The Lyme disease spirochete, Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whether B. burgdorferi spirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawed B. burgdorferi spirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). In contrast to findings in previous studies, where B. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-alpha release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-alpha-inducing activity in known low-, intermediate-, and high-passage B. burgdorferi B31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passage B. burgdorferi spirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells. (+info)
Molecular and evolutionary analysis of Borrelia burgdorferi 297 circular plasmid-encoded lipoproteins with OspE- and OspF-like leader peptides.
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We previously described two OspE and three OspF homologs in Borrelia burgdorferi 297 (D. R. Akins, S. F. Porcella, T. G. Popova, D. Shevchenko, S. I. Baker, M. Li, M. V. Norgard, and J. D. Radolf, Mol. Microbiol. 18:507-520, 1995; D. R. Akins, K. W. Bourell, M. J. Caimano, M. V. Norgard, and J. D. Radolf, J. Clin. Investig. 101:2240-2250, 1998). In this study, we characterized four additional lipoproteins with OspE/F-like leader peptides (Elps) and demonstrated that all are encoded on plasmids homologous to cp32 and cp18 from the B31 and N40 strains, respectively. Statistical analysis of sequence similarities using the binary comparison algorithm revealed that the nine lipoproteins from strain 297, as well as the OspE, OspF, and Erp proteins from the N40 and B31 strains, fall into three distinct families. Based upon the observation that these lipoproteins all contain highly conserved leader peptides, we now propose that the ancestors of each of the three families arose from gene fusion events which joined a common N terminus to unrelated proteins. Additionally, further sequence analysis of the strain 297 circular plasmids revealed that rearrangements appear to have played an important role in generating sequence diversity among the members of these three families and that recombinational events in the downstream flanking regions appear to have occurred independently of those within the lipoprotein-encoding genes. The association of hypervariable regions with genes which are differentially expressed and/or subject to immunological pressures suggests that the Lyme disease spirochete has exploited recombinatorial processes to foster its parasitic strategy and enhance its immunoevasiveness. (+info)
Detection of the agent of human granulocytic ehrlichiosis (HGE) in UK ticks using polymerase chain reaction.
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Nymphal Ixodes ricinus ticks collected from woodland areas in South Wales, UK, were tested using the polymerase chain reaction for the presence both of the causative agent of human granulocytic ehrlichiosis (HGE) and Borrelia burgdorferi. Twenty-two of 60 (37%) ticks were found positive in the PCR for B. burgdorferi and 4/60 (7%) for the HGE agent. One tick was found positive both for B. burgdorferi and HGE agent. Our findings imply the presence of the HGE agent in UK ticks and the finding of a tick apparently containing both pathogens underlines the potential for concurrent infection with HGE agent and B. burgdorferi to occur after a single tick-bite. Based on our observations, we conclude that there may be a need to consider a range of pathogens both in laboratory investigation and clinical management of suspected tick-borne disease in the UK, particularly where there is a clinical presentation atypical of Lyme borreliosis alone. (+info)
Esterases in serum-containing growth media counteract chloramphenicol acetyltransferase activity in vitro.
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The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that of B. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility of E. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. coli bearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo. (+info)
Interaction of Borrelia burgdorferi with peripheral blood fibrocytes, antigen-presenting cells with the potential for connective tissue targeting.
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BACKGROUND: Borrelia Burgdorferi has a predilection for collagenous tissue and can interact with fibronectin and cellular collagens. While the molecular mechanisms of how B. burgdorferi targets connective tissues and causes arthritis are not understood, the spirochetes can bind to a number of different cell types, including fibroblasts. A novel circulating fibroblast-like cell called the peripheral blood fibrocyte has recently been described. Fibrocytes express collagen types I and III as well as fibronectin. Besides playing a role in wound healing, fibrocytes have the potential to target to connective tissue and the functional capacity to recruit, activate, and present antigen to CD4(+) T cells. MATERIALS AND METHODS: Rhesus monkey fibrocytes were isolated and characterized by flow cytometry. B. burgdorferi were incubated with human or monkey fibrocyte cultures in vitro and the cellular interactions analyzed by light and electron microscopy. The two strains of B. burgdorferi studied included JD1, which is highly pathogenic for monkeys, and M297, which lacks the cell surface OspA and OspB proteins. RESULTS: In this study, we demonstrate that B. burgdorferi binds to both human and monkey (rhesus) fibrocytes in vitro. This process does not require OspA or OspB. In addition, the spirochetes are not phagocytosed but are taken into deep recesses of the cell membrane, a process that may protect them from the immune system. CONCLUSIONS: This interaction between B. burgdorferi and peripheral blood fibrocytes provides a potential explanation for the targeting of spirochetes to joint connective tissue and may contribute to the inflammatory process in Lyme arthritis. (+info)
Isolation of Lyme disease Borrelia from puffins (Fratercula arctica) and seabird ticks (Ixodes uriae) on the Faeroe Islands.
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This is the first report on the isolation of Lyme disease Borrelia from seabirds on the Faeroe Islands and the characteristics of its enzootic cycle. The major components of the Borrelia cycle include the puffin (Fratercula arctica) as the reservoir and Ixodes uriae as the vector. The importance of this cycle and its impact on the spread of human Lyme borreliosis have not yet been established. Borrelia spirochetes isolated from 2 of 102 sampled puffins were compared to the borreliae previously obtained from seabird ticks, I. uriae. The rrf-rrl intergenic spacer and the rrs and the ospC genes were sequenced and a series of phylogenetic trees were constructed. Sequence data and restriction fragment length polymorphism analysis grouped the strains together with Borrelia garinii. In a seroepidemiological survey performed with residents involved in puffin hunting on the Faeroe Islands, 3 of 81 serum samples were found to be positive by two commonly used clinical tests: a flagellin-based enzyme-linked immunosorbent assay (ELISA) and Western blotting. These three positive serum samples also had high optical density values in a whole-cell ELISA. The finding of seropositive Faeroe Islanders who are regularly exposed to I. uriae indicate that there may be a transfer of B. garinii by this tick species to humans. (+info)
Rapid and sensitive quantification of Borrelia burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR.
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The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization of B. burgdorferi quantities to the mouse nidogen gene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples. (+info)
Proteome composition and codon usage in spirochaetes: species-specific and DNA strand-specific mutational biases.
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The genomes of the spirochaetes Borrelia burgdorferi and Treponema pallidum show strong strand-specific skews in nucleotide composition, with the leading strand in replication being richer in G and T than the lagging strand in both species. This mutation bias results in codon usage and amino acid composition patterns that are significantly different between genes encoded on the two strands, in both species. There are also substantial differences between the species, with T.pallidum having a much higher G+C content than B. burgdorferi. These changes in amino acid and codon compositions represent neutral sequence change that has been caused by strong strand- and species-specific mutation pressures. Genes that have been relocated between the leading and lagging strands since B. burgdorferi and T.pallidum diverged from a common ancestor now show codon and amino acid compositions typical of their current locations. There is no evidence that translational selection operates on codon usage in highly expressed genes in these species, and the primary influence on codon usage is whether a gene is transcribed in the same direction as replication, or opposite to it. The dnaA gene in both species has codon usage patterns distinctive of a lagging strand gene, indicating that the origin of replication lies downstream of this gene, possibly within dnaN. Our findings strongly suggest that gene-finding algorithms that ignore variability within the genome may be flawed. (+info)