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J Bacteriol 1998 Apr;180(7):1618-1623
Department of Microbiology, University of California Irvine, 92697-4025, USA.
A model of the 66-kDa outer membrane protein (P66) of Lyme disease Borrelia spp. predicts a surface-exposed loop near the C terminus. This region contains an antigen commonly recognized by sera from Lyme disease patients. In the present study, this region of P66 and homologous proteins of other Borrelia spp. were further investigated by using monoclonal antibodies, epitope mapping of P66 of Borrelia burgdorferi, and DNA sequencing. A monoclonal antibody specific for B. burgdorferi bound to the portion of P66 that was accessible to proteolysis in situ. The linear epitope for the antibody was mapped within a variable segment of the surface-exposed region. To further study this protein, the complete gene of Borrelia hermsii for a protein homologous to P66 was cloned. The deduced protein was 589 amino acids in length and 58% identical to P66 of B. burgdorferi. The B. hermsii P66 protein was predicted to have a surface-exposed region in the same location as that of B. burgdorferi's P66 protein. With primers designed on the basis of conserved sequences and PCR, we identified and cloned the same regions of P66 proteins of Borrelia turicatae, Borrelia parkeri, Borrelia coriaceae, and Borrelia anserina. The deduced protein sequences from all species demonstrated two conserved hydrophobic regions flanking a surface-exposed loop. The loop sequences were highly variable between different Borrelia spp. in both sequence and size, varying between 35 and 45 amino acids. Although the actual function of P66 of Borrelia spp. is unknown, the results suggest that its surface-exposed region is subject to selective pressure.
PMID: 9537355, UI: 98196701
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J Bacteriol 1997 Nov;179(21):6837-6842
Department of Microbiology & Molecular Genetics, University of California Irvine, 92697-4025, USA.
A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.
PMID: 9352937, UI: 98012986
Infect Immun 1997 Aug;65(8):3352-3360
Department of Microbiology, University of Texas Health Science Center at San Antonio, 78284, USA.
In mice infected with serotype A but not serotype B of the relapsing fever spirochete Borrelia turicatae, early invasion of the brain occurs. Serotypes A and B are further distinguished by the abundant surface protein they produce: VmpA and VmpB, respectively. Western blotting with monoclonal antibodies, one-dimensional peptide mapping, and partial amino acid sequencing demonstrated regions of the VmpA protein that differed from VmpB. Oligonucleotide primers based on the partial amino acid sequences of unique regions were used to amplify a portion of the VmpA gene (vmpA) by PCR, and the product was used as a probe in Southern blot and Northern blot analyses. These experiments showed that (i) expression of the vmpA sequence was determined at the level of transcription and (ii) the vmpA sequence was in two locations in serotype A and one location in serotype B. The vmpA gene at the expression-linked locus of serotype A was cloned and sequenced. An open reading frame would encode a polypeptide of 214 amino acids. The polypeptide expressed by Escherichia coli was bound by VmA-specific but not VmpB-specific antibody. Primer extension analysis identified a consensus sigma70-type promoter for vmpA at the expression locus. Phylogenetic analysis revealed that VmpA is homologous to small Vmp (Vsp) proteins of B. hermsii and to OspC proteins of B. burgdorferi. These findings indicate that a function of the Vsp-OspC family of proteins of Borrelia spp. may be differential localization in organs, including the brain, during infection.
PMID: 9234797, UI: 97378116
Cell 1997 Apr 18;89(2):275-285
Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, 77030, USA.
We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. This combinatorial variation could potentially produce millions of antigenic variants in the mammalian host.
PMID: 9108482, UI: 97262068
Vaccine 1997 Apr;15(6-7):739-746
Department of Microbiology, University of Texas Health Science Center at San Antonio 78284, USA.
The lipoprotein outer surface protein A (OspA) of the Lyme disease agent. Borrelia burgdorferi, has provided protection to mice and other animals against systemic infection when delivered orally as a recombinant protein in Escherichia coli, bacille Calmette. Guerin or Salmonella typhimurium. In the present study purified recombinant strain B31 OspA or outer surface protein D (OspD), another lipoprotein of B. burgdorferi, were administered either subcutaneously (s.c.) or orally without cell carrier or adjuvant to mice. In comparison to the OspD preparation, the OspA protein was 256-fold more resistant to trypsin. Whereas OspA in the suspension was in regular complexes of 17-25 nm in size, OspD formed amorphous globules of different sizes. Animals received a primary immunization and at least one booster. Mice immunized s.c. with either OspA or OspD had detectable antibodies to B. burgdorferi by enzyme-linked immunosorbent assay (ELISA), growth inhibition assay (GIA) and immunoblot. Delivered orally, OspA but not OspD elicited a specific antibody response, including IgA, as determined by these assays. The geometric mean titre of sera from mice who received 4 micrograms of OspA orally on days 1, 2, 4, 21 and 22 was 1470 by Ig ELISA, 320 by IgA ELISA and 128 by GIA. In infectious challenge experiments with B. burgdorferi strain Sh2-2-82 (OspA+ OspD- ) inoculated intradermally at 100 x the ID 50 all eight mice immunized with the 4 micrograms dose of OspA were protected, none of the mice immunized with the 4 micrograms dose of OspD were protected (P < 0.001 by Fisher exact test). These studies indicate that the lipoprotein OspA provides protection against systemic B. burgdorferi infection when delivered orally as a purified protein.
PMID: 9178476, UI: 97321774
J Infect Dis 1997 Jan;175(1):91-97
Department of Microbiology, University of Texas Health Science Center at San Antonio, USA.
Immunization with recombinant OspA protein of Borrelia burgdorferi protects against experimental Lyme disease. In the present study, mice were injected intramuscularly with plasmid DNA (VR2210) encoding strain B31 OspA. In this vector, the ospA-coding sequence was under transcriptional control of the cytomegalovirus immediate early promoter. For negative and positive controls, mice were immunized with either the plasmid vector without an osp-coding sequence or recombinant OspA protein, respectively. Mice immunized with VR2210 DNA produced OspA-specific antibodies that bound to B. burgdorferi in a whole cell ELISA and inhibited the growth of a homologous strain of B. burgdorferi. Immunization with VR2210 protected mice against challenge with 2 infectious strains of B. burgdorferi, Sh-2-82 and N40. These results indicate that vaccination with plasmid DNA expressing OspA is an efficacious method for providing a protective response against B. burgdorferi infection.
PMID: 8985201, UI: 97138175
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Infect Immun 1997 Jan;65(1):285-292
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284, USA.
Immunodeficient mice infected with Borrelia turicatae, a relapsing fever agent, have a disorder that resembles disseminated Lyme disease. Two serotypes, A and B, differed in their arthritogenicity in both CB-17 SCID and C3H SCID mice. In CB-17 SCID mice infected with serotype A or B, arthritis was assessed by measurement of tibiotarsal diameter, functional ability on a beam walk test, and microscopic assessment of joint inflammation. Serotype B-infected mice had greater joint swelling, functional disability, and leukocytic infiltration in the joints than serotype A-infected mice. Joint swelling and disability peaked at 2 weeks of infection and then decreased, while leukocyte infiltration in the joints persisted. To investigate the basis for the differences in arthritogenicity of serotypes A and B, spirochete burdens in infected mice were measured by quantitative PCR of spirochete DNA in joints, direct immunofluorescence of spirochetes in joints, and counts of spirochetes in the blood. At 2 weeks of infection there were seven times more spirochetes in the joints of serotype B-infected mice than in those of serotype A-infected mice, measured by both quantitative PCR and direct enumeration. Although serotypes A and B had the same infectivity and growth rate in vivo, serotype B spirochetes were eightfold more abundant in the blood than serotype A spirochetes and produced greater fatality in newborn mice. These findings indicate that differences in disease severity in mice infected with serotype A or B are attributable to differences in the spirochete burden in the joints and blood.
PMID: 8975925, UI: 97130049
Infect Immun 1996 Dec;64(12):5111-5116
Department of Microbiology, Umea University, Sweden.
A chromosomally encoded 66-kDa protein (P66) of Borrelia spp. that cause Lyme disease has previously been shown to be associated with the spirochetal outer membrane. A topological model of P66 predicts a surface-exposed fragment which links the N- and C-terminal intramembranous domains of the protein (J. Bunikis, L. Noppa, and S. Bergstrom, FEMS Microbiol. Lett. 131:139-145, 1995). In the present study, an immunogenic determinant of P66 was identified by a comparison of the immunoreactivities of different fragments of P66 generated either by proteolytic treatment of intact spirochetes or as recombinant proteins expressed in Escherichia coli. The immune response to P66 during natural infection was found to be directed against the predicted surface domain which comprises amino acids at positions 454 through 491. A sequence comparison revealed considerable polymorphism of the surface domains of P66 proteins of different Lyme disease-causing Borrelia species. Five sequence patterns of this domain were observed in the B. garinii strains studied. In contrast, sequences of the relevant part of P66 of the B. afzelii and B. burgdorferi sensu stricto isolates studied were identical within the respective species. In immunoblotting, 5 of 17 (29.4%) sera from North American patients with early disseminated or persistent Lyme disease reacted against P66 of B. burgdorferi sensu stricto B31. These sera, however, failed to recognize P66 of B. afzelii and B. garinii, as well as an analog of P66 in the relapsing fever agent, B. hermsii. In conclusion, the topological model of P66 is supported by the demonstration of an apparent surface localization of an immunoreactive domain of this protein. Furthermore, analogous to the plasmid-encoded borrelial outer surface proteins, the predicted surface-exposed portion of chromosomally encoded P66 appears to be antigenically heterogenous.
PMID: 8945554, UI: 97101027
J Bacteriol 1996 Nov;178(22):6635-6639
Department of Microbiology, University of Texas Health Science Center at San Antonio, 78284, USA. abarbour@uci.edu
A linear plasmid of Borrelia burgdorferi had 16,927 bp, a G+C content of 23.1%, a relative deficiency of CpG dinucleotides, and open reading frames A to O. The OrfC and OrfE proteins were similar to hypothetical proteins encoded by circular plasmids of B. burgdorferi. The OrfM and OrfN proteins were similar to replication proteins of circular plasmids of other bacteria.
PMID: 8932323, UI: 97086634
Ann N Y Acad Sci 1996 Oct 25;797:140-150
PMID: 8993358, UI: 97146502
Infection 1996 Mar;24(2):195-202
Dept. of Microbiology and Medicine, University of Texas Health Science Center at San Antonio 78284-7758, USA.
Interest in human and veterinary vaccines against Lyme borreliosis is growing. Both whole cell immunization and subunit vaccines can protect against infection with Borrelia burgdorferi. For development of a human vaccine the focus has been on a subunit vaccine. The most promising candidate is OspA, a major outer membrane lipoprotein of B. burgdorferi sensu lato. Of Osp proteins A through D, OspA shows the least variability between strains in its sequence and in the level of its expression. Borreliae in ticks express OspA. Antibodies to OspA kill borreliae in vitro and provide passive protection in mice. Active immunization of mice with OspA provides protection against challenge by syringe inoculation or tick bite. The lipid moiety of the OspA is necessary for immunogenicity in the absence of a potent adjuvant. A recombinant OspA-based vaccine is already in clinical trials. Although there is compelling evidence that immunization with OspA will provide protection, questions remain regarding the duration of protection from such immunization, the necessity to have a minimum level of neutralizing antibodies at all times for protection, and the relationship of an immune response to OspA and autoimmune features of Lyme borreliosis. The experimental aspects of immunization with Osp-A based constructs and other Lyme vaccine candidates are reviewed and discussed.
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PMID: 8740122, UI: 96311592
J Bacteriol 1996 Feb;178(3):793-800
Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp.
PMID: 8550515, UI: 96146530
Infect Immun 1995 Jun;63(6):2206-2212
Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston 77225, USA.
Borrelias that cause Lyme disease lose the ability to infect and cause disease in laboratory animals following 10 to 16 passages of in vitro culture. In this study, clonal populations of the Sh-2-82 (Sh2) and B31 strains of Borrelia burgdorferi were isolated by subsurface plating on BSK-II agar plates and examined for infectivity in the C3H/HeN mouse model. Mice were injected intradermally with 10(5) B. burgdorferi organisms, and the tibiotarsal joint, heart, and bladder were cultured 2 to 4 weeks postinfection to determine whether viable organisms were present. Clones exhibited either a high-infectivity phenotype, in which cultures were consistently positive at all organ sites, or a low-infectivity phenotype, in which a low proportion of cultures were positive (5 of 40 in a representative experiment). In an Sh2 population that had undergone five in vitro passages, 7 of 10 clones were of the high-infectivity phenotype, and the remaining clones were of the low-infectivity phenotype. The proportion of high-infectivity clones decreased with continued in vitro passage, with only 1 of 10 clones exhibiting the high-infectivity phenotype after 10 passages and 0 of 10 clones yielding positive cultures after 20 passages. Representative high- and low-infectivity clones from passage 5 Sh2 cultures had 50% infectious doses of 1.8 x 10(2) and 1 x 10(5), respectively. Subclones consistently reflected the same infectivity phenotypes as those of the parent clones. The protein profiles and plasmid contents of the high- and low-infectivity clones were compared and exhibited few discernible differences. On the basis of these results, the loss of infectivity during in vitro culture results from the outgrowth of low-infectivity clones and begins to occur within the first five in vitro passages. Further examination of clonal populations may lead to the identification of genetic and protein factors important in the virulence and pathogenicity of Lyme disease borrelias.
PMID: 7768600, UI: 95286265
Infect Immun 1995 Apr;63(4):1573-1580
Department of Microbiology and Medicine, University of Texas Health Science Center at San Antonio 78284.
All Borrelia burgdorferi sensu lato isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of B. burgdorferi lacking Osps were selected with polyclonal or monoclonal antibodies at a frequency of 10(-6) to 10(-5). One mutant that lacked OspA, -B, -C, and -D was further characterized. It was distinguished from the OspA+B+ cells by its (i) autoaggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum and complement sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in mouse skin for the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two types of monoclonal antibodies (MAbs) with growth-inhibiting properties for mutant cells were identified. The first type bound to a 13-kDa surface protein of B. burgdorferi sensu stricto and of B. afzelii. The MIC of the Fab fragment of one MAb of this type was 0.2 micrograms/ml. The second type of MAb to the Osp-less mutant did not bind to B. burgdorferi components by Western blotting (immunoblotting) but did not bind to unfixed, viable cells in immunofluorescence and growth inhibition assays. These studies revealed possible functions Osp proteins in borrelias, specifically serum resistance, and indicated that in the absence of Osp proteins, other antigens are expressed or become accessible at the cell surface.
PMID: 7890424, UI: 95197293
Microbiology 1995 Jan;141( Pt 1):85-93
The flagellin genes from six Borrelia species were cloned, sequenced and characterized at the molecular level. The flagellin genes of two relapsing fever Borrelia species, B. hermsii and B. crocidurae, three Lyme disease genomic species, B. burgdorferi, B. afzelii and B. garinii, and the avian borreliosis agent, B. anserina, were compared and showed an 85-93% sequence identity to each other. Comparison of the fla genes from the different Lyme borreliosis spirochaetes revealed that they were 94-99% identical. Nucleotide sequencing of the fla gene and primer extension on isolated mRNA from both B. hermsii (as transcribed in Escherichia coli) and B. burgdorferi (as transcribed in the natural host) identified the putative transcriptional start points, the ribosomebinding sites and the promoter regions of these genes. The deduced promoter of the Borrelia flagellin gene resembled neither the sigma 70 promoter of prokaryotes, as seen for the genes for the outer-surface proteins A and B in Lyme disease Borrelia and the genes for the variable major proteins 7 and 21 of B. hermsii, nor the sigma 28 consensus promoter region of motility genes from other bacteria. Instead, the promoter of the fla gene in Borrelia has most similarity to the bacteriophage SP01 sigma gp33-34 promoter sequence of Bacillus subtilis.
PMID: 7894724, UI: 95202093
Wien Med Wochenschr 1995;145(7-8):162-165
Abteilung fur Mikrobiologie und Medizin, Universitat von Texas, San Antonio, USA.
Phenomenon of growth inhibition of Borrelia burgdorferi sensu lato (BBSL), the agent of Lyme disease, by antiborrelial antibodies was observed and investigated. Some of the antiborrelial antibodies were bactericidal in the absence of complement and phagocytes. Based on growth inhibition phenomenon we developed and evaluated the function-oriented in vitro growth inhibition assay (GIA). GIA proved to be more strain-specific and a better predictor of protection against infectious challenge than matrix-based assays, such as ELISA and Western blot. Growth inhibition phenomenon was also applied as a tool for selection of antibody-resistant BBSL mutants. All BBSL isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of BBSL with altered or absent Osps were selected with polyclonal or monoclonal antibodies (mAbs) at a frequency of 10(-2) to 10(-6). Based on PAGE and Western blot analysis all examined mutants were catalogued into 4 classes. Some of these mutants were later employed in studies of functional importance of Osps in immunopathogenesis of BBSL. Several studies revealed some possible functions of Osp proteins in borrelias. In one study, Osp-lacking mutant was distinguished from its Osp-bearing parent by autoaggregation and slower growth rate, diminished capacity to adhere to human umbilical vein endothelial cells, decreased plating efficiency on solid medium as well as serum and complement sensitivity. Mutant also was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant cells survived in mouse skin for the same duration as wild type cells.
PMID: 7610664, UI: 95335036
Cell 1994 Sep 9;78(5):867-876
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284-7758.
B. hermsii counters host immunity with multiphasic antigenic variation. This is conferred by interplasmidic and intraplasmidic rearrangements of vmp genes. In several independent events, activation of a silent vmp gene through intraplasmidic deletions but not interplasmidic recombinations was followed by the appearance at its 5' end of multiple mutations that were not present in the silent gene. The prevalence of mutant alleles in postwitch populations increased during infections. Differences between the silent and expressed genes were at the same nucleotides at which vmp pseudogenes differed, suggesting these were templates for postswitch gene conversions. The mechanism of this bacterium to generate diversity, namely, intramolecular deletions followed by mutations in the rearranged gene, mirrors the strategy used by vertebrate hosts to eliminate it.
PMID: 8087853, UI: 94373822
Infect Immun 1994 Jul;62(7):2792-2799
Relapsing fever and Lyme disease spirochetes of the genus Borrelia display at their surfaces abundant lipoproteins: Vmp proteins in Borrelia hermsii and Osp proteins in Borrelia burgdorferi. Vmp and Osp proteins largely determine serotype specificity, and neutralizing antibodies of infected or immunized animals are directed at them. For the present study, we examined B. hermsii serotype 33, which is unique among strain HS1 serotypes in the low frequency of switches to other serotypes during infections and in vitro cultivation. Failing to clone the complete vmp33 gene, we accomplished its further characterization by (i) determining three partial amino acid sequences, (ii) designing oligonucleotide primers based on these amino acid sequences, (iii) cloning and sequencing the central portion of vmp33, and (iv) using outwardly directed primers and the inverse PCR to clone the 5' and 3' ends of the gene and flanking regions. The transcriptional start site was identified by primer extension analysis. Vmp33 was a polypeptide of 211 amino acids; the three partial amino acid sequences were identified in the open reading frame. Vmp33 was found to be more similar to other 20-kDa Vmp proteins of B. hermsii and to OspC proteins of B. burgdorferi than it was to 35- to 39-kDa Vmp proteins of the same strain. Moreover, OspC proteins were more similar to Vmp33 than they were to OspA, -B, or -D proteins of B. burgdorferi. These sequence similarities were consistent with Western blot (immunoblot) findings of cross-reactions between Vmp33 and OspC with anti-Vmp33 and anti-OspC sera. The promoter for the expressed vmp33 gene was found to be different from the expression site for other active vmp genes characterized to date. These results indicate that Vmp33 and other small Vmp's belong with OspC to a genus-wide family of 20-kDa proteins and that expression of these proteins may be coordinated with expression of other Vmp and Osp proteins in Borrelia spp.
PMID: 8005669, UI: 94274293
Mol Microbiol 1994 Jul;13(2):287-299
The relapsing fever agent, Borrelia hermsii, undergoes multiphasic antigenic variation to evade its host's immune response. A frequently observed switch is serotype 7 to 26. Unlike silent vmp genes previously characterized, the transcriptionally silent vmp26 sequence was a pseudogene in lacking a start codon. In serotype 7 the location of the silent vmp26 sequence just downstream of vmp7 on the expression plasmid, as well as on the silent plasmid, was also unique. The demonstration of a predicted circular recombination product in serotype 7 but not serotype 21 populations indicates that the pseudogene was activated by an intramolecular recombination producing a deletion of DNA between 20-nucleotide direct repeats in vmp7 and psi vmp26.
PMID: 7984108, UI: 95075314
FEMS Microbiol Lett 1994 Jun 15;119(3):381-387
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840.
The P39 antigen is a specific, highly conserved, and immunogenic protein of Lyme disease spirochetes, Borrelia burgdorferi sensu lato. The nucleotide sequence of the gene encoding this protein was determined and found to be the first of two tandemly arranged open reading frames located on the spirochete's chromosome. These two open reading frames were designated bmpA for the gene encoding P39 and bmpB for the gene encoding the putative protein ORF2 encoded by the second open reading frame. The nucleic acid sequence identity for the two open reading frames was 62% while their deduced amino acid sequences were 52% identical. Comparison to sequence data bases demonstrated that the deduced amino acid sequences of both P39 and ORF2 were homologous to TmpC, a putative outer or cytoplasmic membrane lipoprotein of the syphilis spirochete, Treponema pallidum.
PMID: 8050720, UI: 94327086
Infect Immun 1993 Sep;61(9):3590-3596
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.
Most Borrelia burgdorferi strains have two major surface proteins, OspA and OspB. In the present study, we selected from a clonal population of infectious B. burgdorferi an OspB escape mutant, identified the genetic basis for this phenotype, and evaluated its functional activities. Selection with the anti-OspB antibody H614 was performed in vitro in medium and extended in vivo in scid mice. Mutants with a truncated OspB protein were selected at a frequency of 1 x 10(-5) to 3 x 10(-5). After no major rearrangements in DNA were detected, sequence analysis of the mutant's ospAB locus revealed a single base change in the consensus ribosomal binding sequence for ospB and a single nucleotide deletion in the ospB gene itself. The effect of these mutations was reduced expression of a truncated OspB protein. When functional abilities of the wild type and mutant were compared, the mutant had a threefold-lower capacity to penetrate a human endothelium umbilical vein cell monolayer. Infectivity of wild-type and mutant cells for scid mice was evaluated by culturing different organs, and the median infectious dose was calculated. The inoculum of mutant cells for infecting the mice was 30- to 300-fold higher than that of wild-type cells. This study shows that reduced size and expression of OspB are associated with lowered virulence of B. burgdorferi. Selection of mutants that to some degree remain infectious is one approach to defining the role of different surface proteins in the pathogenesis of Lyme disease.
PMID: 8359881, UI: 93366412
Trends Microbiol 1993 Sep;1(6):236-239
Dept of Microbiology, University of Texas Health Science Center, San Antonio 78284.
Members of the genus Borrelia may be unique among prokaryotic organisms in having a polyploid genome that is mostly linear. The smaller linear duplex replicons in these organisms have been called plasmids, but there is justification for designating them minichromosomes instead. The antigenic identities of the agents of Lyme disease and relapsing fever are largely determined by these extrachromosomal genes.
PMID: 8137122, UI: 94184809
Antimicrob Agents Chemother 1993 Aug;37(8):1704-1706
Department of Pathology, University of Texas Health Science Center, San Antonio 78284-7881.
The in vitro activities of the macrolide antibiotics clarithromycin, 14-hydroxy-clarithromycin, azithromycin, and erythromycin against 19 isolates of Borrelia burgdorferi were investigated. MICs ranged from 0.003 to 0.03 microgram of clarithromycin per ml, 0.007 to 0.03 microgram of 14-hydroxyclarithromycin per ml, 0.003 to 0.03 microgram of azithromycin per ml, and 0.007 to 0.06 microgram of erythromycin per ml. Time-kill studies using the B31 strain of B. burgdorferi demonstrated a > or = 3-log10-unit killing after 72 h with each of the macrolide antibiotics tested in concentrations representing twice the respective MICs.
PMID: 8215288, UI: 94028805
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