Selective association of outer surface lipoproteins with the lipid rafts of Borrelia burgdorferi.
ABSTRACT: Borrelia burgdorferi contains unique cholesterol-glycolipid-rich lipid rafts that are associated with lipoproteins. These complexes suggest the existence of macromolecular structures that have not been reported for prokaryotes. Outer surface lipoproteins OspA, OspB, and OspC were studied for their participation in the formation of lipid rafts. Single-gene deletion mutants with deletions of ospA, ospB, and ospC and a spontaneous gene mutant, strain B313, which does not express OspA and OspB, were used to establish their structural roles in the lipid rafts. All mutant strains used in this study produced detergent-resistant membranes, a common characteristic of lipid rafts, and had similar lipid and protein slot blot profiles. Lipoproteins OspA and OspB but not OspC were shown to be associated with lipid rafts by transmission electron microscopy. When the ability to form lipid rafts in live B. burgdorferi spirochetes was measured by fluorescence resonance energy transfer (FRET), strain B313 showed a statistically significant lower level of segregation into ordered and disordered membrane domains than did the wild-type and the other single-deletion mutants. The transformation of a B313 strain with a shuttle plasmid containing ospA restored the phenotype shared by the wild type and the single-deletion mutants, demonstrating that OspA and OspB have redundant functions. In contrast, a transformed B313 overexpressing OspC neither rescued the FRET nor colocalized with the lipid rafts. Because these lipoproteins are expressed at different stages of the life cycle of B. burgdorferi, their selective association is likely to have an important role in the structure of prokaryotic lipid rafts and in the organism's adaptation to changing environments. IMPORTANCE Lipid rafts are cholesterol-rich clusters within the membranes of cells. Lipid rafts contain proteins that have functions in sensing the cell environment and transmitting signals. Although selective proteins are present in lipid rafts, little is known about their structural contribution to these domains. Borrelia burgdorferi, the agent of Lyme disease, has lipid rafts, which are novel structures in bacteria. Here, we have shown that the raft-associated lipoproteins OspA and OspB selectively contribute to lipid rafts. A similar but non-raft-associated lipoprotein, OspC, cannot substitute for the role of OspA and OspB. In this study, we have demonstrated that lipoprotein association with lipid rafts is selective, further suggesting a functional adaptation to different stages of the spirochete life cycle. The results of this study are of broader importance and can serve as a model for other bacteria that also possess cholesterol in their membranes and, therefore, may share lipid raft traits with Borrelia.
Project description:Borrelia burgdorferi, a Lyme disease agent, makes different major outer surface lipoproteins at different stages of its mouse-tick infectious cycle. Outer surface protein A (OspA) coats the spirochetes from the time they enter ticks until they are transmitted to a mammal. OspA is required for normal tick colonization and has been shown to bind a tick midgut protein, indicating that OspA may serve as a tick midgut adhesin. Tick colonization by spirochetes lacking OspA is increased when the infecting blood meal is derived from mice that do not produce antibody, indicating that OspA may protect the spirochetes from host antibody, which will not recognize tick-specific proteins such as OspA. To further study the importance of OspA during tick colonization, we constructed a form of B. burgdorferi in which the ospA open reading frame, on lp54, was replaced with the ospC gene or the ospB gene, encoding a mammal-specific or tick-specific lipoprotein, respectively. These fusions yielded a strain that produces OspC within a tick (from the fusion gene) and during early mammalian infection (from the normal ospC locus) and a strain that produces OspB in place of OspA within ticks. Here we show that the related, tick-specific protein OspB can fully substitute for OspA, whereas the unrelated, mammal-specific protein OspC cannot. These data were derived from three different methods of infecting ticks, and they confirm and extend previous studies indicating that OspA both protects spirochetes within ticks from mammalian antibody and serves an additional role during tick colonization.
Project description:Eukaryotic lipid rafts are membrane microdomains rich in cholesterol that contain a selective set of proteins, and have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft-associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC-MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules, and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures.
Project description:A mutant of virulent Borrelia burgdorferi 297 was apparently selected for by long-term storage at 5 degrees C. This mutant was found to lack the plasmid which encodes outer surface protein A (OspA) and OspB. In addition to the loss of the OspA and OspB proteins, the mutant lacked two lipoproteins, of 20 and 7.5 kDa, that were observed in the wild type. Since the mutant was not recovered from the tissues or blood of hamsters injected with the mutant, the mutant was determined to be noninfectious. Hamsters vaccinated with noninfectious mutant 297 were protected completely from challenge with virulent wild-type 297 spirochetes. Prechallenge sera from hamsters vaccinated with mutant 297 lacked antibodies to OspA and OspB, while those from hamsters vaccinated with virulent wild-type 297 or avirulent 297 exhibited antibodies to these proteins. Hamsters vaccinated with virulent wild-type 297 or mutant 297 elicited antibodies to OspC and a 39-kDa protein (P39), whereas hamsters vaccinated with avirulent 297 lacked these antibodies. These results suggest that OspC and/or P39 are important for the development of a protective immune response. Study of this mutant may elucidate factors important to the development of a Lyme disease vaccine.
Project description:To initiate infection, a microbial pathogen must be able to evade innate immunity. Here we show that the Lyme disease spirochete Borrelia burgdorferi depends on its surface lipoproteins for protection against innate defences. The deficiency for OspC, an abundantly expressed surface lipoprotein during early infection, led to quick clearance of B. burgdorferi after inoculation into the skin of SCID mice. Increasing expression of any of the four randomly chosen surface lipoproteins, OspA, OspE, VlsE or DbpA, fully protected the ospC mutant from elimination from the skin tissue of SCID mice; moreover, increased OspA, OspE or VlsE expression allowed the mutant to cause disseminated infection and restored the ability to effectively colonize both joint and skin tissues, albeit the dissemination process was much slower than that of the mutant restored with OspC expression. When the ospC mutant was modified to express OspA under control of the ospC regulatory elements, it registered only a slight increase in the 50% infectious dose than the control in SCID mice but a dramatic increase in immunocompetent mice. Taken together, the study demonstrated that the surface lipoproteins provide B. burgdorferi with an essential protective function against host innate elimination.
Project description:The gene of the immunodominant major protein pC of Borrelia burgdorferi was previously cloned and sequenced (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). pC is abundantly expressed on the outer surface of B. burgdorferi, as demonstrated by immunoelectron microscopy with monoclonal antibody L22 1F8. Accordingly, pC is renamed OspC, by analogy to the outer surface proteins OspA and OspB. Western immunoblot analysis of 45 B. burgdorferi isolates with monoclonal antibodies revealed that OspC is immunologically heterogeneous. Partial sequence analysis of the ospC gene confirmed the protein heterogeneity at the genetic level. We found that the degree of identity between the ospC partial sequences of five strains representing different OspA serotypes was only 63.3 to 85.4%. Immunological heterogeneity was also observed among representatives of the three newly designated genospecies of B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. garinii, and group VS461. Heterogeneity was confirmed for B. garinii at the genetic level. The ospC gene was also cloned from strains that did not express OspC, and antibody-reactive OspC was expressed in Escherichia coli. In addition, OspC-expressing variants were obtained from a nonexpressing strain by plating single colonies on solid medium. These findings confirm that the ospC gene is also present in nonexpressing strains. Because OspC is an immunodominant protein for the early immune response in Lyme borreliosis and was effective as a vaccine in an animal model, the immunological and molecular polymorphisms of ospC and OspC have important implications for the development of diagnostic reagents and vaccines.
Project description:Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.
Project description:Borrelia burgdorferi, the agent of Lyme disease, is unusual as it contains free cholesterol and cholesterol glycolipids. It is also susceptible to complement-independent bactericidal antibodies, such as CB2, a monoclonal IgG1 against outer surface protein B (OspB). We find that the bactericidal action of CB2 requires the presence of cholesterol glycolipids and cholesterol. Ultrastructural, biochemical, and biophysical analysis revealed that the bacterial cholesterol glycolipids exist as lipid raft-like microdomains in the outer membrane of cultured and mouse-derived B. burgdorferi and in model membranes from B. burgdorferi lipids. The order and size of the microdomains are temperature sensitive and correlate with the bactericidal activity of CB2. This study demonstrates the existence of cholesterol-containing lipid raft-like microdomains in a prokaryote, and we suggest that the temperature dependence of B. burgdorferi lipid raft organization may have significant implications in the transmission cycle of the spirochetes which are exposed to a range of temperatures.
Project description:The dimeric OspC/Vsp family surface lipoproteins of Borrelia spirochetes are crucial to the transmission and persistence of Lyme borreliosis and tick-borne relapsing fever. However, the requirements for their proper surface display remained undefined. In previous studies, we showed that localization of Borrelia burgdorferi monomeric surface lipoprotein OspA was dependent on residues in the N-terminal "tether" peptide. Here, site-directed mutagenesis of the B. burgdorferi OspC tether revealed two distinct regions affecting either release from the inner membrane or translocation through the outer membrane. Determinants of both of these steps appear consolidated within a single region of the Borrelia turicatae Vsp1 tether. Periplasmic OspC mutants still were able to form dimers. Their localization defect could be rescued by the addition of an apparently structure-destabilizing C-terminal epitope tag but not by coexpression with wild-type OspC. Furthermore, disruption of intermolecular Vsp1 salt bridges blocked dimerization but not surface localization of the resulting Vsp1 monomers. Together, these results suggest that Borrelia OspC/Vsp1 surface lipoproteins traverse the periplasm and the outer membrane as unfolded monomeric intermediates and assemble into their functional multimeric folds only upon reaching the spirochetal surface.
Project description:We report the cloning and characterization of two outer surface proteins (Osps), designated OspE and OspF, from strain N40 of Borrelia burgdorferi, the spirochetal agent of Lyme disease. The ospE and ospF genes are structurally arranged in tandem as one transcriptional unit under the control of a common promoter. The ospE gene, located at the 5' end of the operon, is 513 nucleotides in length and encodes a 171-amino-acid protein with a calculated molecular mass of 19.2 kDa. The ospF gene, located 27 bp downstream of the stop codon of the ospE gene, consists of 690 nucleotides and encodes a protein of 230 amino acids with a calculated molecular mass of 26.1 kDa. Pulsed-field gel electrophoresis showed that the ospE and ospF genes are located on a 45-kb plasmid. Comparison of the leader sequences of OspE and OspF with those of the four known B. burgdorferi Osps (OspA, OspB, OspC, and OspD) reveals a hydrophobic domain and a consensus cleavage sequence (L-X-Y-C) recognized by signal peptidase II, and [3H]palmitate labeling shows that OspE and OspF are lipoproteins. Immunofluorescence studies demonstrated that both the OspE and OspF proteins are surface exposed. These features are consistent with the finding that OspE and OspF are B. burgdorferi surface lipoproteins.
Project description:Oral vaccination strategies are of interest to prevent transmission of Lyme disease as they can be used to deliver vaccines to humans, pets, and to natural wildlife reservoir hosts of Borrelia burgdorferi. We developed a number of oral vaccines based in E. coli expressing recombinant OspC type K, OspB, BBK32 from B. burgdorferi, and Salp25, Salp15 from Ixodes scapularis. Of the five immunogenic candidates only OspC induced significant levels of antigen-specific IgG and IgA when administered to mice via the oral route. Antibodies to OspC did not prevent dissemination of B. burgdorferi as determined by the presence of spirochetes in ear, heart and bladder tissues four weeks after challenge. Next generation sequencing of genomic DNA from ticks identified multiple phyletic types of B. burgdorferi OspC (A, D, E, F, I, J, K, M, Q, T, X) in nymphs that engorged on vaccinated mice. PCR amplification of OspC types A and K from flat and engorged nymphal ticks, and from heart and bladder tissues collected after challenge confirmed sequencing analysis. Quantification of spirochete growth in a borreliacidal assay shows that both types of spirochetes (A and K) survived in the presence of OspC-K specific serum whereas the spirochetes were killed by OspA specific serum. We show that oral vaccination of C3H-HeN mice with OspC-K induced significant levels of antigen-specific IgG. However, these serologic antibodies did not protect mice from infection with B. burgdorferi expressing homologous or heterologous types of OspC after tick challenge.