Molecular mimicry in Lyme disease: monoclonal antibody H9724 to b. burgdorferi flagellin specifically detects chaperonin-HSP60

A monoclonal antibody (H9724), specific for the 41-kDa flagellar protein of the Lyme disease pathogen Borrelia burgdorferi, cross-reacts with human axons and detects one major protein in human neuroblastoma cell extracts. The homologous cross-reacting protein has now been isolated from calf adrenal and identified as chaperonin-HSP60 by N-terminal sequencing.     

Immunological Properties of Borrelia burgdorferi Isolated from the Ixodes ovatus in Shizuoka,Japan

Three strains of spirochetes (IKA1 to 3) were isolated from the midgut of Ixodes ovatus collected in the Ikawa region of the northern part of Shizuoka, Japan. These isolates had eight flagella, and their size and other morphological features were similar to Borrelia burgdorferi. They showed similar motility and reacted with monoclonal antibody (MAb) H9724 against borrelial flagella and with MAb H5332 against the outer surface protein A. These strains showed similar SDS-PAGE profiles to that of B. burgdorferi strain B31 and P/Bi isolated in the U.S.A. and Europe, respectively. Immunoblot with Lyme disease patient serum showed positive reactions with the flagella (41 Kilodalton, kDa), protein C (20 to 22 kDa), and outer surface protein A (29 kDa) of the isolates. Immunological properties, morphological characteristics, and epidemiological features revealed that these isolates were B. burgdorferi.     

Characterization of Lyme disease spirochetes isolated from ticks and vertebrates in North Carolina

Borrelia burgdorferi isolates obtained from numerous locations and from different hosts in North Carolina, were compared to previously characterized strains of the Lyme disease spirochete and other Borrelia spp. The spirochete isolates were confirmed to be B. burgdorferi sensu stricto based on immunofluorescence (IFA) using a monoclonal antibody to outer surface protein A (Osp A [H5332]) and polymerase chain reaction (PCR) using a species-specific nested primer for a conserved region of the gene that encodes for flagellin. In addition, the isolates tested positive in Western blots with species-specific monoclonal antibodies for outer surface protein A and OspB (84c), and the genus-specific, monoclonal antibody to flagellin (H9724). Infectivity studies with several of these isolates were conducted using Mus musculus and Oryzomys palustris and the isolates exhibited markedly different levels of infectivity. This study demonstrates that B. burgdorferi sensu stricto is present and naturally transmitted on the Outer Banks and in the Coastal Plain and Piedmont regions of North Carolina.     

Acquisition of Borrelia burgdorferi by Ixodes ricinus ticks fed on the European hedgehog,Erinaceus europaeus L

A hedgehog, Erinaceus europaeus, was found to be heavily infested with larval and nymphal Ixodes ricinus in a forest park in Co. Galway, Ireland. A large proportion of the ticks that engorged and detached were infected with the spirochacte, Borrelia burgdorferi, the causative agent of human Lyme borreliosis. The identity of these spirochaetes was confirmed by immunofluorescent assay with B. burgdorferi-specific monoclonal antibody and by polymerase chain reaction test and they were transmitted from the hedgehog to laboratory-reared ticks and from the ticks obtained from the hedgehog to gerbils (Meriones unguiculatus). The high infection rate of the larvae that fed on the hedgehog in comparison with unfed larvae from the same habitat was interpreted as strong evidence that this host species is reservoir competent. Since hedgehogs can evidently feed adult ticks as well as many immature stages, they may well have an important role in the ecology of Lyme borreliosis in some habitats.     

Detection of glycoproteins in Borrelia burgdorferi

The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two major outer surface exposed proteins of 31 kDa and 34 kDa showed to be glycosylated and gel filtration high pressure liquid chromatography (HPLC) of proteins of B. burgdorferi metabolically labeled with ¹⁴C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.Abbreviations N-glycosidase F peptide-N-glycosidase F (EC 3.5.1.52) - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WB Western blotting - HPLC high pressure liquid chromatography - SDS sodium dodecyl sulphate - mAb monoclonal antibody - MIAF mouse immune ascitic fluid - SS Schiff staining - Osp Outer surface protein     

HSP70 is part of the synaptonemal complex in Eimeria tenella

In the current study the expression and ultrastructural localization of heat shock protein 70 (HSP70) was analyzed by immunogold labelling of surface spreads of meiotic chromosomes from Eimeria tenella oocysts. The authors used a previously reported method that overcomes the difficulties of the resistance of Eimeria oocysts to disruption and permits the release of intact meiotic chromosomes. HSP70 was localized at the ultrastructural level using an anti-HSP70 monoclonal antibody in combination with a secondary antibody coupled to colloidal gold. Synaptonemal complexes (SCs) were visualized by means of the surface spreading technique to study both HSP70 expression and the consequences of the lack of HSP70 in the behaviour of the eimerian chromosomes during meiosis. For that purpose E. tenella oocysts were treated with quercetin, a flavonoid that is known to inhibit the synthesis of HSP70. The results showed a close association of HSP70 with the lateral elements (LEs) of the SCs. That association began at the time that SCs were formed and persisted until disassemble. Comparison between distribution of immunogold label over the SCs from non-treated and treated oocysts revealed a decreasing number of gold particles as the concentration of quercetin increased. The current results demonstrated three dose-dependent effects of the quercetin treatment of Eimeria oocysts: a reduction in the HSP70 synthesis; defects in SC formation or desynapsis, and inhibition of sporulation. HSP70, as a structural component of the SCs, may be involved in SC functions such as chromosomal pairing, recombination, or disjunction.     

Lyme disease in transgenic mice expressing the Borrelia burgdorferi flagellin epitope implicated in human neuroborreliosis

Because of an association of human neuroborreliosis with the development of an antibody response against an antigen in neural tissue that cross-reacts with an epitope on the flagellin protein of Borrelia burgdorferi, C3H transgenic mice were created that expressed the flagellin epitope (amino acids 213–224) as a fusion protein with myelin basic protein. The transgenic mice expressed the flagellin epitope selectively in myelinated regions of the nervous system. Both transgenic and non-transgenic mice developed an antibody response to the flagellin epitope during B. burgdorferi infection and both developed arthritis and carditis. However, no lesions were found in the central nervous system of either type of mouse for up to 8 weeks after infection. The data indicate that expression of the flagellin 213–24 epitope in mice does not result in neurologic disease, suggesting that B. burgdorferi flagellin antibodies may not be directly implicated in neuroborreliosis.     

Lyme disease spirochaetes possess an aggrecan‐binding protease with aggrecanase activity

Connective tissues are the most common area of colonization for the Lyme disease spirochaete Borrelia burgdorferi. Colonization is aided by the interaction between numerous bacterial adhesins with components of the extracellular matrix (ECM). Here we describe a novel interaction between B. burgdorferi and the major ECM proteoglycan found in joints, aggrecan. Using affinity chromatography and mass spectrometry we identify two borrelial aggrecan‐binding proteins: the known ECM ligand Bgp (BB0588) and an uncharacterized protease BbHtrA (BB0104). Proteinase K studies demonstrate that BbHtrA is surface exposed. Immunoblots using sera from patients with both early and late Lyme disease establish that BbHtrA is expressed during human disease, immunogenic, and conserved in the three major Lyme disease spirochaete species. Consequences of the interaction between aggrecan and BbHtrA were examined by proteolysis assays. BbHtrA cleaves aggrecan at a site known to destroy aggrecan function and which has been previously observed in the synovial fluid of patients with Lyme arthritis. These data demonstrate that B. burgdorferi possess aggrecan‐binding proteins which may provide the organism with additional capability to colonize connective tissues. Moreover, our studies provide the first evidence that B. burgdorferi possess proteolytic activity which may contribute to the pathogenesis of Lyme arthritis.     

Heat shock protein 60: specific binding of lipopolysaccharide

Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [(3)H]LPS. [(3)H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335-366 of HSP60. Clone 4B9/89 also strongly inhibited [(3)H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331-380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351-370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354-365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.     

HSP60, Bax, apoptosis and the heart

HSP60 has primarily been known as a mitochondrial protein that is important for folding key proteins after import into the mitochondria. It is now clear that a significant amount of HSP60 is also present in the extra-mitochondrial cytosol of many cells. In the heart, this cytosolic HSP60 complexes with Bax, Bak and Bcl-XL, but not with Bcl-2. Reduction in HSP60 expression precipitates apoptosis, but does not alter mitochondrial function. During hypoxia, HSP60 cellular distribution changes, with HSP60 leaving the cytosol, and translocating to the plasma membrane. Total cellular HSP60 does not change until 10 h of reoxygenation; however, release of cytochrome c from the mitochondria occurs prior to reoxygenation, coinciding with the redistribution of HSP60. The changes in HSP60, Bax and cytochrome c during hypoxia can be replicated by ATP depletion. HSP60 has also been shown to accelerate the cleavage of pro-caspase3. Thus, HSP60 has a complex role in apoptosis in the cell. Its binding to Bax under normal conditions suggests a key regulatory role in apoptosis.     

cDNA clones encoding Arabidopsis thaliana and Zea mays mitochondrial chaperonin HSP60 and gene expression during seed germination and heat shock

Mitochondria contain a nuclear-encoded heat shock protein, HSP60, which functions as a chaperonin in the post-translational assembly of multimeric proteins encoded by both nuclear and mitochondrial genes. We have isolated and sequenced full-length complementary DNAs coding for this mitochondrial chaperonin in Arabidopsis thaliana and Zea mays. Southern-blot analysis indicates the presence of a single hsp60 gene in the genome of A. thaliana. There is a high degree of homology at the predicted amino acid levels (43 to 60%) between plant HSP60s and their homologues in prokaryotes and other eukaryotes which indicates that these proteins must have similar evolutionarily conserved functions in all organisms. Northern- and western-blot analyses indicate that the expression of the hsp60 gene is developmentally regulated during seed germination. It is also heat-inducible. Developmental regulation of the (-subunit) of F₁-ATPase, an enzyme complex that is involved in the cyanide-sensitive mitochondrial electron transport system, indicates that imbibed embryos undergo rapid mitochondrial biogenesis through the early stages of germination. Based on the functional role of HSP60 in macromolecular assembly, these data collectively suggest that the presence of higher levels of HSP60 is necessary during active mitochondrial biogenesis, when the need for this protein is greatest in assisting the rapid assembly of the oligomeric protein structures.     

Antibodies against Helicobacter pylori heat shock protein 60 aggravate HSP60-mediated proinflammatory responses

Anti-Helicobacter pylori heat shock protein 60 (HpHSP60) antibodies are usually found in H. pylori-infected patients and are known to be associated with the progression of gastric diseases. However, the effects of these antibodies on the functions of HpHSP60 have not been identified. This study aims to investigate the effects of the interaction between anti-HSP60 antibodies and HpHSP60 on inflammatory responses. Anti-HpHSP60 polyclonal sera and monoclonal antibodies (mAbs) were produced to evaluate their effects on HpHSP60-induced IL-8 and TNF-α activity. The results indicated that anti-HpHSP60 polyclonal sera collected from patients infected with H. pylori or from rabbit and mice immunized with HpHSP60 could significantly enhance HpHSP60-mediated IL-8 and TNF-α secretion from monocytic THP-1 cells. Similar effects were also found with anti-HpHSP60 mAbs. Further analysis revealed that this phenomenon was only carried out by anti-HpHSP60 antibody but not by other non-specific mAbs. Moreover, the non-specific mAbs decreased the synergism of HpHSP60 and anti-HpHSP60 mAbs in proinflammatory cytokine induction. Herein, we have examined the role of anti-HpHSP60 antibody in host immune responses for the first time. This study demonstrated that H. pylori HSP60/mAbs could modulate helicobacterial pathogenesis by increasing IL-8 and TNF-α production. The pathogen-specific antibodies may execute potential immune functions rather than recognize or neutralize microbes.     

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook‐associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non‐motile mutant cells that are unable to assemble flagellar filaments and pentagon‐shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella‐deficient mutants (i.e., in the hook, rod, or MS‐ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.     

The Effect of Manganese-induced Cytotoxicity on mRNA Expressions of HSP27, HSP40, HSP60, HSP70 and HSP90 in Chicken Spleen Lymphocytes in Vitro

The purpose of this study was to investigate the effect of manganese (Mn)-induced cytotoxicity on heat shock proteins in chicken spleen lymphocytes. Lymphocytes were cultured in medium in the absence and presence of MnCl₂ (2?×?10^?4, 4?×?10^?4, 6?×?10^?4, 8?×?10^?4, 10?×?10^?4, and 12?×?10^?4 mmol/L) for 12, 24, 36, and 48 h in vitro. Then, the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 were examined by real-time quantitative PCR. The results showed that the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in all treatment groups at all time points, except mRNA levels of HSP27 at 48 h, had the same tendency. As manganese concentration increased, the mRNA expression of the heat shock proteins first increased and then decreased. In other words, we demonstrated that the mRNA expression of the heat shock proteins was induced at lower concentrations of manganese and was inhibited at higher concentrations. Mn had a dosage-dependent effect on HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression in chicken spleen lymphocytes in vitro.     

Partial Analysis of the Flagellar Antigenic Determinant Recognized by a Monoclonal Antibody to Clostridium tyrobutyricum

In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D -glucose and N-acetyl-glucosamine in a molar ratio of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D -glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The overall stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain α(1→4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.     

Heat Shock Protein 60 (HSP60) Response of <Emphasis Type="Italic">Plationus patulus</Emphasis> (Rotifera: Monogononta) to Combined Exposures of Arsenic and Heavy Metals

Organisms produce stress proteins as a response to natural and anthropogenic environmental changes. Induction of stress proteins has been reported in a variety of aquatic organisms, including rotifers, exposed to pollutants. Past studies on stress protein responses of rotifers have focused on exposure to single toxicants. In this study the rotifer Plationus patulus was exposed singly and in combination to various concentrations of As, Cr, Cu, Ni, Pb, and Zn. Following exposure, total protein was quantified (Bradford method) and stress protein 60 (HSP60) was identified using Western blotting. P. patulus induced HSP60 as a response to single exposures to Cr, Cu, Ni, Pb and Zn. HSP60 expression was increased (2 fold) in rotifers exposed to these single elements at both low and high concentrations as compared to unexposed rotifers. Arsenic exposure resulted in a 2 fold decrease in HSP induction. In rotifers exposed to metal mixtures, HSP60 was induced by the presence of As–Zn, As–Cr–Cu–Pb, As–Cr–Cu, As–Cr–Cu–Ni and As–Cr–Cu–Ni–Pb combinations in the media. HSP60 response to As and heavy metals toxicity depends on the type and number of elements present in the media as well as their concentrations and length of the exposure time.     

The HtrA protease of Borrelia burgdorferi degrades outer membrane protein BmpD and chemotaxis phosphatase CheX

Borrelia burgdorferi, the spirochaetal agent of Lyme disease, codes for a single HtrA protein, HtrABb (BB0104) that is homologous to DegP of Escherichia coli (41% amino acid identity). HtrABb shows physical and biochemical similarities to DegP in that it has the trimer as its fundamental unit and can degrade casein via its catalytic serine. Recombinant HtrABb exhibits proteolytic activity in vitro, while a mutant (HtrABbS198A) does not. However, HtrABb and DegP have some important differences as well. Native HtrABb occurs in both membrane‐bound and soluble forms. Despite its homology to DegP, HtrABb could not complement an E. coli DegP deletion mutant. Late stage Lyme disease patients, as well as infected mice and rabbits developed a robust antibody response to HtrABb, indicating that it is a B‐cell antigen. In co‐immunoprecipitation studies, a number of potential binding partners for HtrABb were identified, as well as two specific proteolytic substrates, basic membrane protein D (BmpD/BB0385) and chemotaxis signal transduction phosphatase CheX (BB0671). HtrABb may function in regulating outer membrane lipoproteins and in modulating the chemotactic response of B. burgdorferi.