CD1b presents self and Borrelia burgdorferi diacylglycerols to human T cells.
ABSTRACT: Lyme disease is a common multisystem disease caused by infection with a tick-transmitted spirochete, Borrelia burgdorferi and related Borrelia species. The monoglycosylated diacylglycerol known as B. burgdorferi glycolipid II (BbGL-II) is a major target of antibodies in sera from infected individuals. Here, we show that CD1b presents BbGL-II to human T cells and that the TCR mediates the recognition. However, we did not detect increased frequency of CD1b-BbGL-II binding T cells in the peripheral blood of Lyme disease patients compared to controls. Unexpectedly, mapping the T cell specificity for BbGL-II-like molecules using tetramers and activation assays revealed a concomitant response to CD1b-expressing APCs in absence of BbGL-II. Further, among all major classes of self-lipid tested, BbGL-II responsive TCRs show strong cross-reactivity to diacylglycerol, a self-lipid antigen with structural similarities to BbGL-II. Extending prior work on MHC and CD1b, CD1c, and CD1d proteins, this study provides evidence for cross-reactive CD1b-restricted T cell responses to bacterial and self-antigens, and identifies chemically defined targets for future discovery of self and foreign antigen cross-reactive T cells.
Project description:Unlike the dominant role of one class II invariant chain peptide (CLIP) in blocking MHC class II, comparative lipidomics analysis shows that human cluster of differentiation (CD) proteins CD1a, CD1b, CD1c, and CD1d bind lipids corresponding to hundreds of diverse accurate mass retention time values. Although most ions were observed in association with several CD1 proteins, ligands binding selectively to one CD1 isoform allowed the study of how differing antigen-binding grooves influence lipid capture. Although the CD1b groove is distinguished by its unusually large volume (2,200 Å(3)) and the T' tunnel, the average mass of compounds eluted from CD1b was similar to that of lipids from CD1 proteins with smaller grooves. Elution of small ligands from the large CD1b groove might be explained if two small lipids bind simultaneously in the groove. Crystal structures indicate that all CD1 proteins can capture one antigen with its hydrophilic head group exposed for T-cell recognition, but CD1b structures show scaffold lipids seated below the antigen. We found that ligands selectively associated with CD1b lacked the hydrophilic head group that is generally needed for antigen recognition but interferes with scaffold function. Furthermore, we identified the scaffolds as deoxyceramides and diacylglycerols and directly demonstrate a function in augmenting presentation of a small glycolipid antigen to T cells. Thus, unlike MHC class II, CD1 proteins capture highly diverse ligands in the secretory pathway. CD1b has a mechanism for presenting either two small or one large lipid, allowing presentation of antigens with an unusually broad range of chain lengths.
Project description:The appearance of group 1 CD1 proteins (CD1a, CD1b and CD1c) on maturing myeloid DC is a key event that converts myeloid DC to effective lipid APC. Here, we show that Borrelia burgdorferi, the causative agent of Lyme disease, triggers appearance of group 1 CD1 proteins at high density on the surface of human myeloid DC during infection. Within human skin, CD1b and CD1c expression was low or absent prior to infection, but increased significantly after experimental infections and in erythema migrans lesions from Lyme disease patients. The induction of CD1 was initiated by borrelial lipids acting through TLR-2 within minutes, but required 3 days for maximum effect. The delay in CD1 protein appearance involved a multi-step process whereby TLR-2 stimulated cells release soluble factors, which are sufficient to transfer the CD1-inducing effect in trans to other cells. Analysis of these soluble factors identified IL-1? as a previously unknown pathway leading to group 1 CD1 protein function. This study establishes that upregulation of group 1 CD1 proteins is an early event in B. burgdorferi infection and suggests a stepwise mechanism whereby bacterial cell walls, TLR activation and cytokine release cause DC precursors to express group 1 CD1 proteins.
Project description:The role of bats in the enzootic cycle of Lyme disease and relapsing fever-causing bacteria is a matter of speculation. In Canada, Borrelia burgdorferi sensu stricto (ss) is the genospecies that is responsible for most cases of Lyme disease in humans. In this study, we determined if big brown bats, Eptesicus fuscus, have been exposed to spirochetes from the genus Borrelia. We collected serum from 31 bats and tested them for the presence of anti-Borrelia burgdorferi antibodies using a commercial enzyme-linked immunosorbent assay (ELISA). We detected cross-reactive antibodies to Borrelia spp. in 14 of 31 bats. We confirmed the ELISA data using a commercial immunoblot assay. Pooled sera from ELISA-positive bats also cross-reacted with Borrelia antigens coated on the immunoblot strips, whereas pooled sera from ELISA-negative bats did not bind to Borrelia spp. antigens. Furthermore, to identify if bat ectoparasites, such as mites, can carry Borrelia spp., we analyzed DNA from 142 bat ectoparasites that were collected between 2003 and 2019. We detected DNA for the Borrelia burgdorferi flaB gene in one bat mite, Spinturnix americanus. The low detection rate of Borrelia burgdorferi DNA in bat ectoparasites suggests that bats are not reservoirs of this bacterium. Data from this study also raises intriguing questions about Borrelia infections in bats, including the role of humoral immunity and the ability of bats to be infected with Borrelia burgdorferi. This study can lead to more sampling efforts and controlled laboratory studies to identify if bats can be infected with Borrelia burgdorferi and the role of bat ectoparasites, such as S. americanus, in the transmission of this spirochete. Furthermore, we outlined reagents that can be used to adapt ELISA kits and immunoblot strips for use with bat sera.
Project description:Multiple circular and linear plasmids of Lyme disease and relapsing fever Borrelia spirochetes carry genes for members of the Bdr (Borrelia direct repeat) protein family. To define their common and divergent attributes, we first comprehensively compared the known homologs. Bdr proteins with predicted sizes ranging from 10.7 to 30. 6 kDa formed five homology groups, based on variable numbers of short direct repeats in a central domain and diverse N- and C-terminal domains. In a further characterization, Western blots were probed with rabbit antisera raised against either of two purified recombinant Bdr proteins from Borrelia burgdorferi B31. The results showed that antibodies cross-react and several Bdr paralogs 19.5 to 30.5 kDa in size are expressed by cultured strain B31 in a temperature-independent manner. In situ proteolysis, immunofluorescence, and growth inhibition assays indicated that Bdr proteins are not surface exposed. Distinct patterns of cross-reacting proteins of 17.5 to 33 kDa were also detected in other B. burgdorferi, Borrelia garinii, and Borrelia afzelii strains as well as in relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae. Last, we examined whether these proteins are antibody targets during Lyme disease. Analysis of 47 Lyme disease patient sera by immunoblotting and enzyme-linked immunosorbent assays showed that 24 (51%) and 20 (43%), respectively, had detectable antibodies to one or more of the Bdr proteins. Together, these data indicate that Bdr proteins constitute a family of cross-reactive Borrelia proteins which are expressed in the course of Lyme disease and in vitro.
Project description:The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.
Project description:Laboratory diagnosis of Lyme disease is based on the serological detection of antibodies against the etiologic agent Borrelia burgdorferi. Current diagnostics are insensitive at detecting early infection, when treatment is most effective. This deficiency results from the limited number of B. burgdorferi antigens expressed in early infection and the use of an insensitive two-tier paradigm, put in place to deal with insufficient specificity associated with the use of whole-protein antigens and/or bacterial lysates as serodiagnostic targets. Whole-protein antigens contain epitopes that are unique to B. burgdorferi as well as cross-reactive epitopes found in other bacteria. One method for overcoming the limitations imposed by cross-reactive epitopes is the use of short peptides containing epitopes unique to B. burgdorferi as antigen targets. This eliminates nonspecific epitopes. Using overlapping peptide libraries, we performed epitope mapping of linear epitopes in oligopeptide permease A2 (OppA2), a member of the oligopeptide permease (Opp) family of peptide transporters, expressed during early B. burgdorferi infection. We identified 9 epitopes, synthesized peptides containing these epitopes, and screened those using panels of blood from patients with early Lyme disease, rheumatoid arthritis (RA), or syphilis or from healthy individuals. Two of the peptides, OppA2 (191-225) (amino acids comprising the peptide are shown in parentheses) and OppA2 (381-400), are highly conserved among the three major pathogenic Borrelia species responsible for most Lyme disease cases in North America and Europe. They detected antibodies in Lyme disease patient sera with sufficient sensitivity and specificity to indicate that they could have value in a serological assay for Lyme disease.
Project description:A growing global health concern, Lyme disease has become the most common tick-borne disease in the United States and Europe. Caused by the bacterial spirochete Borrelia burgdorferi sensu lato (sl), this disease can be debilitating if not treated promptly. Because diagnosis is challenging, prevention remains a priority; however, a previously licensed vaccine is no longer available to the public. Here, we designed a six component vaccine that elicits antibody (Ab) responses against all Borrelia strains that commonly cause Lyme disease in humans. The outer surface protein A (OspA) of Borrelia was fused to a bacterial ferritin to generate self-assembling nanoparticles. OspA-ferritin nanoparticles elicited durable high titer Ab responses to the seven major serotypes in mice and non-human primates at titers higher than a previously licensed vaccine. This response was durable in rhesus macaques for more than 6 months. Vaccination with adjuvanted OspA-ferritin nanoparticles stimulated protective immunity from both B. burgdorferi and B. afzelii infection in a tick-fed murine challenge model. This multivalent Lyme vaccine offers the potential to limit the spread of Lyme disease.
Project description:Epitope mapping of the p66 outer membrane protein of Borrelia burgdorferi revealed that the protein contains numerous cross-reactive linear epitopes recognized by serum antibody in the majority of individuals tested, regardless of Lyme disease history, limiting the usefulness of this antigen in Lyme disease serodiagnostic assays.
Project description:Borrelia burgdorferi sensu lato is a group of spirochetes belonging to the genus Borrelia in the family of Spirochaetaceae. The spirochete is transmitted between reservoirs and hosts by ticks of the family Ixodidae. Infection with B. burgdorferi in humans causes Lyme disease or Lyme borreliosis. Currently, 20 Lyme disease-associated Borrelia species and more than 20 relapsing fever-associated Borrelia species have been described. Identification and differentiation of different Borrelia species and strains is largely dependent on analyses of their genetic characteristics. A variety of molecular techniques have been described for Borrelia isolate speciation, molecular epidemiology, and pathogenicity studies. In this unit, we focus on three basic protocols, PCR-RFLP-based typing of the rrs-rrlA and rrfA-rrlB ribosomal spacer, ospC typing, and MLST. These protocols can be employed alone or in combination for characterization of B. burgdorferi isolates or directly on uncultivated organisms in ticks, mammalian host reservoirs, and human clinical specimens.
Project description:Since Lyme arthritis was first described in the United States, it has now been reported in many countries of Europe. However, very few strains of the causative bacterium, Borrelia burgdorferi, have been isolated from synovial samples. For this reason, different molecular direct typing methods were developed recently to assess which species could be involved in Lyme arthritis in Europe. We developed a simple oligonucleotide typing method with PCR fragments from the flagellin gene of B. burgdorferi sensu lato, which is able to differentiate seven different Borrelia species. Among 10 consecutive PCR-positive patients with Lyme arthritis from the northeastern France, two species were identified in synovial samples: B. burgdorferi sensu stricto in 9 cases and B. garinii in 1 case. Conversely, all B. burgdorferi sensu lato species detected in 10 consecutive PCR-positive biopsies from a second set of patients with erythema migrans from the same geographical area were identified as either B. afzelii or B. garinii (P < 0.001). These results indicate that B. burgdorferi sensu stricto is the principal but not the only Borrelia species involved in Lyme arthritis in northeastern France.