Decorin-binding sites in the adhesin DbpA from Borrelia burgdorferi: a synthetic peptide approach

Lyme disease is caused by the spirochete Borrelia burgdorferi following transmission from infected Ixodes ticks to human hosts. Following colonization of the skin, spirochetes can disseminate throughout the body, resulting in complications that can include ocular, cardiac, neural, and skeletal disease. We have previously shown that B. burgdorferi expresses two closely related decorin-binding adhesins (DbpA and DbpB) of the MSCRAMM (microbial surface component recognizing adhesive matrix molecule) type that can mediate bacterial attachment to extracellular matrices in the host. Furthermore, three Lys residues in DbpA appear to be critical for the binding of DbpA to decorin. We have now characterized the interaction of DbpA and decorin further by using a synthetic peptide approach. We synthesized a panel of peptides that spanned the DbpA sequence and examined their ability to inhibit the binding of intact DbpA to decorin. From these studies, we identified a decorin-binding peptide that lost this activity if the sequence was either scrambled or if a critical Lys residue was chemically modified. A minimal decorin-binding peptide was identified by examining a set of truncated peptides. One peptide is proposed to contain the primary decorin-binding site in DbpA. By comparing the amino acid sequences of 29 different DbpA homologs from different B. burgdorferi sensu lato isolates, we discovered that the identified decorin-binding sequence was quite variable. Therefore, we synthesized a new panel of peptides containing the putative decorin-binding sequence of the different DbpA homologs. All of these peptides were active in our decorin-binding assay, and consensus decorin binding motifs are discussed.     

Borrelia burgdorferi adhesins identified using in vivo phage display

Borrelia burgdorferi, the agent of Lyme disease, disseminates from the site of deposition by Ixodes ticks to cause systemic infection. Dissemination occurs through the circulation and through tissue matrices, but the B. burgdorferi molecules that mediate interactions with the endothelium in vivo have not yet been identified. In vivo selection of filamentous phage expressing B. burgdorferi protein fragments on the phage surface identified several new candidate adhesins, and verified the activity of one adhesin that had been previously characterized in vitro. P66, a B. burgdorferi ligand for beta(3)-chain integrins, OspC, a protein that is essential for the establishment of infection in mammals, and Vls, a protein that undergoes antigenic variation in the mammal, were all selected for binding to the murine endothelium in vivo. Additional B. burgdorferi proteins for which no functions have been identified, including all four members of the OspF family and BmpD, were identified as candidate adhesins. The use of in vivo phage display is one approach to the identification of adhesins in pathogenic bacteria that are not easily grown in the laboratory, or for which genetic manipulations are not straightforward.     

Structural analysis of glycolipids from Borrelia burgdorferi

In this study the lipids of Borrelia burgdorferi, the causative agent of Lyme disease, were analyzed. Lipids comprise about 25-30% of the cell dry weight. The lipid fraction could be separated by HPTLC into 11 components. Staining of these components revealed two glycolipids and two phospholipids. The glycolipids represented about 50% of the total lipids and comprised only galactose as monosaccharide constituents. By means of mass spectrometric and gas chromatographic analysis both glycolipids could be identified as alpha-galactosyl-diacylglycerolipids with different fatty acid compositions. The phospholipids were identified as phosphatidylcholine and phosphatidylglycerol. Immunoassays with sera from patients with Lyme disease showed antibody reactivity only to the glycolipids, which was present in all stages of the disease. Other lipid components seemed to be non-immunogenic in Lyme disease. The glycolipids of B. burgdorferi may be, thus, considered promising candidates for diagnosis and possibly also for vaccination.     

Chemotaxis in Borrelia burgdorferi

Borrelia burgdorferi is a motile spirochete which has been identified as the causative microorganism in Lyme disease. The physiological functions which govern the motility of this organism have not been elucidated. In this study, we found that motility of B. burgdorferi required an environment similar to interstitial fluid (e.g., pH 7.6 and 0.15 M NaCl). Several methods were used to detect and measure chemotaxis of B. burgdorferi. A number of chemical compounds and mixtures were surveyed for the ability to induce positive and negative chemotaxis of B. burgdorferi. Rabbit serum was found to be an attractant for B. burgdorferi, while ethanol and butanol were found to be repellents. Unlike some free-living spirochetes (e.g., Spirochaeta aurantia), B. burgdorferi did not exhibit any observable chemotaxis to common sugars or amino acids. A method was developed to produce spirochete cells with a self-entangled end. These cells enabled us to study the rotation of a single flagellar bundle in response to chemoattractants or repellents. The study shows that the frequency and duration for pausing of flagella are important for chemotaxis of B. burgdorferi.     

全沟硬蜱体内伯氏疏螺旋体的分离及特征

本文报道从黑龙江省全沟硬蜱分离的伯氏疏螺旋体H7株的特征。H7株细胞长9．8—26．5μm,宽0．1 3—0．35μm,有l—11个波,波长1．2—3．Oμm,波幅0．59一1．13μm,两端尖乩每端有7根鞭毛,菌体左旋。体外培养最适温度为31℃,具有2lk、32k、34k主要结构和抗原蛋白,能与新疆及黑龙江莱姆病人血清反应。这些结果表明,该菌株具备伯氏疏螺旋体的特征,它是与其它地区或媒介分离株不同的“亚型”。     

Borrelia burgdorferi sensu lato infection in mosquitoes from Szczecin area

The aim of the study was to determine the level of infection in mosquitoes with spirochetes Borrelia burgdorferi sensu lato in the woody areas of Szczecin. The mosquitoes were collected from May to September 2003. The spirochetes, Borrelia burgdorferi s. l., present in mosquitoes were detected in mosquitoes with indirect immunofluorescence assay (IFA) using rabbit anti-Borrelia burgdorferi antibodies and goat anti-rabbit IgG marked with fluorescein isocyanate (FITC). A total of 1557 females and 58 males were collected. They represented the genera Aedes (63%) and Culex (37%). The infection level of the mosquitoes from the area studied amounted to 1.7%. The results of the present study confirm the potential of these arthropods to spread Lyme borreliosis.     

Requirements for Borrelia burgdorferi plasmid maintenance

Borrelia burgdorferi has multiple linear and circular plasmids that are faithfully replicated and partitioned as the bacterium grows and divides. The low copy number of these replicons implies that active partitioning contributes to plasmid stability. Analyzing the requirements for plasmid replication and partition in B. burgdorferi is complicated by the complexity of the genome and the possibility that products may act in trans. Consequently, we have studied the replication-partition region (bbb10-13) of the B. burgdorferi 26kb circular plasmid (cp26) in Escherichia coli, by fusion with a partition-defective miniF plasmid. Our analysis demonstrated that bbb10, bbb11, and bbb13 are required for stable miniF maintenance, whereas bbb12 is dispensable. To validate these results, we attempted to inactivate two of these genes in B. burgdorferi. bbb12 mutants were obtained at a typical frequency, suggesting that the bbb12 product is dispensable for cp26 maintenance as well. We could not directly measure cp26 stability in the bbb12 mutant, because cp26 carries essential genes, and bacteria that have lost cp26 are inviable. Conversely, we were unable to inactivate bbb10 on cp26 of B. burgdorferi. Our results suggest that bbb12 is dispensable for cp26 maintenance, whereas bbb10, bbb11, and bbb13 play crucial roles in that process.     

Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii.

The growth rate of Borrelia burgdorferi and Borrelia hermsii in BSK II medium prepared with cysteine-free or cysteine-containing (0.185-5.92 mM) CMRL 1066 medium was studied. In media with cysteine-free CMRL 1066, growth of borreliae was detectable, although it was reduced by approximately 80%. Bacterial growth was maximal when the concentration of cysteine in CMRL 1066 reached 1.48 mM, which represents the standard cysteine concentrations of the medium; higher concentrations inhibited the growth of borreliae. Cysteine incorporation, measured by the uptake of radiolabeled cysteine, showed that cysteine enters B. burgdorferi and B. hermsii cells by passive diffusion. Labeling studies of borreliae with [35S]cysteine indicated that B. burgdorferi has several cysteine-containing proteins, including ones at 22, 30 (OspA), and 34 kDa (OspB), whereas B. hermsii showed only two [35S]cysteine-incorporating proteins, at 22 and 24 kDa, which were exposed onto the outer cell surface. In addition, most of the cysteine-incorporating proteins could be biosynthetically radiolabeled when bacterial cells were grown in vitro with [3H]palmitate, and the differences in cysteine incorporation observed between B. burgdorferi and B. hermsii were found to be correlated with differences in lipoproteins.     

Borrelia burgdorferi inhibits human neutrophil functions

Outer surface proteins OspA and OspB are among the most prominent Borrelia burgdorferi surface molecules. We constructed OspAB and OspA complementation mutants of B. burgdorferi Osp-less strain B313 and investigated the role of these surface proteins in the interactions of B. burgdorferi, human neutrophils and the complement system. We found that (1) OspB inhibits the phagocytosis and oxidative burst of human neutrophils at low serum concentrations, whereas OspA induces the oxidative burst in neutrophils; (2) OspB may have an inhibiting role in serum sensitivity and complement activation; (3) all studied strains inhibit the chemotaxis of human neutrophils specifically towards fMLP but not towards C5a, regardless of their Osp expression. These results suggest that although OspA and OspB are co-ordinately transcribed, they differ in their effects on human neutrophil functions. Our findings suggest that B. burgdorferi exploits a wide variety of immune evasion mechanisms, besides previously documented complement resistance, to survive in the vertebrate host.     

Penicillin-binding proteins in Borrelia burgdorferi.

Penicillin-binding proteins were identified in Borrelia burgdorferi membranes. A 94-kilodalton penicillin-binding protein was the first to be labeled with tritiated penicillin and was the first band to disappear in a competition experiment. Its binding ability was destroyed when membranes were preboiled. In addition, several of these penicillin-binding proteins comigrated with bands previously identified as surface proteins.     

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     

Borrelia burgdorferi possesses a collagenolytic activity

Abstract Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi , an invasive spirochete. B. burgdorferi has a predilection for collagenous tissue and one major clinical manifestation of the disease is arthritis. We have identified a collagenolytic activity in B. burgdorferi detergent lysates using iodinated gelatin as well as iodinated pepsinized human collagen types II and IV as protein substrates. In addition, we describe several proteolytic activities in B. burgdorferi with molecular masses greater than 200 kDa on sodium dodecyl sulfate polyacrylamide gels containing copolymerized gelatin. We propose that the collagenolytic activity of B. burgdorferi has a role in invasion, in the pathogenesis of Lyme arthritis, and perhaps also in other manifestations of Lyme borreliosis.     

The many faces of Borrelia burgdorferi

In this review we describe several genetic regulatory mechanisms adopted by the agent of Lyme disease, Borrelia burgdorferi, to sense and adapt to different host and environmental conditions either in vitro or in vivo. This regulation results in the increased or decreased synthesis of several proteins whose levels are believed to play key roles in the ability of B. burgdorferi to cycle between both arthropod and mammalian hosts. Moreover, the differential synthesis of these proteins serves to modulate the response of B. burgdorferito signals in the requisite host and may also, in some cases, function as virulence determinants of this spirochete. Elucidation of these mechanisms will help in the understanding of the pathogenicity of B. burgdorferi as well as aid in identifying proteins that are important during different stages of infection.     

Borrelia burgdorferi infection in Ixodes ricinus from habitats in Denmark

A total of 2647 ticks of the genus Ixodes was sampled by flagging the vegetation in thirty-one sites in eastern Jutland, Denmark. All ticks were identified as Ixodes ricinus Linnaeus. A total of 317 ticks (202 nymphs and 115 adults) from three different sites were examined for the spirochaete Borrelia burgdorferi Johnson et al. by indirect fluorescent antibody staining; the frequency of infection varied from 7% to 22%. It is concluded that I. ricinus, known to be the most common tick in Denmark, is the vector largely responsible for the transmission of B. burgdorferi in this country.     

Isolation of Borrelia burgdorferi from Peromyscus leucopus in Oklahoma.

Borrelia burgdorferi was isolated from a field-caught Peromyscus leucopus from central Oklahoma (USA). The strain was identified as B. burgdorferi by reaction with monoclonal antibody H5332 specific for the outer surface protein OspA of B. burgdorferi. This represents the first isolation of B. burgdorferi from a wild mouse outside of the normal range of the known vectors Ixodes dammini and I. pacificus.     

Functional properties of Borrelia burgdorferi recA

Functions of the Borrelia burgdorferi RecA protein were investigated in Escherichia coli recA null mutants. Complementation with B. burgdorferi recA increased survival of E. coli recA mutants by 3 orders of magnitude at a UV dose of 2,000 microJ/cm(2). The viability at this UV dose was about 10% that provided by the homologous recA gene. Expression of B. burgdorferi recA resulted in survival of E. coli at levels of mitomycin C that were lethal to noncomplemented hosts. B. burgdorferi RecA was as effective as E. coli RecA in mediating homologous recombination in E. coli. Furthermore, E. coli lambda phage lysogens complemented with B. burgdorferi recA produced phage even in the absence of UV irradiation. The level of phage induction was 55-fold higher than the level in cells complemented with the homologous recA gene, suggesting that B. burgdorferi RecA may possess an enhanced coprotease activity. This study indicates that B. burgdorferi RecA mediates the same functions in E. coli as the homologous E. coli protein mediates. However, the rapid loss of viability and the absence of induction in recA expression after UV irradiation in B. burgdorferi suggest that recA is not involved in the repair of UV-induced damage in B. burgdorferi. The primary role of RecA in B. burgdorferi is likely to be a role in some aspect of recombination.     

Efficient targeted mutagenesis in Borrelia burgdorferi

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A(1). The utility of the coumermycin-resistant gyrB(r) gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.