- Email: [email protected]
Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo
ELSEVIER
MICROBIOLOGY LETTERS FEMS Microbiology Letters 145 (1996) 309-314
Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo Lieselotte Aron a) Clara Toth ‘, Henry P. Godfrey b, Felipe C. Cabello av* a Department of Microbiology and Immunology, New York Medical College, Basic Science Building, Valhalla, NY 10595, USA ’ Department of Pathology, New York Medical College, Basic Science Building, Valhalla, NY 10595, USA
Received 10 April 1996; revised 10 June 1996; accepted 30 August 1996
Abstract
A clone containing a 6.4 kb Borrelia burgdorferi chromosomal DNA insert reacted only with sera from patients with Lyme disease and not with any normal human or rabbit sera. Restriction enzyme analysis indicated that this DNA fragment was located on the B. burgdorferi chromosomal map between rpoB and p22A; its direction of transcription was towards p22A. Sequence analysis suggests that LA006 encodes six proteins: three previously described immunodominant lipoproteins of the 39 kDa Bmp protein family, BmpA, BmpB and BmpC; a 51 kDa MgtE magnesium transporter protein; a 16 kDa protein kinase C inhibitor; and a 56 kDa protein with similarity to an uncharacterized Escherichiu coli chromosomal open reading frame. Keywords: Borrelia burgdorferi; Mg‘+ transporter; Protein kinase Cl inhibitor
1. Introduction Lyme disease, caused by the spirochete Borrelia is the most common arthropod-borne disease in the United States [l]. The structure that primarily interacts with the mammalian immune system in B. burgdorferi infections is composed of an outer sheath which contains a variety of proteins [2]. Many of these proteins have been purified and characterized, and the possible protective role of mammalian immune responses against them analyzed [2]. However, little is known about the role of any B. burgdorferi antigen in infection of ticks or produc-
burgdorferi,
* Corresponding author. Tel.: +l (914) 993 4182; Fax: +l (914) 993 4176; E-mail: [email protected]
tion of disease in mammals during the different stages of Lyme disease. A strategy for discovering B. burgdorferi gene products expressed in infected human beings is to screen a B. burgdorferi expression library with sera from patients with Lyme disease. The expression library should optimally be prepared from a low passage strain and steps should be taken to insure cloning of all B. burgdorferi chromosomal and plasmid genes [3]. In addition, sera used for screening should be extensively absorbed to remove antibodies to crossreacting antigens of Gram-negative and other bacteria [3,4]. Application of this approach resulted in the discovery of BmpC, a new, chromosomally encoded member of the immunodominant 39 kDa protein family [3]. Extension of these studies has now permitted characterization of a region of DNA in the B.
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et al. I FEMS
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chromosome containing open reading frames (ORF) for three of the four members of the 39 kDa family and for two other genes with virulence potential, a protein kinase C inhibitor (PKCI) and an Mg2+ transporter and has permitted location of the region in the B. burgdorferi chromosome. These findings suggest that genes encoding virulence properties of B. burgdorferi may be clustered in well defined regions in the chromosome as appears to be the case with other pathogens [5-71.
2. Materials and methods 2. I. B. burgdorferi library A B. burgdorferi hgtl 1 library was prepared by standard methods [8,9]. DNA extractions, restriction enzyme analysis, elution of DNA fragments, ligation, transformation and selection of recombinant clones, primer synthesis and DNA sequencing were also performed by standard methods [8,9]. Cloning efficiency in preparing the B. burgdorferi hgt 11 library was 1 x 10” bacteriophages/yg of B. burgdorferi DNA; 70% of the phages in the library were recombinants with inserts of 2-7X lo3 bp.
Letters
145 11996)
309-314
necessary to insure inclusion of all B. burgdorferi chromosomal and plasmid genes at least once [9,11]. The large number of plaques screened also insured against over-representation of recombinants preferentially containing genes from high copy number plasmids [9,11]. One highly reactive bacteriophage clone from the library, LA006, was selected for further studies [3]. 2.3. DNA sequence analysis Automated DNA sequencing was done using the double-stranded dideoxy method in an Applied Biosystems model 373A DNA sequencer. DNA sequences were aligned and analyzed with AssemblyLIGN and MacVector software (IBI, New Haven, CT). DNA and deduced amino acid sequences were compared to entries in the non-redundant GenBank database at the National Center for Biotechnology Information using the BLAST algorithm [12]. 2.4. Nucleotide sequence accession number The nucleotide sequence of B. burgdorferi LA006 was deposited in the GenBank database with accession number U49938.
2.2. Sera and immunoscreening 3. Results The antibody pool used to screen the B. burgdorferi library [3,9] contained serum and synovial fluid of 16 patients with confirmed Lyme disease and sera from rabbits immunized by repeated injections of B. burgdorferi 297 strain sonicates [3]. Lyme disease patients had erythema migrans, carditis, neurologic disease or arthritis, and elevated anti-B. burgdorferi titers by ELISA and FIAX. Human and rabbit sera were passed several times through rabbit and human IgG-Sepharose columns, respectively, before pooling; the antibody pool was repeatedly absorbed with whole Escherichiu coli Y 1090 r- , bacterial membranes and ribosomal fractions, and soluble proteins covalently bound to Sepharose [lo]. The resulting reagent did not react with E. coli Y 1090 r- antigens on dot blotting. 8000 plaques of the unamplified IPTG-induced library were screeened with the extensively absorbed anti-B. burgdorferi serum pool, over 2.5 times more than the minimum of 3000 clones
3.1. DNA sequence analysis of the LA006 clone DNA sequencing of 47 recombinant clones in pUC19 containing overlapping segments of the original 6.4 kb B. burgdorferi DNA insert of LA006 produced the complete sequence of this DNA fragment including the entire bmpC gene (6930 bp) and a bmpC
bw’A
bmp8
ORF4
ORF5EglEl
&I &I 5012 5594
OzE.(PKCI)
5978
EcoRI 6950
Fig. 1. Location of ORF and restriction enzyme sites in LA006 DNA segment from B. burgdorferi chromosome. Direction of translation shown by arrow. PvuI site at position 5394 corresponds to Pvul at position 390 on the B. burgdorferi chromosoma1 map [7], placing LAO06 between rpoB and p22A on the B. burgdorferi chromosome.
L. Arm et al. IFEMS
A.
Microbiology Letters 145 (1996) 309-314
311
MgtE Mg” transporter MIDIDELRIFLKEKSYSKIKEKFLKHDSFDIAEALKRLNGTELILLYRFLPKKIAVETFSNFDQSTKNKL
Bb Bf
- 70
PS
Bb Bf PS
ANSFTNKEISEMIDELNLDDVIDLLEEVPANWQRFLASSTEENRGIINKFLSYSDDSAGSIVTIEWEL
Bb Bf PS
KEDFl'@GKALDYI&RVAKTKXD@fTYYITDD~ KWhKQVIKIBDXXL&KD TAVE@$EVIGRLZ&E%PDABT%%YLYWl%ERE Kpqmqm-E s1AD.!3mxKKNL IEHLDGEQIPTYL~ILSVKS@LA#DB
MTKBFIRV MSQSYLPV
Bb Bf PS Bb
PII~~SSTFg&TIBSNYQNLMLSLWI;ASFIPLLMDT RAPWII~FOLIT~IGQFEETLEAIVLLAAFIP~S~~S~VA~G~~~ ~~~~~K~FEEQLEAAIS~~~IGTG(~NSQ~ITSTL~~S~A
Bb Bf PS
VFtlCEICVSILV~3&ABVNFLR;LVFFVAPHHSDKLKIAFWSSCLMVSLTVAKILGGLLPIVA#&BR&D MLKRELGTGLMLGFZCMLVIAG-IVTVMHGNMM----LAFIVGISIFLTXiSIATIIGTIVPLIINKMELD ~~STSFLVAVTIGAAALI~WILGVGA-----EVTIWSLTIVAITMWSAIVSSII
Bb Bf PS
P%?T.,MAGPLITTIAUAIT@!A%~AKWVLVStiV H~sGPFITT~NDILG~IYFS~ATALL~EYL PXVVSAPFIATFIDGTGCZIYFEIAICLVMTEFA
B.
I%%!='NDE: -210 sqsAKTD - 77 -qAPDQs -77
s;ILV?mwN SSFQAAm DEkUlAQLQGAT'kPLEEPYLAT~ITLWRK
Bf PS
SGNAGSQASkLIIREfFFK
-280 -145 -146 -350 -215 -2i6 -420 -280 -281
-454 -312 -314
Protein kinase C inhibitor
Bb Hi
-
MDRLIRRTFYREQTMAEE
SY Mh Man
cow Rat
SC Cab
Maize
MSSEKEAALRRLDDS
Bb
KRGmLvNLE
Hi
SY Mb Man
cow Rat SC Cab Maize Bb SC
-140 - 8 8
BDLSYLIVKAREFALQEIKKLGAT-DDESLLGHLMIVGKX!AADLGLNK-DDESLLGHLMIVGKX!AADL-DDESLLGHUIIVGKKCAADLGLKK--@RMWNE@
EERHIBILGYL
LLAKLEGSD
-144
57 70 58 60 70 70 70 64 55 70
-126 -130 -114 -111 -126 -126 -126 -127 -113 -129
L. Arm
312
et al. IFEMS
Microbiology
Letters 145 (1996)
309-314
Fig. 2. Alignment of deduced amino acid sequences of B. burgdorferi ORF (LA006) and homologous proteins. A: MgtE MgZf transporter (ORF 5). B: protein kinase C inhibitor (ORF 6). Amino acid residues identical to ORF 5 or ORF 6 occurring at the same position are indicated by shading. Bb, B. burgdorferi; Bf, Bacillusjirmus; Cab, Chinese cabbage; Hi, Hemophilus injuenzae; Mh, Mycoplasma hyorhinis; Ps, Providencia stuartii; SC, Saccharomyces cerevisiae; Sy, Synechococcus sp. t
schematic map of the insert is shown in Fig. 1. The PvuI site at position 5394 corresponds to the single PvuI site at about 390 kbp on the B. burdorferi chromosomal map between P22A and rpoB ([5], I. Schwartz, personal communication). Taken together with the position of the bmpA gene in the same map, the direction of transcription in the chromosomal DNA fragment cloned in LA006 is established as towards p22A in the B. burgdorferi chromosome [3,5,13]. The recently reported fourth member of the family of genes encoding for 39 kDa proteins, bmpD, maps at the 5’ end of bmpC [14]. Sequence analysis indicates that LA006 contains full ORF for BmpA and BmpB [3,13], and for three additional deduced proteins 3’ to bmpC, bmpA and bmpB. These proteins have molecular weights of 55716 Da (ORF 4; p1, 7.23), 50773 Da (ORF 5; p1, 4.86), and 15867 Da (ORF 6; p1, 8.68) (Fig. 2). ORF 4 begins at nt3525, is 1422 nucleotides long and would encode a protein of 474 amino acids. It has a potential ribosomal binding site AGAGGA at nt3512, could be initiated with TTG and terminated with TGA. This ORF also has a potential starter codon ATG at nt3537. ORF 5 begins at nt5003, is 1362 nucleotides long and would encode a protein of 454 amino acids. It has a potential ribosomal binding site GGAGGC at nt4991, is initiated with a ATG and terminated with TAA. ORF 6 begins at nt6418, is 417 nucleotides long and would encode a protein of 139 amino acids. It has a ribosomal binding site AGGATA at nt6406, is initiated with ATG and terminated with TAA. These ORF are preceded by regions that could function as canonical promoters corresponding to -35 and -10 consensus sequences at nt3460 and nt3480 for ORF 4, nt4954 and nt4975 for ORF 5, and nt6351 and nt6375 for ORF 6. 3.2. DNA sequence homology of ORF 4, ORF 5 and ORF 6
Comparison
of the deduced amino acid sequences
to entries in the database at GenBank using the BLAST algorithm [12] indicates that ORF 4 is homologous (32% identity) with an E. coli chromosomal ORF for an uncharacterized 88 kDa protein. ORF 5 has highly significant identity with a magnesium transporter protein, MgtE [ 151, found in several bacterial species (31% of identity with Bacillus firmus, 34% with Providencia stuartiz) (Fig. 2). ORF 6 has highly significant identity with protein kinase C inhibitors from eubacteria (55% identity with Hemophilus infhzenzae, 52% identity with the cyanobacterium Synechococcus sp., 51% identity with Mycoplasma hyorhinis), mammals (49% identity with human, cow and rat), and plants (46% identity with Saccharomyces cerevisiae, 52% identity with maize, 55% identity with Chinese cabbage) (Fig. 2).
4. Discussion We have developed a screening method to detect immunogenic antigens of B. burgdorferi expressed in vivo that has permitted identification, cloning and DNA sequencing of a cluster of possible virulencerelated genes of B. burgdorferi. This and recently published evidence suggest that this cluster of genes contains four members of the Bmp family of membrane lipoproteins located at approximately 390 kba of the B. burgdorferi chromosome [3,13,14]. These lipoproteins are important immunogens in Lyme disease [3,13]. Their preferential expression at 35°C and not at lower temperatures [16] and their immunogenicity in mice following infection by tick bite [17] could indicate involvement in evasion of mammalian host defenses and proliferation in host tissues. Location of the genes for the 4 Bmp proteins in tandem in the chromosome of B. burgdorferi and their shared homology and overlapping transcriptional signals also suggest coordinated differential regulation of in vivo expression [3,13]. Of other ORF on this DNA segment, protein kinase C inhibitor may also be involved in B. burgdorferi evasion of host defenses
L. Aron et al. I FEMS Microbiology Letters 145 (1996) 309-314
since protein kinase C mediates a wide variety of processes in the host immune and inflammatory responses and is clearly involved in phagocytosis and bacterial killing [l&-20]. MgtE magnesium transporter may enhance B. burgdorferi survival under low Mg2+ concentrations found in phagolysosomes; a similar scenario has been suggested for Salmonella within epithelial cell vacuoles [21]. Clustering of six genes that are potentially expressed in vivo in response to environmental clues is consistent with an evolutionary advantage of such a spatial arrangement [6,7]. While we do not know whether all these B. burdorferi proteins are expressed in vivo, we have already expressed recombinant BmpC in E. coli and have determined that the recombinant molecule reacts with sera from patients with Lyme disease (M. Alekshun, L. Aron, H. Godfrey, I. Schwarz and F.C. Cabello, manuscript in preparation). We are in the process of characterizing the immunological reactivity of other gene products encoded by the gene cluster with this sera.
Acknowledgments This work was supported by grants from the New York State Tick-Borne Disease Institute (F.C.C.) and the New York Medical College Lyme Disease Fund (F.C.C, H.P.G.), and by grants from the National Institutes of Health: R43 AI36004 (F.C.C.) and ROl CA34141 and AI37014 (H.P.G.). We would like to thank Drs. E. Ash and A. Weinstein for providing Lyme disease patient sera.
References [1] Anonymous (1995) Lyme disease. United States, 1994. Morbid. Mortal. Week Rep. 44, 459-462. [2] Nocton, J.J. and Steere, A.C. (1995) Lyme disease. Adv. Intern. Med. 40, 69-115. [3] Aron, L., Alekshun, M., Perlee, L., Schwartz, I., Godfrey, H.P. and Cabello, F.C. (1994) Cloning and DNA sequence analysis of bmpC, a gene encoding a potential membrane lipoprotein of Borrelia burgdorferi. FEMS Microbial. Lett. 123, 75-82. [4] Coleman, J.L. and Benach, J.L. (1992) Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria. J. Infect. Dis. 165, 658-656. [5] Casjens, S., Delang, M., Ley, III, H.L., Rosa, P. and Huang,
313
W.M. (1995) Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order. J. Bacterial. 177, 2769-2780. [6] Blum, G., Ott, M., Lischewski, A., Ritter, A., Imrich, H., Tschape, H. and Hacker, J. (1994) Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen. Infect. Immun. 62, 606-614. [7] McDaniel, T.K., Jarvis, K.G., Donnenberg, M.S. and Kaper, J.B. (1995) A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc. Natl. Acad. Sci. USA 92, 16641668. [8] Aguero, J., Mora, G., Mroczenski-Wildey, M.J., Femandez, M.E., Aron, L. and Cabello, F.C. (1987) Cloning, expression and characterization of the 36 kDa Salmonella typhi in Escherichia coli. Microb. Pathog. 3, 399407. [9] Mierendorf, R.C., Persy, C. and Young, R.A. (1987) Gene isolation by screening hgtll libraries. Meth. Enzymol. 152, 458-469. [lo] Aron-Hott, L., Faundez, G., Gonzalez, C., Roessler, E. and Cabello, F.C. (1993) Lipolysaccharide-independent radioimmunoprecipitation and identification of structural and in viva induced immunogenic surface proteins of Salmonella typhi in typhoid. Vaccine 11, l&17. [Ill Jendrisok, J., Young, R. and Engel, J.D. (1987) Cloning cDNA into hgtl0 and hgtll. Meth. Enzymol. 152, 359-371. 1121Altshul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403410. [I31 Simpson, W.J., Cieplak, W., Schrumpf, M.E., Barbour, A.G. and Schwan, T.G. (1994) Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen. FEMS Microbial. Lett. 119, 381-388. R., Povinelli, L. and Philipp, M.T. (1996) Mo1141 Ramamoorthy, lecular characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of genes encoding membrane proteins of Borrelia burgdorferi. Infect. Immun. 64, 1259-1264. 1151 Smith, R.L., Thompson, L.J. and Maguire, M.E. (1995) Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from BaciZlus firmus OF4. J. Bacterial. 177, 1233-1238. of 1161 Cluss, R.G. and Boothby, J.T. (1990) Thermoregulation protein synthesis in Borrelia burgdorferi. Infect. Immun. 58, 1038-1042. [I71 Golde, W.T., Kappel, K.J., Duquesne, G., Feron, C., Plainchamp, D., Capiau, C. and Lobet, Y. (1994) Tick transmission of Borrelia burgdorferi to inbred strains of mice induces and antibody response to p39 but not to outer surface protein A. Infect. Immun. 62, 2625-2627. M.J., Harrison, P.T., Floto, R.A. and Allen, 1181 Hutchinson, J.M. (1995) Fey receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent. Eur. J. Immunol. 25, 481-487. v91 Indik, Z.K., Park, J.-G., Pan, X.Q. and Schreiber, A.D. (1995) Induction of phagocytosis by a protein tyrosine kinase. Blood 85, 1175-l 180.
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[20] Zheng, L., Zomerdijk, T.P.L., Aarnoudse, C., Van Furth, R. and Nibbering, P.H. (1995) Role of protein kinase C isozymes in F, receptor-mediated intracellular killing of Staphylococcus aweus by human monocytes. J. Immunol. 155, 776-784.
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[21] Garcia-Del Portillo, F., Foster, J.W., Maguire, M.E. and Finlay, B.B. (1992) Characterization of the micro-environment of Salmonella typhimurium-containing vacuoles within MDCK epithelial cells. Mol. Microbial. 6, 3289-3297.
MICROBIOLOGY LETTERS FEMS Microbiology Letters 145 (1996) 309-314
Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo Lieselotte Aron a) Clara Toth ‘, Henry P. Godfrey b, Felipe C. Cabello av* a Department of Microbiology and Immunology, New York Medical College, Basic Science Building, Valhalla, NY 10595, USA ’ Department of Pathology, New York Medical College, Basic Science Building, Valhalla, NY 10595, USA
Received 10 April 1996; revised 10 June 1996; accepted 30 August 1996
Abstract
A clone containing a 6.4 kb Borrelia burgdorferi chromosomal DNA insert reacted only with sera from patients with Lyme disease and not with any normal human or rabbit sera. Restriction enzyme analysis indicated that this DNA fragment was located on the B. burgdorferi chromosomal map between rpoB and p22A; its direction of transcription was towards p22A. Sequence analysis suggests that LA006 encodes six proteins: three previously described immunodominant lipoproteins of the 39 kDa Bmp protein family, BmpA, BmpB and BmpC; a 51 kDa MgtE magnesium transporter protein; a 16 kDa protein kinase C inhibitor; and a 56 kDa protein with similarity to an uncharacterized Escherichiu coli chromosomal open reading frame. Keywords: Borrelia burgdorferi; Mg‘+ transporter; Protein kinase Cl inhibitor
1. Introduction Lyme disease, caused by the spirochete Borrelia is the most common arthropod-borne disease in the United States [l]. The structure that primarily interacts with the mammalian immune system in B. burgdorferi infections is composed of an outer sheath which contains a variety of proteins [2]. Many of these proteins have been purified and characterized, and the possible protective role of mammalian immune responses against them analyzed [2]. However, little is known about the role of any B. burgdorferi antigen in infection of ticks or produc-
burgdorferi,
* Corresponding author. Tel.: +l (914) 993 4182; Fax: +l (914) 993 4176; E-mail: [email protected]
tion of disease in mammals during the different stages of Lyme disease. A strategy for discovering B. burgdorferi gene products expressed in infected human beings is to screen a B. burgdorferi expression library with sera from patients with Lyme disease. The expression library should optimally be prepared from a low passage strain and steps should be taken to insure cloning of all B. burgdorferi chromosomal and plasmid genes [3]. In addition, sera used for screening should be extensively absorbed to remove antibodies to crossreacting antigens of Gram-negative and other bacteria [3,4]. Application of this approach resulted in the discovery of BmpC, a new, chromosomally encoded member of the immunodominant 39 kDa protein family [3]. Extension of these studies has now permitted characterization of a region of DNA in the B.
0378-1097/96/%12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00355-2
310
L. Arm
et al. I FEMS
Microbiology
burgdorferi
chromosome containing open reading frames (ORF) for three of the four members of the 39 kDa family and for two other genes with virulence potential, a protein kinase C inhibitor (PKCI) and an Mg2+ transporter and has permitted location of the region in the B. burgdorferi chromosome. These findings suggest that genes encoding virulence properties of B. burgdorferi may be clustered in well defined regions in the chromosome as appears to be the case with other pathogens [5-71.
2. Materials and methods 2. I. B. burgdorferi library A B. burgdorferi hgtl 1 library was prepared by standard methods [8,9]. DNA extractions, restriction enzyme analysis, elution of DNA fragments, ligation, transformation and selection of recombinant clones, primer synthesis and DNA sequencing were also performed by standard methods [8,9]. Cloning efficiency in preparing the B. burgdorferi hgt 11 library was 1 x 10” bacteriophages/yg of B. burgdorferi DNA; 70% of the phages in the library were recombinants with inserts of 2-7X lo3 bp.
Letters
145 11996)
309-314
necessary to insure inclusion of all B. burgdorferi chromosomal and plasmid genes at least once [9,11]. The large number of plaques screened also insured against over-representation of recombinants preferentially containing genes from high copy number plasmids [9,11]. One highly reactive bacteriophage clone from the library, LA006, was selected for further studies [3]. 2.3. DNA sequence analysis Automated DNA sequencing was done using the double-stranded dideoxy method in an Applied Biosystems model 373A DNA sequencer. DNA sequences were aligned and analyzed with AssemblyLIGN and MacVector software (IBI, New Haven, CT). DNA and deduced amino acid sequences were compared to entries in the non-redundant GenBank database at the National Center for Biotechnology Information using the BLAST algorithm [12]. 2.4. Nucleotide sequence accession number The nucleotide sequence of B. burgdorferi LA006 was deposited in the GenBank database with accession number U49938.
2.2. Sera and immunoscreening 3. Results The antibody pool used to screen the B. burgdorferi library [3,9] contained serum and synovial fluid of 16 patients with confirmed Lyme disease and sera from rabbits immunized by repeated injections of B. burgdorferi 297 strain sonicates [3]. Lyme disease patients had erythema migrans, carditis, neurologic disease or arthritis, and elevated anti-B. burgdorferi titers by ELISA and FIAX. Human and rabbit sera were passed several times through rabbit and human IgG-Sepharose columns, respectively, before pooling; the antibody pool was repeatedly absorbed with whole Escherichiu coli Y 1090 r- , bacterial membranes and ribosomal fractions, and soluble proteins covalently bound to Sepharose [lo]. The resulting reagent did not react with E. coli Y 1090 r- antigens on dot blotting. 8000 plaques of the unamplified IPTG-induced library were screeened with the extensively absorbed anti-B. burgdorferi serum pool, over 2.5 times more than the minimum of 3000 clones
3.1. DNA sequence analysis of the LA006 clone DNA sequencing of 47 recombinant clones in pUC19 containing overlapping segments of the original 6.4 kb B. burgdorferi DNA insert of LA006 produced the complete sequence of this DNA fragment including the entire bmpC gene (6930 bp) and a bmpC
bw’A
bmp8
ORF4
ORF5EglEl
&I &I 5012 5594
OzE.(PKCI)
5978
EcoRI 6950
Fig. 1. Location of ORF and restriction enzyme sites in LA006 DNA segment from B. burgdorferi chromosome. Direction of translation shown by arrow. PvuI site at position 5394 corresponds to Pvul at position 390 on the B. burgdorferi chromosoma1 map [7], placing LAO06 between rpoB and p22A on the B. burgdorferi chromosome.
L. Arm et al. IFEMS
A.
Microbiology Letters 145 (1996) 309-314
311
MgtE Mg” transporter MIDIDELRIFLKEKSYSKIKEKFLKHDSFDIAEALKRLNGTELILLYRFLPKKIAVETFSNFDQSTKNKL
Bb Bf
- 70
PS
Bb Bf PS
ANSFTNKEISEMIDELNLDDVIDLLEEVPANWQRFLASSTEENRGIINKFLSYSDDSAGSIVTIEWEL
Bb Bf PS
KEDFl'@GKALDYI&RVAKTKXD@fTYYITDD~ KWhKQVIKIBDXXL&KD TAVE@$EVIGRLZ&E%PDABT%%YLYWl%ERE Kpqmqm-E s1AD.!3mxKKNL IEHLDGEQIPTYL~ILSVKS@LA#DB
MTKBFIRV MSQSYLPV
Bb Bf PS Bb
PII~~SSTFg&TIBSNYQNLMLSLWI;ASFIPLLMDT RAPWII~FOLIT~IGQFEETLEAIVLLAAFIP~S~~S~VA~G~~~ ~~~~~K~FEEQLEAAIS~~~IGTG(~NSQ~ITSTL~~S~A
Bb Bf PS
VFtlCEICVSILV~3&ABVNFLR;LVFFVAPHHSDKLKIAFWSSCLMVSLTVAKILGGLLPIVA#&BR&D MLKRELGTGLMLGFZCMLVIAG-IVTVMHGNMM----LAFIVGISIFLTXiSIATIIGTIVPLIINKMELD ~~STSFLVAVTIGAAALI~WILGVGA-----EVTIWSLTIVAITMWSAIVSSII
Bb Bf PS
P%?T.,MAGPLITTIAUAIT@!A%~AKWVLVStiV H~sGPFITT~NDILG~IYFS~ATALL~EYL PXVVSAPFIATFIDGTGCZIYFEIAICLVMTEFA
B.
I%%!='NDE: -210 sqsAKTD - 77 -qAPDQs -77
s;ILV?mwN SSFQAAm DEkUlAQLQGAT'kPLEEPYLAT~ITLWRK
Bf PS
SGNAGSQASkLIIREfFFK
-280 -145 -146 -350 -215 -2i6 -420 -280 -281
-454 -312 -314
Protein kinase C inhibitor
Bb Hi
-
MDRLIRRTFYREQTMAEE
SY Mh Man
cow Rat
SC Cab
Maize
MSSEKEAALRRLDDS
Bb
KRGmLvNLE
Hi
SY Mb Man
cow Rat SC Cab Maize Bb SC
-140 - 8 8
BDLSYLIVKAREFALQEIKKLGAT-DDESLLGHLMIVGKX!AADLGLNK-DDESLLGHLMIVGKX!AADL-DDESLLGHUIIVGKKCAADLGLKK--@RMWNE@
EERHIBILGYL
LLAKLEGSD
-144
57 70 58 60 70 70 70 64 55 70
-126 -130 -114 -111 -126 -126 -126 -127 -113 -129
L. Arm
312
et al. IFEMS
Microbiology
Letters 145 (1996)
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Fig. 2. Alignment of deduced amino acid sequences of B. burgdorferi ORF (LA006) and homologous proteins. A: MgtE MgZf transporter (ORF 5). B: protein kinase C inhibitor (ORF 6). Amino acid residues identical to ORF 5 or ORF 6 occurring at the same position are indicated by shading. Bb, B. burgdorferi; Bf, Bacillusjirmus; Cab, Chinese cabbage; Hi, Hemophilus injuenzae; Mh, Mycoplasma hyorhinis; Ps, Providencia stuartii; SC, Saccharomyces cerevisiae; Sy, Synechococcus sp. t
schematic map of the insert is shown in Fig. 1. The PvuI site at position 5394 corresponds to the single PvuI site at about 390 kbp on the B. burdorferi chromosomal map between P22A and rpoB ([5], I. Schwartz, personal communication). Taken together with the position of the bmpA gene in the same map, the direction of transcription in the chromosomal DNA fragment cloned in LA006 is established as towards p22A in the B. burgdorferi chromosome [3,5,13]. The recently reported fourth member of the family of genes encoding for 39 kDa proteins, bmpD, maps at the 5’ end of bmpC [14]. Sequence analysis indicates that LA006 contains full ORF for BmpA and BmpB [3,13], and for three additional deduced proteins 3’ to bmpC, bmpA and bmpB. These proteins have molecular weights of 55716 Da (ORF 4; p1, 7.23), 50773 Da (ORF 5; p1, 4.86), and 15867 Da (ORF 6; p1, 8.68) (Fig. 2). ORF 4 begins at nt3525, is 1422 nucleotides long and would encode a protein of 474 amino acids. It has a potential ribosomal binding site AGAGGA at nt3512, could be initiated with TTG and terminated with TGA. This ORF also has a potential starter codon ATG at nt3537. ORF 5 begins at nt5003, is 1362 nucleotides long and would encode a protein of 454 amino acids. It has a potential ribosomal binding site GGAGGC at nt4991, is initiated with a ATG and terminated with TAA. ORF 6 begins at nt6418, is 417 nucleotides long and would encode a protein of 139 amino acids. It has a ribosomal binding site AGGATA at nt6406, is initiated with ATG and terminated with TAA. These ORF are preceded by regions that could function as canonical promoters corresponding to -35 and -10 consensus sequences at nt3460 and nt3480 for ORF 4, nt4954 and nt4975 for ORF 5, and nt6351 and nt6375 for ORF 6. 3.2. DNA sequence homology of ORF 4, ORF 5 and ORF 6
Comparison
of the deduced amino acid sequences
to entries in the database at GenBank using the BLAST algorithm [12] indicates that ORF 4 is homologous (32% identity) with an E. coli chromosomal ORF for an uncharacterized 88 kDa protein. ORF 5 has highly significant identity with a magnesium transporter protein, MgtE [ 151, found in several bacterial species (31% of identity with Bacillus firmus, 34% with Providencia stuartiz) (Fig. 2). ORF 6 has highly significant identity with protein kinase C inhibitors from eubacteria (55% identity with Hemophilus infhzenzae, 52% identity with the cyanobacterium Synechococcus sp., 51% identity with Mycoplasma hyorhinis), mammals (49% identity with human, cow and rat), and plants (46% identity with Saccharomyces cerevisiae, 52% identity with maize, 55% identity with Chinese cabbage) (Fig. 2).
4. Discussion We have developed a screening method to detect immunogenic antigens of B. burgdorferi expressed in vivo that has permitted identification, cloning and DNA sequencing of a cluster of possible virulencerelated genes of B. burgdorferi. This and recently published evidence suggest that this cluster of genes contains four members of the Bmp family of membrane lipoproteins located at approximately 390 kba of the B. burgdorferi chromosome [3,13,14]. These lipoproteins are important immunogens in Lyme disease [3,13]. Their preferential expression at 35°C and not at lower temperatures [16] and their immunogenicity in mice following infection by tick bite [17] could indicate involvement in evasion of mammalian host defenses and proliferation in host tissues. Location of the genes for the 4 Bmp proteins in tandem in the chromosome of B. burgdorferi and their shared homology and overlapping transcriptional signals also suggest coordinated differential regulation of in vivo expression [3,13]. Of other ORF on this DNA segment, protein kinase C inhibitor may also be involved in B. burgdorferi evasion of host defenses
L. Aron et al. I FEMS Microbiology Letters 145 (1996) 309-314
since protein kinase C mediates a wide variety of processes in the host immune and inflammatory responses and is clearly involved in phagocytosis and bacterial killing [l&-20]. MgtE magnesium transporter may enhance B. burgdorferi survival under low Mg2+ concentrations found in phagolysosomes; a similar scenario has been suggested for Salmonella within epithelial cell vacuoles [21]. Clustering of six genes that are potentially expressed in vivo in response to environmental clues is consistent with an evolutionary advantage of such a spatial arrangement [6,7]. While we do not know whether all these B. burdorferi proteins are expressed in vivo, we have already expressed recombinant BmpC in E. coli and have determined that the recombinant molecule reacts with sera from patients with Lyme disease (M. Alekshun, L. Aron, H. Godfrey, I. Schwarz and F.C. Cabello, manuscript in preparation). We are in the process of characterizing the immunological reactivity of other gene products encoded by the gene cluster with this sera.
Acknowledgments This work was supported by grants from the New York State Tick-Borne Disease Institute (F.C.C.) and the New York Medical College Lyme Disease Fund (F.C.C, H.P.G.), and by grants from the National Institutes of Health: R43 AI36004 (F.C.C.) and ROl CA34141 and AI37014 (H.P.G.). We would like to thank Drs. E. Ash and A. Weinstein for providing Lyme disease patient sera.
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