Lyme borreliosis is a tick-born disease caused by the spirochete Borrelia burgdorferi. The aim of this research was molecular typing of different strains of B. burgdorferi and protective efficacy of outer surface protein in animal model. Seven strains of B. burgdorferi isolated from skin and CSF of Lyme disease patients were compared using PCR-RFLP as well as DNA and amino acid sequences. The gene coding for Outer Surface Protein A (OspA) was cloned and recombinant protein was expressed. Immunization experiment was conducted on gerbils with purified recombinant OspA protein. The OspA gene sequence of B. afzelii and B. garinii showed 83.76% similarity and calculated amino acid identity of 84% with each other and 82.5% similarity to B31 strain. DK6 was 100% similar to Pbi, 99.6% to Ptrob isolate and 88% to B31 strain. Cloning and expression of OspA from DK6 strain in E. coli has shown lipoprotein with molecular weight of 32 kDa. Immuno blot of recombinant protein from DK6 with rabbit anti DK1 revealed strong reaction with OspA from DK6 strain. Immunization experiment in gerbils with three doses of rOspA protein revealed that all immunized animals either with OspA fusion protein alone or combined with adjuvant were protected against infection with DK1 or DK6 pathogenic strains.

ABSTRACT Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis , and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae . Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis , was sequenced from 18 rifampin-resistant Legionella isolates representing four species ( L. bozemanii , L. longbeachae , L. micdadei , and L. pneumophila ), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae .

Self proteins are processed and presented by APCs in the same way as foreign proteins. Presentation of fragments derived from self proteins does not, however, lead to Th cell stimulation because of T cell tolerance. In this study, a novel approach was used to investigate whether B cell tolerance toward a self Ag could be due to the absence of this Th cell recognition. The highly conserved nonimmunogenic protein ubiquitin was used as a model protein. Two modified ubiquitin molecules were constructed with ubiquitin segments exchanged either with the T cell epitope, OVA(325-336), which binds to the mouse A(d) MHC class II molecule, or with the T cell epitope, hen egg lysozyme(50-61), which binds to the A(k) molecule. Mice were immunized with the resulting proteins. Both modified proteins elicited strong autoantibody responses toward soluble native ubiquitin, demonstrating that insertion of a single foreign T cell epitope can overcome the B cell nonresponsiveness. The T cell regulatory role of one of the inserted foreign T cell epitopes in ubiquitin was studied, and at least two different Th cell specificities were found to operate in the response. The T cells were directed against: 1) the inserted epitope, and 2) a combination of the inserted epitope and parts of the neighboring ubiquitin regions. Therefore, the absence of T cell help seems to be an important reason for B cell tolerance toward self proteins.

To study the structure and function of the Legionella flagellum, we screened a genomic L. micdadei library in Escherichia coli for expression of the flagellin (Fla) subunit. One recombinant clone, JM105 (pHI5588), producing a truncated Fla protein of 40.5 kDa was identified. The plasmid pHI5588 carried a L. micdadei DNA insert of 5 kb, containing ca 95% of the fla gene. The complete DNA sequence of the L. micdadei fla gene was obtained by combining sequence data from pHI5588 with results using a polymerase chain reaction‐based system for genome walking (vectoretle PCR). The L. micdadei fla gene shared a high degree of homology with other flagellin genes in the amino‐ and carboxy termini, whereas the central region was found to be nonconserved. The fla sequence will facilitate the cloning of Fla proteins from other Legionella species and the study of flagella in the pathogenesis of Legionnaires' disease.

By alignment of GroEL amino acid sequences from four distantly related bacteria two highly conserved domains were identified. Two oligonucleotides complementary to the conserved domains were designed based on the preferred Pseudomonas aeruginosa codon usage. The primers were used in the PCR to amplify a 900-base fragment of the P. aeruginosa groEL gene. The fragment was sequenced and the partial GroEL sequence was expanded by vectorette PCR upstream and downstream to cover the complete P. aeruginosa groE operon. The same technique was used to sequence the Burkholderia cepacia (formerly Pseudomonas cepacia) groE operon and the region immediately upstream of groES. The B. cepacia groE operon is preceded by typical -10 and -35 heat shock expression signals. A total of 2041 and 2139 bp was sequenced from P. aeruginosa and B. cepacia respectively. Each revealed two open reading frames encoding two proteins with a predicted molecular mass of 10 and 57 kDa, corresponding to GroES and GroEL respectively. The GroEL proteins show an interspecies amino acid homology of 71%, and 73% with E. coli GroEL. Both GroEL proteins are 52% homologous to the corresponding human mitochondrial GroEL protein. The sequence data confirm the existence of highly conserved structures, which could be functionally important for the concerted action of GroEL and GroES in the folding and assembly of other proteins, and possibly in the initiation of autoimmune diseases.

Quantitative crossed immunoelectrophoresis was used to evaluate the antigenic similarity of Pseudomonas aeruginosa and Pseudomonas cepacia GroEL proteins. We found that the two proteins showed 75% identity. By using a panel of monoclonal antibodies against the P. aeruginosa GroEL protein, we identified 10 monoclonal antibodies which cross-reacted with the P. cepacia GroEL protein and 21 monoclonal antibodies which recognized type-specific epitopes on the P. aeruginosa GroEL protein. In crossed immunoelectrophoresis two different fractions of GroEL reactive material could be resolved. These fractions showed a reaction of partial identity. Examination of the two immunoprecipitates by Western blotting, showed that both fractions consisted of anti-60 kDa GroEL reactive protein. One fraction, in addition, contained LPS with a characteristic 'ladder' reaction in modified Western blotting. We therefore conclude that this fraction represents a complex between LPS and GroEL.

The success of an invading organism must depend on several cytoplasmic, surface-associated and secreted factors. The technical difficulties in handling pathogenic spirochetes like Treponema pallidum and Borrelia burgdorferi have made it difficult to define specific factors involved in entry and long-term survival. The problem of defining virulence factors has been attacked by several strategies: T. pallidum secretes a number of immunogenic low molecular mass proteins. The most predominant are of molecular weight 15.5 and 22 kDa. Preliminary data suggest that antibodies against these proteins induce protective immunity in rabbits experimentally infected with T. pallidum. Many potentially important surface-associated antigens of T. pallidum have now been cloned and characterized. Two of these, TpD and TpE, are lipoproteins which exhibit characteristic size heterogeneity. The apparent molecular weight of TpE from T. pallidum and T. pertenue are different. The clinical symptoms in syphilis and yaws are very different, but sequence analysis of TpE has shown that the TpE proteins are indeed very similar in the two strains. This observation makes it unlikely that heterogeneity of TpE can account for the different clinical symptoms of syphilis and yaws. Sequence data for another newly sequenced surface-associated antigen of T. pallidum (molecular weight 41 kDa) indicate that this protein is involved in glucose transport and chemotaxis/motility. Intracellular factors like the molecular chaperonin GroEL have been documented both in treponemes and borreliae. This stress protein is involved in cellular repair processes and folding/assembly of protein subunits. Indirect evidence suggests that GroEL affects the ability of spirochetes to survive in the stressful environment of the infected host. Several lines of evidence suggest that the Osp proteins of Borrelia are important for host/parasite interaction. Further support for this idea has come from studies of a series of monoclonal antibodies against OspA. A monoclonal antibody against OspA (9B3D) is able to block attachment of B. burgdorferi to a cell monolayer. Borrelia loses infectivity after several passages in vitro. The loss of pathogenicity is associated with loss of specific plasmids and proteins. One of the low-passage-associated proteins (Lap30) has been cloned and sequenced. Lap30 is a lipoprotein encoded by a 38-kb plasmid, not present in high passage B. burgdorferi. Aberrant immunological processes induced by the lipopolysaccharide component of Treponema hyodysenteriae could explain the dramatic intestinal lesions in swine dysenteriae. But analysis by TLC reveals that the LPS of this treponeme is different from classical Salmonella LPS.(ABSTRACT TRUNCATED AT 400 WORDS)

A polymerase chain reaction (PCR) was developed for identification of Borrelia burgdorferi in biological specimens. The diagnostic efficiency was compared with that of in vitro culture. A primer set specifying a 791-bp DNA fragment of the B. burgdorferi B31 flagellin gene was used. Amplified DNA sequences were analyzed by agarose gel electrophoresis, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and Southern blot hybridization with a 32P-labeled probe. By using purified B. burgdorferi DNA, the detection limit of the assay was approximately 0.002 pg of DNA, corresponding to one copy of the B. burgdorferi genome. By using in vitro-cultivated B. burgdorferi without prior DNA purification as the template DNA, 2 to 20 organisms could be detected. A 791-bp DNA fragment was amplified from all of 18 different B. burgdorferi strains tested, as well as from Borrelia hermsii and Borrelia anserina but not from Treponema pallidum. The efficacy of the PCR assay was evaluated on spleen, renal, and urinary bladder tissue specimens from eight experimentally infected gerbils. Specimens from the same organs were cultured in BSK medium in parallel. Of 24 organs, 21 (88%) were PCR positive and 17 (71%) were culture positive. All culture-positive specimens were also PCR positive. Compared with B. burgdorferi cultivation, PCR had at least a comparable diagnostic sensitivity, it was less laborious, and results were available within 1 to 2 days.

After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid pBA6004 revealed a high degree of homology (71%) to the L. pneumophila mip gene, with the predicted secondary structures of the two Mip proteins following the same pattern. Southern hybridization experiments, with the plasmid pBA6004 as the probe, suggested that the mip gene of L. micdadei has extensive homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins).

Structural analysis by electronmicroscopy and pulsed field agarose gel electrophoresis of the genome of Borrelia burgdorferi has revealed a 1000 kilobase (kb) chromosome and a unique type of extra chromosomal linear DNA, as well as supercoiled circular DNA. The plasmid DNA ranged in size from 1.5 to 50 kb. Gene mapping of the ospA and ospB genes have placed them on the 49 kb linear plasmid molecule. The osp operon of B. burgdorferi has been extensively characterized at the molecular level.

A library of Treponema pallidum DNA was established in the direct selection vector pUN121. Six clones carrying a gene coding for a 33-kilodalton T. pallidum flagellum subunit were identified by colony hybridization with an oligodeoxynucleotide probe based on the N-terminal amino acid sequence of this subunit. An open reading frame of 286 amino acids with the expected N-terminal sequence and absence of cysteine residues was identified. The deduced protein had a calculated molecular weight of 31.3 kilodaltons. We propose to name this flagellar subunit FlaB2. FlaB2 shows a significant amino acid homology with flagellins of several remotely related bacterial species. This homology was most pronounced corresponding to the C-terminal and N-terminal parts of the protein, whereas the central region was variable.

To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15–25 kilobase pairs (kb)) were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli ‐absorbed anti‐L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly with a monospecific rabbit antiserum raised against L. micdadei“common antigen” (CA), and an additional 13 K L. micdadei protein. The region encoding these two proteins from the 17 kb recombinant plasmid (pBA 2) was subcloned in pBGS 18+. The DNA sequence of the CA encoding region in the 2.7 kb subcloned fragment will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram‐negative species, as well as to CA structure and possible function.

The isolation of Borrelia burgdorferi flagella and an enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin G (IgG) and IgM to the B. burgdorferi flagellum are described. The diagnostic performance of the flagellum ELISA for serodiagnosis of Lyme disease was compared with the performance of a traditional whole cell B. burgdorferi sonic extract ELISA. We examined sera and cerebrospinal fluid (CSF) from 56 patients with lymphocytic meningoradiculitis (Bannwarth's syndrome), the most frequent secondary-stage manifestation of Lyme disease in Europe. Two hundred healthy individuals and patients with aseptic meningitis, encephalitis, Guillain-Barré syndrome, and syphilis served as controls. The flagellum ELISA was significantly more sensitive than the sonic extract ELISA. The diagnostic sensitivities were increased from 41.1 to 76.8% (P less than 0.01) for IgG and from 35.7 to 67.9% (P less than 0.05) for IgM detection in serum. The increase in sensitivity was most pronounced in patients with a short duration of disease (less than 20 days after onset). The diagnostic specificity increased for IgG detection but was almost unaltered for IgM. The flagellum ELISA did not improve the diagnostic sensitivity of measuring antibodies to borreliae in CSF, most likely owing to the low level of unspecific antibodies in CSF compared with serum. The cross-reactivity of sera and CSF from patients with syphilis decreased significantly. The flagellum antigen of B. burgdorferi shows no strain variation, is easy to purify in sufficient quantity, and is therefore a suitable reference antigen for routine serodiagnosis of Lyme disease.

In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae recombinants were frequently found expressing an immunodominant 65 kDa protein antigen. In this study polyclonal and monoclonal antibodies against the 65 kDa antigen were found to react with a variety of different bacteria. Furthermore it is shown that the 65 kDa mycobacterial protein belongs to the family of antigens previously designated ‘common antigen’ due to their presence in a large variety of bacterial species. The molecular weight of this common antigen in 17 bacterial species was determined and it varied from 59 to 65 kDa. These bacteria included Gram-negative, Gram-positive and archae-bacteria, indicating that this antigen consists of members of an evolutionary well-conserved protein family. The 65 kDa protein was located in the cytoplasmic fraction of both Escherichia coli K12 and M. bovis BCG. Its function for the bacterial cell is presently unknown. The immunological relatedness of this common antigen to the MbaA protein might indicate a role in the etiology of rheumatoid arthritis, as was recently suggested for the mycobacterial 65 kDa antigen.

By crossed immunoelectrophoresis and Western blotting (immunoblotting), it was shown that Borrelia burgdorferi expresses the 60-kilodalton Common Antigen (CA) that is cross-reactive with an equivalent antigen in a wide range of remotely related bacteria. B. burgdorferi CA is strongly immunogenic. A B. burgdorferi genomic library was constructed by using a plasmid cloning system. Escherichia coli recombinants were screened for expression of immunodominant B. burgdorferi antigens. One of the recombinant clones expressed the 60-kilodalton CA of B. burgdorferi. The DNA region encoding B. burgdorferi CA was localized on a 2.3-kilobase fragment of the plasmid pKH1. CA may have pathogenetic implications in Lyme borreliosis, since the CA of mycobacteria recently has been shown to play a role in the etiology of experimental autoimmune arthritis. The extensive cross-reactivity of this antigen may account for the low diagnostic specificity of the currently used serological tests in Lyme borreliosis.

A library of Treponema pallidum DNA was constructed using a cosmid cloning system. Sixteen hundred Escherichia coli recombinant clones were generated covering the T. pallidum genome with a probability of 99%. Three hundred of the clones were screened for expression of T. pallidum antigens by a modified rocket immunoelectrophoresis technique using a polyspecific antiserum to T. pallidum. One clone was identified which produced the 'common antigen' (CA) of T. pallidum (Tp-4). CA shares epitopes with antigens present in more than 50 different bacterial species, but nothing is known about its structure, function and localization. The recombinant E. coli clone will be of value for a structural analysis of the CA gene.

AbstractAs the first step in a cooperative effort to standardize the identification of the polypeptides of Treponema pallidum subsp. pallidum, the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) results obtained in 16 laboratories were compared. Although it was possible to correlate the positions of 16 of the major polypeptide bands, the cross‐identification of many of the polypeptides was ambiguous, particularly in the low molecular weight range. Two‐dimensional electrophoresis provided an improved means of separating and characterizing T. pallidum polypeptides as isolated molecular species. An approach to the unambiguous identification of treponemal polypeptides was outlined which will utilize two‐dimensional electrophoresis in combination with specific properties attributable to individual proteins, including reactivity with monoclonal antibodies or monospecific antisera, biochemical and structural properties, and sequence information. To demonstrate the feasibility of this approach, two‐dimensional electrophoresis in conjunction with immunoperoxidase staining was used to specifically identify three cloned T. pallidum proteins.

Affinity chromatographic procedures are difficult to scale up from the analytical to the preparative level when the ligand used for purification is a limiting factor. A versatile, computer-controlled affinity chromatographic system is described which permits automatic repetition of the purification process and sophisticated control functions based on the ultra-violet absorbance of fluid passing through the affinity column. The system has been used for automation and scaling up of the purification of Plasmodium falciparum exoantigens.