LuxS-mediated quorum sensing in Borrelia burgdorferi,the lyme disease spirochete

The establishment of Borrelia burgdorferi infection involves numerous interactions between the bacteria and a variety of vertebrate host and arthropod vector tissues. This complex process requires regulated synthesis of many bacterial proteins. We now demonstrate that these spirochetes utilize a LuxS/autoinducer-2 (AI-2)-based quorum-sensing mechanism to regulate protein expression, the first system of cell-cell communication to be described in a spirochete. The luxS gene of B. burgdorferi was identified and demonstrated to encode a functional enzyme by complementation of an Escherichia coli luxS mutant. Cultured B. burgdorferi responded to AI-2 by altering the expression levels of a large number of proteins, including the complement regulator factor H-binding Erp proteins. Through this mechanism, a population of Lyme disease spirochetes may synchronize production of specific proteins needed for infection processes.     

Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus ticks removed from humans

A total of 131 Ixodes ricinus (51 females, 1 male and 79 nymphs) removed from persons living in Southern Germany were investigated by immunofluorescence assay for the presence of Borrelia burgdorferi with a polyvalent rabbit immune serum and monoclonal antibodies specific for outer surface proteins (Osp) A or C. Borreliae were detectable in 48 (36.6%) of the ticks. Infection rates of these adults and nymphs were significantly higher than infection rates of unfed ticks from Southern Germany. Borreliae in 31.3% (n = 15) of the infected ticks expressed solely OspA, solely OspC in 12.5% (n = 6), and both OspA and OspC in 39.6% (n = 19) of ticks, while in 16.7% (n = 8) of ticks neither were expressed. Presentation of OspC by B. burgdorferi in I. ricinus was correlated with tick weight: in females, OspC was detectable only in ticks with a minimum weight of about 3.5 mg, and in nymphs weighing at least 1 mg. These results indicate that in I. ricinus removed from humans OspC is up-regulated during the blood meal of the tick, but in most ticks OspA is still detectable and might even be present in the absence of OspC expression in the midgut and salivary glands of nearly fully engorged nymphal ticks. Furthermore, we found strong evidence that borreliae expressing solely OspA while in the salivary glands can cause Lyme borreliosis. Our findings indicate that during tick feeding, humans are exposed to borreliae that may express either OspA or OspC or both, or lack both OspA and C. These findings suggests that, at the minimum, both OspA and C should be considered as vaccine candidates for prophylaxis of Lyme borreliosis in Europe. Received: 28 July 1998     

Identification of Borrelia burgdorferi outer surface proteins

Several Borrelia burgdorferi outer surface proteins have been identified over the past decade that are up-regulated by temperature- and/or mammalian host-specific signals as this spirochete is transmitted from ticks to mammals. Given the potential role(s) that these differentially up-regulated proteins may play in B. burgdorferi transmission and Lyme disease pathogenesis, much attention has recently been placed on identifying additional borrelial outer surface proteins. To identify uncharacterized B. burgdorferi outer surface proteins, we previously performed a comprehensive gene expression profiling analysis of temperature-shifted and mammalian host-adapted B. burgdorferi. The combined microarray analyses revealed that many genes encoding known and putative outer surface proteins are down-regulated in mammalian host-adapted B. burgdorferi. At the same time, however, several different genes encoding putative outer surface proteins were found to be up-regulated during the transmission and infection process. Among the putative outer surface proteins identified, biochemical and surface localization analyses confirmed that seven (Bb0405, Bb0689, BbA36, BbA64, BbA66, BbA69, and BbI42) are localized to the surface of B. burgdorferi. Furthermore, enzyme-linked immunosorbent assay analysis using serum from tick-infested baboons indicated that all seven outer surface proteins identified are immunogenic and that antibodies are generated against all seven during a natural infection. Specific antibodies generated against all seven of these surface proteins were found to be bactericidal against B. burgdorferi, indicating that these newly identified outer surface proteins are prime candidates for analysis as second-generation Lyme disease vaccinogens.     

The OspE-related proteins inhibit complement deposition and enhance serum resistance of Borrelia burgdorferi, the lyme disease spirochete

Borrelia burgdorferi, the Lyme disease spirochete, binds the host complement inhibitors factor H (FH) and FH-like protein 1 (FHL-1). Binding of FH/FHL-1 by the B. burgdorferi proteins CspA and the OspE-related proteins is thought to enhance resistance to serum-mediated killing. While previous reports have shown that CspA confers serum resistance in B. burgdorferi, it is unclear whether the OspE-related proteins are relevant in B. burgdorferi serum resistance when OspE is expressed on the borrelial surface. To assess the role of the OspE-related proteins, we overexpressed them in a serum-sensitive CspA mutant strain. OspE overexpression enhanced serum resistance of the CspA-deficient organisms. Furthermore, FH was more efficiently bound to the B. burgdorferi surface when OspE was overexpressed. Deposition of complement components C3 and C5b-9 (the membrane attack complex), however, was reduced on the surface of the OspE-overexpressing strain compared to that on the CspA mutant strain. These data demonstrate that OspE proteins expressed on the surface of B. burgdorferi bind FH and protect the organism from complement deposition and subsequent serum-mediated destruction.     

Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus.

A total of 472 field-collected Ixodes ricinus ticks from southern Germany were investigated by immunofluorescence for the presence of Borrelia burgdorferi with a polyvalent rabbit immune serum and with monoclonal antibodies specific for outer surface proteins A and C (OspA and OspC, respectively). Borreliae were detected in 90 ticks with the polyvalent immunofluorescence assay. Infection rates in adults (females, 20.2%; males, 25.2%) were significantly higher than in nymphs (12.1%). OspA was detected in 77 ticks and OspC was detected in only 1 tick with the respective monoclonal antibodies. We therefore conclude that B. burgdorferi in unfed I. ricinus ticks usually expresses OspA and very rarely OspC.     

Differential expression of outer surface proteins A and C by individual Borrelia burgdorferi in different genospecies

Previous studies with different Borrelia burgdorferi sensu stricto (s.s.) strains revealed that temperature as well as cocultivation with tick cells modulates the expression of outer surface proteins (Osp) A and C. We investigated the effects of temperature and of interaction with tick cells in culture on the expression of OspA and OspC of the B. afzelii clones cPKo97 and cPKo345 in comparison to the B. burgdorferi s.s. strain N40. To follow the dynamics of Osp expression of single borreliae we used indirect immunofluorescence microscopy with double staining of OspA and OspC. Clone PKo345 always showed expression of only OspA, regardless the conditions it was subjected to. Sequencing of the ospC gene disclosed a insertion leading to a stop codon after base 222 and inability to produce OspC. In cPKo97 and N40 OspC is down-regulated at lower temperatures and up-regulated at higher temperatures, which was especially pronounced on cocultivation with tick cells. Borreliae adherent to tick cells showed greater OspA expression compared to the nonadherent ones, an indication that OspA might play a role as adhesin for tick cells. Interestingly, cPKo97 and N40 displayed different patterns of Osp expression: cPKo97 simultaneously presents OspA and OspC on single borreliae, while N40 has either OspA or OspC on single cells. Adaptation of OspC expression in cPKo97 seems to occur by up- or down-regulation of this protein on single borreliae, as shown by alternating intensities of OspC expression at different temperatures. In contrast, N40 seem to consist of two subsets of borreliae one expressing only OspA and the other only OspC, and change in temperature results in growth benefit for one of these subtypes. Our findings indicate that, regarding OspA and OspC expression, response to temperature and cocultivation with tick cells of B. afzelii is comparable to B. burgdorferi s.s., but the mode of regulation seems phenotypically different. Further European isolates should be investigated for OspA and OspC regulation, especially in the face of vaccine development for the European situation. Received: 19 July 2000     

Cystitis induced by infection with the Lyme disease spirochete, Borrelia burgdorferi, in mice.

Previous studies have demonstrated that the urinary bladder is a consistent source for isolating the Lyme disease spirochete, Borrelia burgdorferi, from both experimentally infected and naturally exposed rodents. We examined histopathologic changes in the urinary bladder of different types of rodents experimentally infected with Lyme spirochetes, including BALB/c mice (Mus musculus), nude mice (M. musculus), white-footed mice (Peromyscus leucopus), and grasshopper mice (Onychomys leucogaster). Animals were inoculated intraperitoneally, subcutaneously, or intranasally with low-passaged spirochetes, high-passaged spirochetes, or phosphate-buffered saline. At various times after inoculation, animals were killed and approximately one-half of each urinary bladder and kidney were cultured separately in BSK-II medium while the other half of each organ was prepared for histologic examination. Spirochetes were cultured from the urinary bladder of all 35 mice inoculated with low-passaged spirochetes while we were unable to isolate spirochetes from any kidneys of the same mice. The pathologic changes observed most frequently in the urinary bladder of the infected mice were the presence of lymphoid aggregates, vascular changes, including an increase in the number of vessels and thickening of the vessel walls, and perivascular infiltrates. Our results demonstrate that nearly all individuals (93%) of the four types of mice examined had a cystitis associated with spirochetal infection.     

Resistance to tick-borne spirochete challenge induced by Borrelia burgdorferi strains that differ in expression of outer surface proteins.

Hamsters were immunized with thimerosal-killed Borrelia burgdorferi 297 or a mutant of 297 (M297) that lacks the 49-kb linear plasmid and expression of outer surface proteins A and B (OspA and OspB). Ixodes scapularis nymphs infected with either the B. burgdorferi sensu stricto strain 297 or JMNT, similar in OspA and OspB but differing in OspC expression, were used to evaluate protection. In a homologous challenge, 24 hamsters were vaccinated, 8 each with 297 or M297 and 8 sham (adjuvant)-vaccinated controls. Hamsters vaccinated with either bacterin were completely protected against a natural tick bite or subcutaneous (s.c.) inoculation of 297. Borreliae were effectively eliminated from 80 to 90% of the 297-infected ticks that fed on four hamsters immunized with the 297 bacterin. Cultures of spirochetes isolated from the ticks that remained infected were infectious and induced joint inflammation in naive hamsters. There was no reduction of strain 297 spirochetes in ticks that fed on four hamsters immunized with M297, but the hamsters were protected. Results with the M297 bacterin indicate that proteins other than OspA or OspB can protect hamsters against a tick challenge without eliminating B. burgdorferi in the tick. In a heterologous challenge, 36 hamsters were vaccinated, 12 with each bacterin and 12 controls. None of the hamsters immunized with either bacterin were protected from a challenge involving JMNT-infected ticks, while two of four were protected against an s.c. challenge. Hamsters challenged s.c. with strain 297 spirochetes were protected. There was partial elimination of JMNT spirochetes in ticks that fed on the group of four hamsters immunized with the 297 bacterin, and infection rates were reduced by 50 to 60%. JMNT spirochetes reisolated from the ticks that fed on 297-vaccinated hamsters also remained infectious for hamsters. In the JMNT-infected ticks that fed on four M297-immunized hamsters, there was no decline in the proportion of infected ticks. Destruction of spirochetes in ticks that fed on the hamsters vaccinated with the 297 bacterin suggests that antibodies to OspA and OspB may have been responsible, since the mutant did not induce this activity.     

Characterization of outer membranes isolated from Borrelia burgdorferi, the Lyme disease spirochete.

The lack of methods for isolating Borrelia burgdorferi outer membranes (OMs) has hindered efforts to characterize borrelial surface-exposed proteins. Here we isolated OMs by immersion of motile spirochetes in hypertonic sucrose followed by isopycnic ultracentrifugation of the plasmolyzed cells. The unilamellar vesicles thus obtained were shown to be OMs by the following criteria: (i) they contained OspA and OspB; (ii) they did not contain flagellin, NADH oxidase activity, or the 60-kDa heat shock protein; and (iii) their morphology by freeze-fracture electron microscopy was identical to that of OMs of intact organisms. Consistent with previous studies which employed immunoelectron microscopy and detergent-based solubilization of B. burgdorferi OMs, only small proportions of the total cellular content of OspA or OspB were OM associated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fluorography of OMs from spirochetes metabolically radiolabeled with [3H]palmitate or 35S-amino acids demonstrated that the OMs contained both nonlipidated and lipidated proteins. This fractionation procedure was also used to isolate OMs from virulent and avirulent isolates of the well-characterized B. burgdorferi N40 strain. SDS-PAGE fluorography revealed that OMs from the two isolates differed with respect to both nonlipoprotein and lipoprotein constituents. When whole cells, protoplasmic cylinders, and OMs were immunoblotted against sera from mice persistently infected with B. burgdorferi N40, the majority of antibody reactivity was directed against intracellular proteins. The availability of isolated OMs should facilitate efforts to elucidate the complex relationship(s) between B. burgdorferi membrane composition and Lyme disease pathogenesis.     

Occurrence of severe destructive lyme arthritis in hamsters vaccinated with outer surface protein A and challenged with Borrelia burgdorferi

Arthritis is a frequent and major complication of infection with Borrelia burgdorferi sensu stricto. The antigens responsible for the induction of arthritis are unknown. Here we provide direct evidence that a major surface protein, outer surface protein A (OspA), can induce arthritis. Hamsters were vaccinated with 30, 60, or 120 microg of recombinant OspA (rOspA) in aluminum hydroxide and challenged with B. burgdorferi sensu stricto isolate 297 or C-1-11. Swelling of the hind paws was detected in 100, 100, and 50% of hamsters vaccinated with 30, 60, or 120 microg of rOspA, respectively. In addition, arthritis developed in 57% of hamsters vaccinated with a canine rOspA vaccine after infection with B. burgdorferi sensu stricto. When the canine rOspA vaccine was combined with aluminum hydroxide, all vaccinated hamsters developed arthritis after challenge with B. burgdorferi sensu stricto. Histopathologic examination confirmed the development of severe destructive arthritis in rOspA-vaccinated hamsters challenged with B. burgdorferi sensu stricto. These findings suggest that rOspA vaccines should be modified to eliminate epitopes of OspA responsible for the induction of arthritis. Our results are important because an rOspA vaccine in aluminum hydroxide was approved by the Food and Drug Administration for use in humans.     

Detection of Borrelia burgdorferi infection in Ixodes dammini ticks with the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify DNA sequences of the etiologic agent of Lyme disease, Borrelia burgdorferi, and was applied to the detection of the spirochete in its tick vector. The target for PCR amplification was the OSP-A gene of strain B31; analysis of isolates from different geographical areas indicated that this gene could be used to identify most North American isolates. These methods were extended to the analysis of colony-derived and field-collected Ixodes dammini. OSP-A-specific sequences were identified in 15 of 15 colony-derived nymphal ticks that had fed previously on an infected animal; no such amplification products were detected in 8 control ticks. Segregated midgut tissues of field-collected adult and nymphal ticks from Nantucket Island, Mass., and the Crane Reserve, Ipswich, Mass., were examined by both direct fluorescent-antibody (DFA) staining and PCR. The DFA technique identified 16 infected ticks of 30 paired specimens; 15 of these specimens were positive by PCR. One specimen was positive by PCR that was DFA negative. Both live whole ticks and desiccated dead specimens were suitable for this analysis. Because only five ticks are suitable for DFA analysis, the use of PCR may extend the range of specimens that can be analyzed for the presence of the Lyme spirochete.     

Evidence for vaccine synergy between Borrelia burgdorferi decorin binding protein A and outer surface protein A in the mouse model of lyme borreliosis

Mice immunized with either the predominantly vector-stage lipoprotein outer surface protein A (OspA) or the in vivo-expressed lipoprotein decorin binding protein A (DbpA) are protected against Borrelia burgdorferi challenge. DbpA-OspA combinations protected against 100-fold-higher challenge doses than did either single-antigen vaccine and conferred significant protection against heterologous B. burgdorferi, B. garinii, and B. afzelii isolates, suggesting that there is synergy between these two immunogens.     

Molecular analysis of neutralizing epitopes on outer surface proteins A and B of Borrelia burgdorferi.

The neutralizing epitopes of the major outer surface proteins A and B (OspA and OspB) of Borrelia burgdorferi B31 were investigated by epitope mapping using overlapping synthetic peptides, encompassing full-length OspA and OspB, and antiborrelial monoclonal antibodies (MAbs). OspA MAb N4B12 and OspB MAbs N5G5, W7C2, and P4D1 displayed a complement-independent antiborrelial activity, and complement failed to enhance the antiborrelial activity, as measured by a sensitive colorimetric assay. A combination of N4B12 with N5G5 displayed a higher antiborrelial activity than did the MAbs individually. OspA MAbs B3G11 and L3B5, however, exhibited a significant antiborrelial activity only in the presence of complement. Epitope mapping showed that B3G11 bound to one OspA synthetic peptide with the sequence of amino acids 247 to 256 (QYDSNGTKLE) and produced more than sixfold-higher reactivity than with other sequences, as measured by an enzyme-linked immunosorbent assay. OspB MAb N5G5 bound to an OspB peptide with the sequence of amino acids 211 to 220 (TLKREIEKDG), yielding at least threefold-higher reactivity than with other sequences. These two peptide sequences were found to contain neutralizing epitopes. Other MAbs had weak binding activities with the synthetic peptides, and their specific epitopes remain to be further analyzed. Thus, this study demonstrated both complement-independent and complement-dependent antiborrelial MAbs and identified the linear epitopes on OspA and OspB capable of inducing neutralizing antibody responses.     

Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi

We were unable to demonstrate the presence of the classic enterobacterium-type lipopolysaccharide in the cells of the Lyme disease spirochete, Borrelia burgdorferi B31. This finding was primarily based on chemical analysis and the absence of free lipid A upon mild acid hydrolysis of the appropriate cell extracts. These results do not preclude the possible existence of an unusual lipopolysaccharide-like compound(s) in B. burgdorferi.     

Expression and gene sequence of outer surface protein C of Borrelia burgdorferi reisolated from chronically infected mice.

OspC from Borrelia burgdorferi reisolated from mice persistently infected with cloned spirochetes was examined. In all cases, the sequence of the ospC gene was identical to that of the original inoculant. We conclude that variation of ospC is not necessary for evasion of the host immune system.     

Lipopeptides of Borrelia burgdorferi outer surface proteins induce Th1 phenotype development in alphabeta T-cell receptor transgenic mice.

Induction of the appropriate T helper cell (Th) subset is crucial for the resolution of infectious diseases and the prevention of immunopathology. Some pathogens preferentially induce Th1 or Th2 responses. How microorganisms influence Th phenotype development is unknown. We asked if Borrelia burgdorferi, the spirochete which causes Lyme arthritis, can promote a cytokine milieu in which T cells which are not specific for B. burgdorferi are induced to produce proinflammatory cytokines. Using alphabeta T-cell receptor transgenic mice as a source of T cells with a defined specificity other than for B. burgdorferi, we found that B. burgdorferi induced Th1 phenotype development in ovalbumin-specific transgenic T cells. Small synthetic lipopeptides corresponding to the N-terminal sequences of B. burgdorferi outer surface lipoproteins had similar effects. B. burgdorferi and its lipopeptides induced host cells to produce interleukin-12. When the peptides were used in delipidated form, they did not induce Th1 development. These findings may be of pathogenic importance, since it is currently assumed that a Th2-mediated antibody response is protective against B. burgdorferi. Bacteria associated with reactive arthritis, namely, Yersinia enterocolitica, Shigella flexneri, and Salmonella enteritidis, had different effects. The molecular definition of pathogen-host interactions determining cytokine production should facilitate rational therapeutic interventions directing the host response towards the desired cytokine response. Here, we describe small synthetic molecules capable of inducing Th1 phenotype development.     

Temporal analysis of the antigenic composition of Borrelia burgdorferi during infection in rabbit skin

The numbers of host-adapted Borrelia burgdorferi (HAB) organisms in rabbit skin were assessed by real-time PCR over the first 3 weeks of infection. Maximal numbers were found at day 11, while spirochete numbers decreased by more than 30-fold by day 21. The antigenic composition of HAB in skin biopsy samples was determined by use of a procedure termed hydrophobic antigen tissue Triton extraction. Immune sera from rabbits, sera from chronically infected mice, and monospecific antiserum to the antigenic variation protein, VlsE, were used to probe parallel two-dimensional immunoblots representing each time point. Individual proteins were identified using either specific antisera or by matching protein spots to mass spectrometry-identified protein spots from in vitro-cultivated Borrelia. There were significant changes in the relative expression of a variety of known and previously unrecognized HAB antigens during the 21-day period. OspC and the outer membrane proteins OspA and OspB were prominent at the earliest time point, day 5, when the antigenic variation protein VlsE was barely detected. OspA and OspB were not detected after day 5. OspC was not detected after day 9. VlsE was the most prominent antigen from day 7 through day 21. BmpA, ErpN, ErpP, LA7, OppA-2, DbpA, and an unidentified 15-kDa protein were also detected from day 7 through day 21. Immunoblot analysis using monospecific anti-VlsE revealed the presence of prominent distinct VlsE lower forms in HAB at days 9, 11, and 14; however, these lower forms were no longer detected at day 21. This marked diminution in VlsE lower forms paralleled the clearance of the spirochete from skin.