ABSTRACT
A hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin (an 18-kDa fragment) and a 59-kDa fragment (lacking the N-terminal part) of the 83-kDa protein has been constructed by genetic engineering. It was expressed as a nonfusion protein of an apparent molecular weight of 77,000 in Escherichia coli. The suitability of this new antigen for the diagnosis of Lyme disease was tested by immunoblotting; for comparison, the recombinant variable region of the flagellin, the 18-kDa fragment (p18), and the whole recombinant 83-kDa protein (p83), both expressed in E. coli, were used. A total of 120 serum samples from various stages of Lyme disease, which were positive in two serological assays, a passive hemagglutination assay and an indirect immunofluorescence assay, were tested. By indirect immunofluorescence, 74 samples were positive for immunoglobulin G (IgG) antibodies and 72 were positive for IgM antibodies. Of these serum samples, 69 of 74 (93%) contained IgG antibodies against p18 and/or p83, and IgG antibodies were detected by the hybrid protein in 67 (90%) samples. IgM antibodies against p18 and/or p83 were detected in 60 of 72 (83%) serum samples, and 57 (79%) serum samples were reactive with the hybrid protein. Twenty serum samples of patients with a history of syphilis and 40 serum samples, negative in routine B. burgdorferi serology, were tested as controls. The hybrid protein, made up of specific epitopes of an early (p18) and late (p83) antigen, is recognized by almost the same number of patient serum samples as the individual antigens.
Subject(s)
Antigens, Bacterial , Borrelia burgdorferi Group/immunology , Flagellin/immunology , Lyme Disease/diagnosis , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Base Sequence , Borrelia burgdorferi Group/genetics , Cloning, Molecular , DNA Primers/genetics , Escherichia coli/genetics , Flagellin/chemistry , Flagellin/genetics , Genes, Bacterial , Humans , Immunodominant Epitopes/genetics , Lyme Disease/immunology , Molecular Sequence Data , Molecular Weight , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Serologic TestsABSTRACT
A total of 17 B. burgdorferi isolates from various sources were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, restriction enzyme analysis, Southern hybridization with probes complementary to unique regions of evolutionarily conserved genes (16S rRNA and fla), and direct sequencing of in vitro polymerase chain reaction-amplified fragments of the 16S rRNA gene. Three groups were distinguished on the basis of phenotypic and genotypic traits, the latter traced to the nucleotide sequence level.
Subject(s)
Borrelia burgdorferi Group/genetics , RNA, Ribosomal, 16S/genetics , Bacterial Proteins/analysis , Base Sequence , Blotting, Southern , Borrelia burgdorferi Group/isolation & purification , DNA Fingerprinting , DNA Restriction Enzymes/metabolism , DNA, Bacterial , Electrophoresis, Polyacrylamide Gel , Genes, Bacterial , Genotype , Molecular Sequence Data , Phenotype , Polymerase Chain ReactionABSTRACT
The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis.
Subject(s)
Borrelia burgdorferi Group/genetics , Flagellin/genetics , Genes, Bacterial , Amino Acid Sequence , Base Sequence , Blotting, Northern , Chromosome Mapping , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Epitopes/isolation & purification , Gene Expression , Molecular Sequence Data , Sequence Homology, Nucleic Acid , Transcription, Genetic , Transformation, BacterialABSTRACT
The 41 kDa flagellar protein of Borrelia burgdorferi appears to be an immunodominant antigen producing an early and strong response in most, if not all, individuals during infection in humans. It would represent a very good antigen for serodiagnosis of Lyme disease, if its crossreactivity with flagella of other bacteria was low. To gain information on this point we isolated the B. burgdorferi flagellin by preparative two-dimensional electrophoresis for N-terminal amino acid analysis. By comparing the N-terminal amino acid sequences of flagellar proteins from other eubacteria we found that the first six out of twenty nine amino acids were identical to the Treponema pallidum and Treponema phagedenis 'class B' flagellins. All 29 N-terminal residues exhibited a moderate inter-genus homology (44-55%), in contrast to the high degree (67-95%) of inter-species conservation of the treponemal 'class B' flagellar N-terminal sequences. There was little similarity to other flagellins except the B. subtilis flagellar protein.
