The enolase of Borrelia burgdorferi is a plasminogen receptor released in outer membrane vesicles.

The agent of Lyme disease, Borrelia burgdorferi, has a number of outer membrane proteins that are differentially regulated during its life cycle. In addition to their physiological functions in the organism, these proteins also likely serve different functions in invasiveness and immune evasion. In borreliae, as well as in other bacteria, a number of membrane proteins have been implicated in binding plasminogen. The activation and transformation of plasminogen into its proteolytically active form, plasmin, enhances the ability of the bacteria to disseminate in the host. Outer membrane vesicles of B. burgdorferi contain enolase, a glycolytic-cycle enzyme that catalyzes 2-phosphoglycerate to form phosphoenolpyruvate, which is also a known plasminogen receptor in Gram-positive bacteria. The enolase was cloned, expressed, purified, and used to generate rabbit antienolase serum. The enolase binds plasminogen in a lysine-dependent manner but not through ionic interactions. Although it is present in the outer membrane, microscopy and proteinase K treatment showed that enolase does not appear to be exposed on the surface. However, enolase in the outer membrane vesicles is accessible to proteolytic degradation by proteinase K. Samples from experimentally and tick-infected mice and rabbits as well as from Lyme disease patients exhibit recognition of enolase in serologic assays. Thus, this immunogenic plasminogen receptor released in outer membrane vesicles could be responsible for external proteolysis in the pericellular environment and have roles in nutrition and in enhancing dissemination.

Phylogenetic analysis of a virulent Borrelia species isolated from patients with relapsing fever.

Multilocus sequence analysis (MLSA) was used to clarify the taxonomic status of a virulent Borrelia organism previously isolated from patients with relapsing fever and from ticks in Spain that is designated the Spanish relapsing fever (SRF) Borrelia. This species has been used extensively in experimental infection models because of its continued virulence. Seven genes were amplified to analyze the phylogenetic relationships among several Spanish isolates of SRF Borrelia and other relapsing fever Borrelia species. The genes targeted in this study included rrs and flaB, which have commonly been used in phylogenetic studies; the rrf-rrl intergenic spacer (IGS), which is highly discriminatory; and four additional genes, p66, groEL, glpQ, and recC, which are located on the chromosome and which have therefore evolved in a clonal way. The species included in this study were Borrelia duttonii, B. recurrentis, B. crocidurae, and B. hispanica as Old World Borrelia species and B. turicatae and B. hermsii as New World Borrelia species. The results obtained by MLSA of the SRF Borrelia on the basis of 1% of the genomic sequence data analyzed confirmed that the SRF Borrelia isolates are B. hispanica. However, the prototype isolates of B. hispanica used in this study have an uncertain history and display unique phenotypic characteristics that are not shared with the SRF Borrelia. Therefore, we propose to use strain SP1, isolated from a relapsing fever patient in 1994 in southern Spain, as the type strain for B. hispanica.

The important and diverse roles of antibodies in the host response to Borrelia infections.

Antibodies are of critical importance in the host response to tick-borne Borrelia species that cause relapsing fever and Lyme disease. Recent studies on the role of various B cell subsets in the host response to Borrelia, complement-independent, bactericidal antibodies, and diagnostics led to this review that focuses on the array of functions that antibodies to Borrelia can perform.

Cutting edge: the spirochetemia of murine relapsing fever is cleared by complement-independent bactericidal antibodies.

Abs are the major effectors of host defense against infections with BORRELIA: Bactericidal murine mAbs and their Fabs destroy B. burgdorferi, the agent of Lyme disease, and relapsing fever Borrelia in the absence of complement. These in vitro observations led to the expansion of a search for functionally similar Abs in vivo. In this study, we demonstrate that functionally unique IgM Abs develop in vivo and are responsible for the elimination of spirochetemia in murine models of relapsing fever, without the assistance of complement. Mice deficient in the fifth or third component of complement can clear the spirochetemia, whereas B cell-deficient mice cannot. The B cell-deficient mice developed spirochetemia that was an order of magnitude higher and persisted for a longer period of time in comparison to the wild-type mice. Additionally, B cell-deficient mice passively immunized with immune IgM and with immune serum were protected from challenge.

Borrelia spirochetes upregulate release and activation of matrix metalloproteinase gelatinase B (MMP-9) and collagenase 1 (MMP-1) in human cells.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, stimulated human peripheral blood monocytes to release pro-matrix metalloproteinase-9 (gelatinase B; pro-MMP-9) and active matrix metalloproteinase-1 (collagenase-1; MMP-1). Human neutrophils also released pro-MMP-9 and a 130-kDa protein with gelatinolytic activity in response to live B. burgdorferi. In addition, U937 cells and human keratinocyte cells were also stimulated to release pro-MMP-9 under the same conditions. However, human umbilical vein endothelial cells (HUVECs) released pro-MMP-9 and pro-MMP-2 in a constitutive manner and were not influenced by live spirochetes. MMPs produced by human monocytes also enhanced the penetration of B. burgdorferi through extracellular matrix component barriers in vitro. Plasmin stabilized on the surface of the Lyme disease spirochete was shown to activate pro-MMP-9 to its active form. This active form was also observed in the plasma of mice infected with a relapsing fever borrelia. These results suggest that borreliae can upregulate MMPs and possibly mediate an activation cascade initiated by plasmin bound to the microbial surface. MMPs may play a role in dissemination of the Lyme disease spirochete and in the pathogenesis of Borrelia infection.

Borrelia burgdorferi and other bacterial products induce expression and release of the urokinase receptor (CD87).

The urokinase-type plasminogen activator receptor (uPAR, CD87) is a highly glycosylated 55- to 60-kDa protein anchored to the cell membrane through a glycosylphosphatidylinositol moiety that promotes the acquisition of plasmin on the surface of cells and subsequent cell movement and migration by binding urokinase-type plasminogen activator. uPAR also occurs in a soluble form in body fluids and tumor extracts, and both membrane and soluble uPAR are overexpressed in patients with tumors. uPAR may be a factor in inflammatory disorders as well. We investigated whether Borrelia burgdorferi could stimulate up-regulation of cell membrane uPAR in vitro. B. burgdorferi, purified native outer surface protein A, and a synthetic outer surface protein A hexalipopeptide stimulated human monocytes to up-regulate membrane uPAR as measured by immunofluorescence/FACS and Western blot. The presence of soluble uPAR in culture supernatants, measured by Ag capture ELISA, was also observed. LPS from Salmonella typhimurium and lipotechoic acid from Streptococcus pyogenes also induced the up-regulation of both membrane and soluble uPAR protein by monocytes. Up-regulation of uPAR was induced by conditioned medium from B. burgdorferi/monocyte cocultures. The up-regulation of uPAR by B. burgdorferi was concomitant with an increase in uPAR mRNA, indicating that synthesis was de novo. The expression and release of uPAR in response to B. burgdorferi and other bacterial components suggests a role in the pathogenesis of Lyme disease as well as in other bacterial infections.

The generation of enzymatically active plasmin on the surface of spirochetes.

The spirochete Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted to the host by a feeding Ixodid tick. The spirochete subsequently disseminates through the skin, enters the bloodstream, and becomes systemic. A potential mechanism for this invasiveness was identified with the discovery that B. burgdorferi can bind components of the plasminogen activation system (PAS). The methodology for analyzing the generation of enzymatically active plasmin on the surface of this organism is given, and applied to measure spirochete viability, strain differences, and breakdown of extracellular matrix (ECM) macromolecules. Plasmin acquisition by B. burgdorferi was measured photometrically by a specific chromogenic substrate. The growth of B. burgdorferi in culture was not affected by the presence of active plasmin on the spirochete surface. Plasmin-coated B. burgdorferi degraded the purified (ECM) components fibronectin, laminin, and vitronectin, but not collagen. The addition of B. burgdorferi with surface plasmin to a radiolabeled, native ECM resulted in degradation of noncollagenous protein, as measured by release of solubilized radioactivity. Breakdown of purified ECM components or native ECM did not occur after exposure to untreated spirochetes or spirochetes treated with uPA or PLG alone. These results provide in vitro evidence that enzymatically active plasmin on the surface of B. burgdorferi may be partially responsible for its invasiveness.

A bactericidal monoclonal antibody elicits a change in its antigen, OspB of Borrelia burgdorferi, that can be detected by limited proteolysis.

mAb CB2, directed against outer surface protein B (OspB), causes bacteriolysis of Borrelia burgdorferi in the absence of complement. How this happens is unknown. We examined the effect of mAb binding on OspB tertiary structure by using limited proteolysis to probe changes in protein conformation. Truncated OspB (tOspB) that lacked N-terminal lipid was cleaved by four enzymes: trypsin, endoproteinase Arg-C, endoproteinase Asp-N, and endoproteinase Glu-C. CB2 affected the cleavage by trypsin and Arg-C, but not by AspN or Glu-C. None of the enzymes cleaved CB2 under these conditions. Both trypsin and Arg-C cleaved tOspB near the N-terminus; CB2 slowed the rate of cleavage, but did not affect the identity of the sites cleaved. Irrelevant mAb had no effect, indicating that the effect was specific. CB2 was active against tOspB of strain B31, but not against tOspB of strain BEP4, to which it does not bind, suggesting that binding was required to elicit the effect on cleavage. With trypsin, CB2 showed a maximal effect at 8 mol of tOspB to 1 mol of mAb. At this ratio, not enough CB2 was present to bind all the tOspB; therefore, either CB2 shows turnover or CB2 acts by binding tOspB and effecting a change in this tOspB such that it, in turn, propagates the effect in other molecules of tOspB. Regardless of the mechanism, these data show that CB2 elicits a change in tOspB that can be measured by its reduced susceptibility to protease cleavage.

The relapsing fever spirochaete, Borrelia crocidurae, activates human endothelial cells and promotes the transendothelial migration of neutrophils.

The blood-borne, erythrocyte-aggregating Borrelia crocidurae, the causative agent of African relapsing fever, have been shown to induce severe cellular lesions in mice. In this paper, we present the first report of how the endothelium is stimulated during an African relapsing fever B. crocidurae infection. B. crocidurae co-incubated with cultured human umbilical vein endothelial cells (HUVECs) activated endothelium in such way that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose- and time-dependent fashion, as determined by a whole-cell enzyme-linked immunosorbent assay (ELISA). The upregulation was reduced by treatment that killed the bacteria, suggesting that viability is important for the stimulation of HUVECs by B. crocidurae. Furthermore, conditioned medium from HUVECs stimulated with B. crocidurae contained interleukin (IL)-8, which is a chemotactic agent for neutrophils. Activation of HUVECs by B. crocidurae resulted in migration of subsequently added neutrophils across the endothelial monolayers, and this migration was inhibited by antibodies to IL-8. The activation of endothelium by B. crocidurae may constitute a key pathophysiological mechanism in B. crocidurae-induced vascular damage.

Use of the plasminogen activation system by microorganisms.

The use of host-derived PAS components by invasive bacteria is an increasingly recognized mechanism for acquisition of extracellular proteolytic activity. This overview summarizes the pertinent contributions to this field and is divided into three parts: (1) the PAS, (2) the interaction of bacteria that produce their own plasminogen activators with the host's PAS, and (3) the interaction of bacteria that do not produce their own plasminogen activators but use plasminogen activators supplied by the host. The significance of these mechanisms in relation to the invasive potentials of the various organisms is discussed.

Plasmin-coated borrelia Burgdorferi degrades soluble and insoluble components of the mammalian extracellular matrix.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, binds plasminogen in vitro. Exogenously provided urokinase-type plasminogen (PLG) activator (uPA) converts surface-bound PLG to enzymatically active plasmin. In this study, we investigated the capacity of a B. burgdorferi human isolate, once complexed with plasmin, to degrade purified extracellular matrix (ECM) components and an interstitial ECM. In a modified enzyme-linked immunosorbent assay using immobilized, soluble ECM components, plasmin-coated B. burgdorferi degraded fibronectin, laminin, and vitronectin but not collagen. Incubation of plasmin-coated organisms with biosynthetically radiolabeled native ECM resulted in breakdown of insoluble glycoprotein, other noncollagenous proteins, and collagen, as measured by release of solubilized radioactivity. Radioactive release did not occur with untreated spirochetes or spirochetes treated with uPA or PLG alone. Kinetic and inhibition studies suggested that the breakdown of collagen was indirect and due to prior disruption of supportive ECM proteins. B. burgdorferi is an invasive bacterial pathogen that may benefit by use of the host's plasminogen activation system. The results of this study have identified mechanisms in which the spirochete can use this borrowed proteolytic activity to enhance invasiveness.

Seroprevalence and seroconversion for tick-borne diseases in a high-risk population in the northeast United States.

PURPOSE: To determine the prevalence of serologic reactivity, the 1-year incidence of seroconversion, and the frequency of multiple infections, and their associations with symptoms in a group of volunteers at high risk for tick-borne infections in New York state. METHODS: We performed a seroepidemiologic study of Lyme borreliosis, 2 of the ehrlichioses, Rocky Mountain spotted fever, and babesiosis among 671 participants who lived or worked in a high-risk area (mainly in eastern Long Island, New York) for tick-borne diseases. Sera were collected in the winters of 1994 and 1995. Signs and symptoms of tick-borne disease were monitored monthly by mail and telephone. Lyme borreliosis serologies were done by enzyme-linked immunosorbent assay and Western blot. Rocky Mountain spotted fever serologies were initially screened using Dip-S-Ticks, followed by specific indirect immunofluorescence. Ehrlichiosis serologies were determined by epifluorescent microscopy, as were antibodies to Babesia microti. RESULTS: Of the 671 participants, 88 (13%) had antibodies to > or = 1 tick-borne organisms, including 34 (5% of the total) with antibodies to Borrelia burgdorferi. Twenty-seven participants had evidence of exposure to B. burgdorferi at baseline. Seven participants (1%) seroconverted during the course of the study, 5 of whom were symptomatic for Lyme borreliosis. Antibodies to spotted fever group rickettsiae were seen in 28 participants (4%), 22 of whom were positive at baseline and 6 of whom seroconverted during the observation period. None of the seropositive patients had any symptoms or signs of infection. Twenty-four participants (3%) had serologic evidence of exposure to Ehrlichia (all but one to Ehrlichia equi); 5 (0.7%) seroconverted during the observation period, including 3 subjects who were asymptomatic. Antibodies to B. microti were seen in 7 participants (1%), including one asymptomatic seroconversion during the year of observation. There was evidence of possible dual infection in 5 patients. CONCLUSION: In a high-risk population, there was evidence of exposure to 5 tick-borne pathogens; however, many infections were asymptomatic, and coinfections were rare.

The plasminogen activation system enhances brain and heart invasion in murine relapsing fever borreliosis.

The role of the plasminogen activation system (PAS) was investigated during the course of infection of a relapsing fever Borrelia species in plasminogen-deficient (plg -/-) and control (plg +/+ and plg +/-) mice. Subcutaneous inoculation of 10(4) spirochetes resulted in a peak spirochetemia five days after infection with 20-23 x 10(6) organisms per milliliter of whole blood in all mice, indicating that the PAS had no effect on the development of this phase of the infection. Anemia, thrombocytopenia, hepatitis, carditis, and splenomegaly were noted in all mice during and immediately after peak spirochetemia. Fibrin deposition in organs was noted in plg -/- mice but not in controls during these stages. Significantly greater spirochetal DNA burdens were consistently observed in the hearts and brains of control mice 28-30 days after infection, as determined by PCR amplification of this organism's flagellin gene (flaB), followed by quantitative densitometry. Furthermore, the decreased spirochetal load in brains of plg -/- mice was associated with a significant decrease in the degree of inflammation of the leptomeninges in these mice. These findings indicate a role for the PAS in heart and brain invasion by relapsing fever Borrelia, resulting in organ injury.

Functional heterogeneity in the antibodies produced to Borrelia burgdorferi.

Antibodies to outer surface molecules of Borrelia burgdorferi (Osp) that have a bactericidal action in the absence of complement have been described. These antibodies are primarily monoclonal to antigenic determinants in OspA and OspB. One of these, CB2, is an IgG1 monoclonal antibody that recognizes an epitope in the carboxy terminus of OspB. The specificity of CB2 is critically dependent on the presence of a lysine (Lys) residue in position 253, not only for binding but also for killing the spirochete. This antibody has been used successfully to select escape variants or mutants that are missing the Lys residue either by a mutation or by deletion as a result of premature stop codons. Other antibodies to OspA, OspB, and p39 have also been characterized with similar properties. Another important feature of CB2 is that its bactericidal action is not dependent on agglutination, since Fab fragments of the whole immunoglobulin molecule can also kill in the absence of complement synergy. The killing action of CB2 is not inhibited by protease inhibitors, and is dependent on the presence of calcium. Upon contact with Borrelia burgdorferi, CB2 causes lysis of the outer membrane and the formation of a spheroplast. The bactericidal mechanism of this antibody is not known. The sequence of the heavy and light chain variable regions of CB2 have striking homology to murine antibodies of the autoimmune repertoire, and some of these antibodies have catalytic properties. In general, catalytic antibodies have enzymatic rates of acceleration that are significantly less than those of proteolytic enzymes. If CB2 were a catalytic antibody, its substrate specificity may be expected to be broader. CB2 does not cleave recombinant OspB, nor does it cleave other protein substrates. Its killing activity is not dependent on proteolysis. Because the bactericidal action of CB2 involves the destruction of the outer membrane, it is possible that a phospholipase could be associated with the mechanism. The mobility of spirochetal lipids is altered after incubation with CB2, and the bactericidal activity is reduced in the presence of phospholipase inhibitors. These studies suggest that the bactericidal mechanism of CB2, and other similar antibodies, is novel.

Borrelia burgdorferi adherence and injury to undifferentiated and differentiated neural cells in vitro.

The role of outer surface proteins (Osp) A and B and length of time in culture on the adhesion and cytotoxicity of Borrelia burgdorferi to C6 glioma and PC-12 pheochromocytoma cells was investigated using 6 different spirochete strains in an ELISA. Statistically significant differences in adhesion between OspB mutants and parental isolates were not seen, yet clear differences in adhesion were noted between low- and high-passage isolates. Polar adhesion and penetration by the tips of spirochetes resulted in the formation of surface cavities and blebs. Adhesion of spirochetes to C6 and to undifferentiated PC-12 cells did not result in significant cytotoxicity, but adhesion of spirochetes to PC-12 cells differentiated with nerve growth factor resulted in a loss of confluence of the monolayer and cytotoxicity at high spirochete-to-cell ratios. These results demonstrate that B. burgdorferi can induce damage to neural cells directly.

Plasminogen is required for efficient dissemination of B. burgdorferi in ticks and for enhancement of spirochetemia in mice.

The role of the host plasminogen activation system in transmission of and invasion by Borrelia burgdorferi, the tick-borne spirochetal agent of Lyme disease, was investigated using plasminogen (Plg)-knockout mice. PLG was not detected in spirochetes from unfed ticks, but binding occurred as ticks fed on the host's blood. Plasminogen activators were derived from the host blood meal. PLG was required for efficient dissemination of B. burgdorferi within the tick and for enhancement of spirochetemia in mice but was not critical for transmission and infection. These results provide evidence for a bacterium using a vertebrate protease to disseminate in an invertebrate vector and underscores the interplay among vector, pathogen, and host in promoting the life cycle and disease.

Characterization of the physiological requirements for the bactericidal effects of a monoclonal antibody to OspB of Borrelia burgdorferi by confocal microscopy.

A confocal microscopy study was undertaken to characterize the bactericidal effects of the Fab fragments of CB2, an immunoglobulin G1kappa murine monoclonal antibody, to an epitope in the carboxy region of the outer surface protein B (OspB) of Borrelia burgdorferi. Simultaneous direct labeling of both fixed and live spirochetes with fluorochrome-labeled Fab-CB2 and 11G1, and an immunoglobulin Mkappa monoclonal antibody to OspA, showed that OspA and OspB seem to colocalize in dead spirochetes but do not appear to be physically associated when the organisms are alive. A polar bleb composed of a Fab-CB2-OspB complex, followed by incorporation of 11G1-OspA, precedes the formation of a spheroplast. The spheroplasts contain both OspA and OspB and are a terminal stage in the bactericidal process induced by Fab-CB2. Outer membrane destabilization by Fab-CB2, but not cell wall or cytoplasmic membrane alterations, was demonstrated experimentally by the sequential treatment of spirochetes with Fab-CB2 and monoclonal antibodies to flagellin and DnaK. The action of Fab-CB2 is epitope specific, as another monoclonal antibody to an epitope in the amino terminus of OspB was not bactericidal. The bactericidal effect of Fab-CB2 is not dependent on the induction of spirochetal proteases but is dependent on the presence of Ca2+ and Mg2+. Supplementation of Ca2(+)- and Mg2(+)-free medium with these cations restored the bactericidal effects of Fab-CB2. The mechanism by which a Fab fragment of an antibody destroys a bacterium directly may represent a novel form of antibody-organism interaction.

A mouse model of Borrelia meningitis after intradermal injection.

Both young and adult C3H/HeN mice developed meningitis within 3 weeks of intradermal inoculation with a newly identified uncultivable Borrelia species, an agent of human relapsing fever. Meningoencephalitis with perivascular infiltrates and plexitis developed at approximately 25 days after inoculation. Infiltrates were composed of B and plasma cells and monocytes. This model recreated the meningitis associated with spirochetal infections through an intradermal route of infection.

Glycolytic enzyme operon of Borrelia burgdorferi: characterization and evolutionary implications.

The genes encoding three enzymes of the glycolytic pathway have been identified and sequenced completely in Borrelia burgdorferi sensu stricto and partially in B. hermsii. They are clustered on the chromosome into an operon with a single putative promoter and are arranged downstream of this promoter in the following order: gapdh (glyceraldehyde-3-phosphate dehydrogenase), pgk (phosphoglycerate kinase), and tpi (triosephosphate isomerase). gapdh and pgk are separated by 19 bp of intergenic sequence and pgk and tpi are separated by only 1 bp. Each of the three genes contains a putative RBS 6-7 bp upstream of each respective translational (ATG) start codon. The deduced protein encoded by gapdh consists of 335 amino acids (aa) with a predicted MW of 36,400, that of pgk is 393 aa (MW of 42,156) and that of tpi is 290 aa (MW of 27,683). The aa sequences of each of the three enzymes share 58.4% (GAPDH), 52.8% (PGK) and 46.1% (TPI) identity with respective enzymes from other prokaryotic organisms. Phylogenetic analyses based on these universal and conserved proteins support the hypothesis that spirochetes are an ancient and distinct eubacterial phylum.