable that synthetic peptides recognized in vitro may be different from the natural epitopes generated by endogenous processing of the chlamydial proteins that elicit the HLA-B27-restricted T-cell responses in ReA patients. To investigate this issue we focused on two predicted epitopes (6). Stable transfectants expressing the corresponding chlamydial proteins fused to green fluorescent protein (GFP) were generated in a B*2705-positive cell line. The endogenous processing and presentation of the predicted epitopes or other peptides from the same bacterial protein were analyzed by comparative immunoproteomics analysis of the B*2705-bound peptide repertoires from transfected and untransfected cells and sequencing of peptides differentially presented on the bacterial protein transfectant.     

Aetiology and pathogenesis of reactive arthritis: role of non-antigen-presenting effects of HLA-B27

Spondyloarthropathies are inflammatory diseases closely associated with human leukocyte antigen (HLA)-B27 by unknown mechanisms. One of these diseases is reactive arthritis (ReA), which is typically triggered by Gram-negative bacteria, which have lipopolysaccharide as an integral component of their outer membrane. Several findings in vivo and in vitro obtained from patients with ReA and from different model systems suggest that HLA-B27 modulates the interaction between ReA-triggering bacteria and immune cells by a mechanism unrelated to the antigen presentation function of HLA-B27. In this review we piece together a jigsaw puzzle from the new information obtained from the non-antigen-presenting effects of HLA-B27.     

Epidemiology of sexually transmitted Chlamydia trachomatis infections in male patients

868 male urethritis patients were studied for the presence of chlamydiae in the 1981-1986 period. 36% of NGU, 31.3% of gonococcal urethritis and 58.8% of PGU urethritis patients presented C. trachomatis infections as detected by cell culture inoculation. Chlamydial infection was recorded more often in the 21-30 years age group (30.8%), in unmarried patients (70.6%), as well as in people with low educational degree (46.6% of cases), 42.3% of the C. trachomatis cases were already confronted with one or more urethritis episodes. 24.7% of patients have been subjected to a previous antichlamydial treatment.     

Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na⁺-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27⁺ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.     

Detection and frequency of Chlamydia trachomatis DNA in synovial samples from Tunisian patients with reactive arthritis and undifferentiated oligoarthritis

We aimed to determine the frequency of Chlamydia trachomatis DNA in the synovial compartment of 34 arthritic patients. Chlamydia trachomatis DNA was detected using a nested PCR targeting the cryptic plasmid, the 16S rRNA gene and the outer membrane protein 1 gene. The presence of serum immunoglobulin (Ig)G and IgA antibodies against C. trachomatis was studied by a microimmunofluorescence assay and by an enzyme-linked immunosorbent assay, respectively. Synovial samples from 20 of 34 (59%) patients [nine with reactive arthritis (ReA), seven with undifferentiated oligoarthritis (UOA), two with rheumatoid arthritis and two with osteoarthritis] were positive for at least one C. trachomatis DNA sequence by nested PCR. The high sensitivity results most likely from the combination of a standardized automated MagNA Pure extraction method, PCR targeting three different C. trachomatis genes and the screening for C. trachomatis in synovial tissue and fluid samples. There was no correlation between the presence of C. trachomatis DNA in the joint and a Chlamydia -specific serologic response. Our data support that PCR is the method of choice to establish the diagnosis of Chlamydia -induced arthritis in patients with ReA. We suggest that this diagnosis might also be considered in C. trachomatis -positive patients previously classified as UOA.     

Identification of endogenously presented peptides from Chlamydia trachomatis with high homology to human proteins and to a natural self-ligand of HLA-B27

A strategy for the stable expression of proteins, or large protein fragments, from Chlamydia trachomatis into human cells was designed to identify bacterial epitopes endogenously processed and presented by HLA-B27. Fusion protein constructs in which the green fluorescent protein gene was placed at the 5'-end of the bacterial DNA primase gene or some of its fragments were transfected into B*2705-C1R cells. One of these constructs, including residues 90-450 of the bacterial protein, was stably and efficiently expressed. Mass spectrometry-based comparative analysis of HLA-B27-bound peptide pools led to identification of three HLA-B27 ligands differentially presented in the transfectant cells. Sequencing of these peptides confirmed that they were derived from the bacterial DNA primase. One of them, spanning residues 211-221, showed 55% sequence identity with a known self-ligand of HLA-B27 derived from its own molecule. The other two bacterial ligands, P-(112-121) and P-(112-122), were derived from the same region and differed in length by one residue at the C terminus. Both peptides showed >50% identity with multiple human protein sequences that possessed the optimal peptide motifs for HLA-B27 binding. Thus, expression of proteins from arthritogenic bacteria in HLA-B27-positive human cells allows identifying bacterial peptides that are endogenously processed and presented by HLA-B27 and show molecular mimicry with known self-ligands of this molecule and human proteins.     

Chlamydia trachomatis infections in infants.

In recent years considerable progress has been made in understanding chlamydial infections. The spectrum of pediatric Chlamydia trachomatis infection includes neonatal inclusion conjunctivitis, infantile pneumonia, occasional respiratory or genital tract infections in older children and sexually transmitted diseases in adolescents. The role of maternal chlamydial infection in prematurity and in perinatal death is currently an area of active study. We outline the current knowledge of the biologic characteristics of C. trachomatis, the epidemiologic features of chlamydial infection, and the clinical aspects, diagnosis and treatment of neonatal chlamydial infections.     

Common intra-articular T cell expansions in patients with reactive arthritis: identical beta-chain junctional sequences and cytotoxicity toward HLA-B27

Spondyloarthropathies constitute a group of autoimmune diseases of special interest because of their tight association with the MHC class I molecule HLA-B27 and the bacterial triggering of some clinical forms called reactive arthritis (ReA). One current hypothesis is the presentation by HLA-B27 of a so-called arthritogenic peptide to T cells. To better focus on the relevant T cell populations within the joint, we performed an extensive beta-chain T cell repertoire analysis of synovial fluid compared with PBL in seven patients, four of whom were characterized as having ReA triggered by Yersinia enterocolitica, Chlamydia trachomatis, or Shigella sonnei. Analysis of the size diversity of the beta-chain complementarity-determining region 3 (CDR3) allowed us to evaluate the degree of T cell clonality in the samples. Oligoclonal T cell expansions were frequently observed in the joint. In one patient, CDR3 amino acid sequences of major expansions using two different BV genes were identical. One dominant T cell expansion and several CDR3 amino acid sequences were identical in two different patients. Furthermore, one sequence was identical with a sequence reported independently in a Salmonella-induced ReA patient. Together, these data indicate a surprisingly high degree of conservation in the T cell responses in recent-onset ReA triggered by different micro-organisms. A CD8+ synovial line expressing shared clonotypes was established and reacted toward several B*2705 lymphoblastoid cell lines, therefore supporting a molecular mimicry phenomenon at the T cell level in the disease mechanism.     

Role of binding pockets for amino-terminal peptide residues in HLA-B27 allorecognition.

The peptide binding site of HLA-B27 and other class I Ag consists of a series of pockets that bind peptide side chains. Two of these pockets interact with the amino-terminal peptide residue (pocket A) and with the highly conserved second residue (pocket B). In this study, the role of pockets A and B in HLA-B27-specific T cell allorecognition has been analyzed. Four HLA-B27 mutants with single or double changes in pocket B (24T----A, 45E----M, 67C----V, and 24,67T,C----A,V) and three mutants with single changes in pocket A (163E----T, 167W----S, and 171Y----H) were constructed by site-directed mutagenesis and expressed in HMy2.C1R cells after DNA-mediated gene transfer. These transfectants were used as target cells in cytotoxicity assays with a series of HLA-B27-specific CTL. All the mutations analyzed affected allorecognition by a significant proportion of the CTL tested, but no single change abrogated recognition by all CTL. The global effects of each mutation on allorecognition were comparable to one another, except for the effect of the change at position 67, which was smaller. The behavior of individual CTL with the mutants was very diverse, ranging from CTL that did not recognize most of the mutants to CTL recognizing all of them. Thus, some alloreactive CTL can withstand drastic alterations in pockets A and B. Two CTL showed heteroclytic effects towards the V67 and M45 mutants. CTL behavior with the H171 mutant was closely parallel to that with the B*2703 subtype, having a single Y----H change at position 59. This parallelism correlates with the similar role of Tyr59 and Tyr171 in establishing hydrogen bonds with the amino termini of HLA-B27-bound peptides. The results demonstrate that altering the structure of pockets that interact with the amino-terminal first and second residues of HLA-B27-bound peptides significantly affects recognition by alloreactive CTL, and they strongly suggest widespread peptide involvement in HLA-B27 allorecognition.     

Chlamydia trachomatis: identification of susceptibility markers for ocular and sexually transmitted infection by immunogenetics

The aim of this review is to present a concise overview of all data available on the immunogenetics of Chlamydia trachomatis infections, both sexually transmitted urogenital and ocular infections. Currently, candidate gene approaches are used to identify genes related to the susceptibility to and severity of C. trachomatis infections. The main focus in the review will be on data obtained by the study of human cohorts.     

Zinc and Chlamydia trachomatis

Zinc was noted to have significant effects upon the infection of McCoy cells by each of two strains of Chlamydia trachomatis. With a high or low Chlamydia inoculant, the number of infected cells increased up to 200% utilizing supplemental zinc (up to 1 X 10(-4) M) in the inoculation media compared with standard Chlamydia cultivation media (8 X 10(-6) M zinc). Ferric chloride and calcium chloride did not effect any such changes. Higher concentrations of zinc, after 2 hr of incubation with Chlamydia, significantly decreased the number of inclusions. This direct effect of zinc on the Chlamydia remained constant after further repassage of the Chlamydia without supplemental zinc, suggesting a lethal effect of the zinc. Supplemental zinc (up to 10(-4)M) may prove to be a useful addition to inoculation media to increase the yield of culturing for Chlamydia trachomatis. Similarly, topical or oral zinc preparations used by people may alter their susceptivity to Chlamydia trachomatis infections.     

Breakdown of CTL tolerance to self HLA-B*2705 induced by exposure to Chlamydia trachomatis

There is a strong association between seronegative arthritis and HLA B27, but it is still unresolved whether the contribution of B27 to disease pathogenesis is solely as a restriction element for an arthritogenic peptide, or whether B27 itself serves as an autoantigen. This study uses transgenic rats to address the question as to whether exposure to an arthritogenic pathogen can alter tolerance to B27. Unlike their nontransgenic counterparts, B27-transgenic rats are tolerant of B27 immunization using either B27(+) splenocytes or plasmid DNA and do not develop anti-B27 CTL. However, if splenocytes from such immunized animals are exposed to Chlamydia in vitro, CTL are generated that lyse B27(+) targets. No killing was seen with targets transfected with control B7, B14, B40, or B44. This phenomenon was not observed with immunization by nontransgenic splenocytes, or HLA-A2 DNA alone. Using targets expressing mutated B27, we show that the epitope for autoreactive CTL recognition of B27 involves the Lys(70) amino acid residue in the alpha(1) domain of the MHC class I molecule. The generation of CTL with specificity for B27 under these conditions demonstrates that tolerance to B27 can be subverted by CHLAMYDIA: This indicates a dynamic interrelationship between the pathogen and B27, which may have important implications for B27-related spondyloarthropathies triggered by intracellular bacteria.     

The glycyl-tRNA synthetase of Chlamydia trachomatis.

Aminoacyl-tRNA synthetases specifically charge tRNAs with their cognate amino acids. A prototype for the most complex aminoacyl-tRNA synthetases is the four-subunit glycyl-tRNA synthetase from Escherichia coli, encoded by two open reading frames. We examined the glycyl-tRNA synthetase gene from Chlamydia trachomatis, a genetically isolated bacterium, and identified only a single open reading frame for the chlamydial homolog (glyQS). This is the first report of a prokaryotic glycyl-tRNA synthetase encoded by a single gene.