Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.
The diversity of peptides displayed by class I human leukocyte antigen (HLA) plays an essential role in T cell immunity. The peptide repertoire is extended by various posttranslational modifications, including proteasomal splicing of peptide fragments from distinct regions of an antigen to form nongenomically templated cis-spliced sequences. Previously, it has been suggested that a fraction of the immunopeptidome constitutes such cis-spliced peptides; however, because of computational limitations, it has not been possible to assess whether trans-spliced peptides (i.e., the fusion of peptide segments from distinct antigens) are also bound and presented by HLA molecules, and if so, in what proportion. Here, we have developed and applied a bioinformatic workflow and demonstrated that trans-spliced peptides are presented by HLA-I, and their abundance challenges current models of proteasomal splicing that predict cis-splicing as the most probable outcome. These trans-spliced peptides display canonical HLA-binding sequence features and are as frequently identified as cis-spliced peptides found bound to a number of different HLA-A and HLA-B allotypes. Structural analysis reveals that the junction between spliced peptides is highly solvent exposed and likely to participate in T cell receptor interactions. These results highlight the unanticipated diversity of the immunopeptidome and have important implications for autoimmunity, vaccine design, and immunotherapy.
Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis and Goodpasture’s disease, is associated with particular Human Leukocyte Antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigated the molecular mechanism of Goodpasture’s disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T cell self-epitope derived from the α3 chain of Type IV collagen (α3135-145)1–4. While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR152. We show that autoreactive α3135-145-specific T cells expand in patients with Goodpasture’s disease and, in α3135-145-immunized HLA-DR15 transgenic mice, α3135-145-specific T cells infiltrate the kidney and mice develop Goodpasture’s disease. HLA-DR15 and HLA-DR1 exhibited distinct peptide repertoires and binding preferences and presented the α3135-145 epitope in different binding registers. HLA-DR15-α3135-145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLA-DR1-α3135-145 tetramer+ T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4+Foxp3+ regulatory T cells (Tregs) expressing tolerogenic cytokines. HLA-DR1-induced Tregs confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15+ and HLA-DR1+ healthy human donors displayed altered α3135-145-specific TCR usage, HLA-DR15-α3135-145 tetramer+ Foxp3− Tconv and HLA-DR1-α3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes, and patients with Goodpasture’s disease display a clonally expanded α3135-145-specific CD4+ T cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative abundance of self-epitope specific Tregs that leads to protection or causation of autoimmunity.
T cell receptors (TCRs) recognize antigens presented by major histocompatibility complex (MHC) and MHC class I–like molecules. We describe a diverse population of human γδ T cells isolated from peripheral blood and tissues that exhibit autoreactivity to the monomorphic MHC-related protein 1 (MR1). The crystal structure of a γδTCR–MR1–antigen complex starkly contrasts with all other TCR–MHC and TCR–MHC-I-like complex structures. Namely, the γδTCR binds underneath the MR1 antigen-binding cleft, where contacts are dominated by the MR1 α3 domain. A similar pattern of reactivity was observed for diverse MR1-restricted γδTCRs from multiple individuals. Accordingly, we simultaneously report MR1 as a ligand for human γδ T cells and redefine the parameters for TCR recognition.
We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.DOI: http://dx.doi.org/10.7554/eLife.07661.001
The HLA-DRB1 locus is strongly associated with rheumatoid arthritis (RA) susceptibility, whereupon citrullinated selfpeptides bind to HLA-DR molecules bearing the shared epitope (SE) amino acid motif. However, the differing propensity for citrullinated/noncitrullinated self-peptides to bind given HLA-DR allomorphs remains unclear. Here, we used a fluorescence polarisation assay to determine a hierarchy of binding affinities of 34 self-peptides implicated in RA against three HLA-DRB1 allomorphs (HLA-DRB1*04:01/*04:04/*04:05) each possessing the SE motif. For all three HLA-DRB1 allomorphs, we observed a strong correlation between binding affinity and citrullination at P4 of the bound peptide ligand. A differing hierarchy of peptide-binding affinities across the three HLA-DRB1 allomorphs was attributable to the b-chain polymorphisms that resided outside the SE motif and were consistent with sequences of naturally presented peptide ligands. Structure determination of eight HLA-DR4-self-epitope complexes revealed strict conformational convergence of the P4-Cit and surrounding HLA b-chain residues. Polymorphic residues that form part of the P1 and P9 pockets of the HLA-DR molecules provided a structural basis for the preferential binding of the citrullinated selfpeptides to the HLA-DR4 allomorphs. Collectively, we provide a molecular basis for the interplay between citrullination of self-antigens and HLA polymorphisms that shape peptide-HLA-DR4 binding affinities in RA. Sources for the primary RA-associated autoantigens may be from the site of disease including articular cartilage and synovial fluids (12-14) but others may be derived from blood plasma or surrounding mucosal tissues that are susceptible to inflammation (2). These proteins could undergo PTMs during numerous physiologic processes including infection, apoptosis and cellular stress. Some of the best-characterised autoantigens that bind ACPAs are citrullinated vimentin, fibrinogen, α-enolase and Type II collagen, which are present at high levels in the joint synovium (3,15).One of the key inherited risk factors that contribute to ACPA positive RA is the human leukocyte antigen (HLA) class II loci, namely HLA-DRB1, which encodes the HLA class II antigen presenting molecules (16-21). The antigen-binding groove of the HLA class II molecule can accommodate peptide ligands that vary in length, but the main pockets that interact most strongly with the bound peptide are P1, P4, P6, P7 and P9, which can accommodate the side chains of the peptide residues 1, 4, 6, 7 and 9 (22,23). A conserved amino acid sequence QKRAA, QRRAA, or RRRAA in position 70-74 of the HLA-DRB1 chain, known as the shared epitope (SE) motif, is highly prevalent (~90%) among ACPA seropositive patients (11,16). This SE motif defines the P4 pocket of the high-risk HLA-DRB1 RA-associated allomorphs. Subsequent GWAS studies have shown that two polymorphisms encoding β-chain residues at positions 11 and 13 at the base of the P4 pocket are also strongly associated with RA susceptibility (16)....
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