Constitutive expression of outer surface protein C diminishes the ability of Borrelia burgdorferi to evade specific humoral immunity

The Lyme disease spirochete Borrelia burgdorferi reduces the expression of outer surface protein C (OspC) in response to the development of an anti-OspC humoral response, leading to the hypothesis that the ability to repress OspC expression is critical for the pathogen to proceed to chronic infection. B. burgdorferi was genetically modified to constitutively express OspC by introducing an extra ospC copy fused with the borrelial flagellar gene (flaB) promoter. Such a genetic modification did not reduce infectivity or pathogenicity in severe combined immunodeficiency mice but resulted in clearance of infection by passively transferred OspC antibody. Spirochetes with constitutive ospC expression were unable to establish chronic infections in immunocompetent mice unless they had undergone very destructive mutations in the introduced ospC copy. Two escape mutants were identified; one had all 7 bp deleted between the putative ribosome-binding site and the start codon, ATG, causing a failure in translational initiation, and the other mutant had an insertion of 2 bp between nucleotides 315 and 316, resulting in a nonsense mutation at codon 108. Thus, the ability of B. burgdorferi to repress ospC expression during mammalian infection allows the pathogen to avoid clearance and to preserve the integrity of the important gene for subsequent utilization during its enzootic life cycle.     

Temporal changes in outer surface proteins A and C of the lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice

The Lyme disease-associated spirochete, Borrelia burgdorferi, is maintained in enzootic cycles involving Ixodes ticks and small mammals. Previous studies demonstrated that B. burgdorferi expresses outer surface protein A (OspA) but not OspC when residing in the midgut of unfed ticks. However, after ticks feed on blood, some spirochetes stop making OspA and express OspC. Our current work examined the timing and frequency of OspA and OspC expression by B. burgdorferi in infected Ixodes scapularis nymphs as they fed on uninfected mice and in uninfected I. scapularis larvae and nymphs as they first acquired spirochetes from infected mice. Smears of midguts from previously infected ticks were prepared at 12- or 24-h intervals following attachment through repletion at 96 h, and spirochetes were stained for immunofluorescence for detection of antibodies to OspA and OspC. As shown previously, prior to feeding spirochetes in nymphs expressed OspA but not OspC. During nymphal feeding, however, the proportion of spirochetes expressing OspA decreased, while spirochetes expressing OspC became detectable. In fact, spirochetes rapidly began to express OspC, with the greatest proportion of spirochetes having this protein at 48 h of attachment and then with the proportion decreasing significantly by the time that the ticks had completed feeding. In vitro cultivation of the spirochete at different temperatures showed OspC to be most abundant when the spirochetes were grown at 37 degrees C. Yet, the synthesis of this protein waned with continuous passage at this temperature. Immunofluorescence staining of spirochetes in smears of midguts from larvae and nymphs still attached or having completed feeding on infected mice demonstrated that OspA but not OspC was produced by these spirochetes recently acquired from mice. Therefore, the temporal synthesis of OspC by spirochetes only in feeding ticks that were infected prior to the blood meal suggests that this surface protein is involved in transmission from tick to mammal but not from mammal to tick.     

Borrelia burgdorferi regulates expression of complement regulator-acquiring surface protein 1 during the mammal-tick infection cycle

During the natural mammal-tick infection cycle, the Lyme disease spirochete Borrelia burgdorferi comes into contact with components of the alternative complement pathway. B. burgdorferi, like many other human pathogens, has evolved the immune evasion strategy of binding two host-derived fluid-phase regulators of complement, factor H and factor H-like protein 1 (FHL-1). The borrelial complement regulator-acquiring surface protein 1 (CRASP-1) is a surface-exposed lipoprotein that binds both factor H and FHL-1. Analysis of CRASP-1 expression during the mammal-tick infectious cycle indicated that B. burgdorferi expresses this protein during mammalian infection, supporting the hypothesized role for CRASP-1 in immune evasion. However, CRASP-1 synthesis was repressed in bacteria during colonization of vector ticks. Analysis of cultured bacteria indicated that CRASP-1 is differentially expressed in response to changes in pH. Comparisons of CRASP-1 expression patterns with those of other infection-associated B. burgdorferi proteins, including the OspC, OspA, and Erp proteins, indicated that each protein is regulated through a unique mechanism.     

The relapsing fever spirochete Borrelia hermsii contains multiple, antigen-encoding circular plasmids that are homologous to the cp32 plasmids of Lyme disease spirochetes

Borrelia hermsii, an agent of tick-borne relapsing fever, was found to contain multiple circular plasmids approximately 30 kb in size. Sequencing of a DNA library constructed from circular plasmid fragments enabled assembly of a composite DNA sequence that is homologous to the cp32 plasmid family of the Lyme disease spirochete, B. burgdorferi. Analysis of another relapsing fever bacterium, B. parkeri, indicated that it contains linear homologs of the B. hermsii and B. burgdorferi cp32 plasmids. The B. hermsii cp32 plasmids encode homologs of the B. burgdorferi Mlp and Bdr antigenic proteins and BlyA/BlyB putative hemolysins, but homologs of B. burgdorferi erp genes were absent. Immunoblot analyses demonstrated that relapsing fever patients produced antibodies to Mlp proteins, indicating that those proteins are synthesized by the spirochetes during human infection. Conservation of cp32-encoded genes in different Borrelia species suggests that their protein products serve functions essential to both relapsing fever and Lyme disease spirochetes. Relapsing fever borreliae replicate to high levels in the blood of infected animals, permitting direct detection and possible functional studies of Mlp, Bdr, BlyA/BlyB, and other cp32-encoded proteins in vivo.     

Borrelia burgdorferi in Tick Cell Culture Modulates Expression of Outer Surface Proteins A and C in Response to Temperature

The Lyme disease spirochete Borrelia burgdorferi sensu stricto downregulates outer surface protein A (OspA) and upregulates outer surface protein C (OspC) during tick feeding. The switching of these proteins correlates with increased spirochetal infectivity for the mammal. We examined the effect of temperature on differential expression of OspA and OspC by B. burgdorferi cocultivated with a cell line isolated from the vector tick Ixodes scapularis. The effect of incubation at 31, 34, or 37 degrees C on expression of OspA and OspC by B. burgdorferi JMNT and N40 was analyzed by indirect fluorescent-antibody microscopy, polyacrylamide gel electrophoresis, and immunoblotting. The amount of OspA relative to the amount of flagellin was highest in spirochetes cocultivated with tick cells at 31 degrees C and declined with increasing temperature in both strains. OspC production was enhanced in spirochetes cocultivated with tick cells at 37 degrees C. Spirochetes grown axenically in BSK-H medium also produced more OspC at 37 degrees C, but OspA content was not appreciably affected by temperature. Our findings indicate that temperature, along with cultivation in a tick cell culture system, plays a role in the differential expression of OspA and enhances differential expression of OspC by spirochetes.     

Outer surface protein C (OspC), but not P39, is a protective immunogen against a tick-transmitted Borrelia burgdorferi challenge: evidence for a conformational protective epitope in OspC.

Outbred mice were immunized with the soluble fraction of a crude Escherichia coli lysate containing either recombinant outer surface protein C (OspC or P39 of Borrelia burgdorferi B31 (low passage). Following seroconversion, the mice were challenged with an infectious dose of B. burgdorferi B31 via the natural transmission mode of tick bite. Three mice immunized with P39 were not protected; however, all 12 of the recombinant OspC-immunized mice were protected from infection as assayed by culture and serology. Although OspC has been shown to be a protective immunogen against challenge with in vitro-cultured borrelia administered by needle, this study is the first to demonstrate OspC effectiveness against tick-borne spirochetes. Following feeding, all ticks still harbored B. burgdorferi, suggesting that the mechanism of protection is not linked to destruction of the infectious spirochete within the tick. In a separate experiment, groups of four mice were immunized with protein fractions from B. burgdorferi B31 purified by preparative gel electrophoresis in an attempt to identify potential protective antigens. Many of these mice developed high-titer-antibody responses against OspC, but curiously the mice were susceptible to B. burgdorferi infection via tick bite. These results suggest that the protective epitope(s) on OspC is heat sensitive/conformational, a finding which has implications in vaccine development.     

Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.     

Cultivation at 37°C enhances Borrelia burgdorferi sensu stricto infectivity for hamsters

Borrelia burgdorferi sensu stricto (s.s.), the causative agent of Lyme disease in North America is transmitted to the mammalian host by ticks belonging to the genus, Ixodes. Antibodies to several spirochetal proteins, most notably outer surface protein C (OspC), have been observed in early infection in both humans and laboratory animals. Thus, the expression of these proteins have been postulated to play a role in tick transmission and spirochetal infectivity for the mammalian host. B. burgdorferi strain JMNT was induced to produce increased levels of OspC by cultivation in BSK medium at 37 degrees C. To diminish expression of OspC, spirochetes were cultivated at 31 degrees C. Spirochetes shifted down from 37 degrees C to 31 degrees C or up from 31 degrees C to 37 degrees C for 1 week contained equivalent amounts of OspC. To evaluate spirochetal infectivity, hamsters were inoculated subcutaneously with 1 x 10(4) or 1 x 10(6) spirochetes grown at the above-mentioned temperatures. Hamsters inoculated with spirochetes expressing high amounts of OspC all became infected, irrespective of the inoculum size. None of the hamsters inoculated with 1 x 10(4) spirochetes grown at 31 degrees C or in cultures shifted down from 37 degrees C to 31 degrees C were infected. All infected hamsters, confirmed by isolation of spirochetes in ear and/or bladder cultures, had an antibody response to OspC. In contrast, all non-infected hamsters lacked antibodies to OspC. We conclude that cultivation of spirochetes at 37 degrees C enhances their infectivity for hamsters. This study also suggests there is a correlation between enhancement of OspC expression and spirochetal infectivity for hamsters.     

Serologic diagnosis of Lyme borreliosis by using enzyme-linked immunosorbent assays with recombinant antigens

Class-specific enzyme-linked immunosorbent assays (ELISAs) with purified recombinant antigens of Borrelia burgdorferi sensu stricto and Western blot analyses with whole cells of this spirochete were used to test human sera to determine which antigens were diagnostically important. In analyses for immunoglobulin M (IgM) antibodies, 14 (82%) of 17 serum samples from persons who had erythema migrans reacted positively by an ELISA with one or more recombinant antigens. There was frequent antibody reactivity to protein 41-G (p41-G), outer surface protein C (OspC), and OspF antigens. In an ELISA for IgG antibodies, 13 (87%) of 15 serum samples had antibodies to recombinant antigens; reactivity to p22, p39, p41-G, OspC, and OspF antigens was frequent. By both ELISAs, serum specimens positive for OspB, OspE, and p37 were uncommon. Analyses of sera obtained from persons who were suspected of having human granulocytic ehrlichiosis (HGE) but who lacked antibodies to ehrlichiae revealed IgM antibodies to all recombinant antigens of B. burgdorferi except OspB and IgG antibodies to all antigens except OspE. Immunoblotting of sera from the study group of individuals suspected of having HGE reaffirmed antibody reactivity to multiple antigens of B. burgdorferi. There was minor cross-reactivity when sera from healthy subjects or persons who had syphilis, oral infections, or rheumatoid arthritis were tested by ELISAs with p37, p41-G, OspB, OspC, OspE, and OspF antigens. Although the results of class-specific ELISAs with recombinant antigens were comparable to those recorded for assays with whole-cell antigen and for individuals with confirmed clinical diagnoses of Lyme borreliosis, immunoblotting is still advised as an adjunct procedure, particularly when there are low antibody titers by an ELISA.     

Influence of cultivation media on genetic regulatory patterns in Borrelia burgdorferi

Barbour-Stoenner-Kelly II (BSKII) medium and BSKH medium both are routinely used for the cultivation of Borrelia burgdorferi. However, heretofore there have been no studies to compare how these two media affect gene expression patterns in virulent B. burgdorferi. In the present study, we found that some B. burgdorferi strain 297 genes (e.g., ospA, mlp-7A, mlp-8, p22, and lp6.6) that typically are regulated by temperature or pH displayed their predicted pattern of expression when B. burgdorferi was cultivated in BSKH medium; this was not true when spirochetes were cultivated in conventional BSKII medium. The results suggest that BSKH medium is superior to BSKII medium for gene expression studies with B. burgdorferi.     

CD14 mediates cross talk between mononuclear cells and fibroblasts for upregulation of matrix metalloproteinase 9 by Borrelia burgdorferi

Lyme disease is an infection caused by a tick-borne spirochete, Borrelia burgdorferi. Matrix metalloproteinase 9 (MMP-9) was selectively upregulated in the erythema migrans skin lesions of patients with acute Lyme disease. In this study, the mechanism of upregulation of MMP-9 was investigated in vitro and in vivo. The concentrations of MMP-9 and soluble CD14 were markedly elevated in serum from patients with acute Lyme disease and were also upregulated in U937 cells by B. burgdorferi in a time- and concentration-dependent manner. MMP-9 mRNA was expressed at baseline in fibroblasts in the presence or absence of B. burgdorferi. However, when fibroblasts were incubated with supernatants from U937 cells with B. burgdorferi or recombinant CD14, the expression of MMP-9 was significantly increased. This effect was completely abolished by the anti-CD14 antibody. These data suggest that the upregulation of MMP-9 by B. burgdorferi involves the CD14 pathway in infiltrating inflammatory cells. Fibroblasts could be recruited to amplify local production of MMP-9 by acquiring CD14 from macrophages.