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Infection and Immunity, September 2000, p. 4992-5001, Vol. 68, No. 9
Laboratory of Human Bacterial Pathogenesis,
National Institute of Allergy and Infectious Disease, National
Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana
59840
Received 17 March 2000/Returned for modification 11 May
2000/Accepted 28 May 2000
A lipoprotein gene family first identified in Borrelia
burgdorferi strain 297, designated 2.9 LP and
recently renamed mlp, was found on circular and
linear plasmids in the genome sequence of B. burgdorferi
strain B31-M1. Sequence analyses of the B31 mlp genes and
physically linked variant gene families indicated that mlp
gene heterogeneity is unique and unrelated to location or linkage to
divergent sequences. Evidence of recombination between B31
mlp alleles was also detected. Northern blot analysis of
cultured strain B31 indicated that the mlp genes were not
expressed at a temperature (23°C) characteristic of that of ticks in
the environment. In striking contrast, expression of many
mlp genes increased substantially when strain B31 was
shifted to 35°C, a temperature change mimicking that
occurring in the natural transmission cycle of the spirochete from tick
to mammal. Primer extension analysis of the mlp mRNA transcripts suggested that sigma 70-like promoters are involved in
mlp expression during temperature shift conditions.
Antibodies were made against strain B31 Mlp proteins within the first 4 weeks after experimental mouse infection. Importantly, Lyme disease patients also had serum antibodies reactive with purified recombinant Mlp proteins from strain B31, a result indicating that humans are
exposed to Mlp proteins during infection. Taken together, the data
indicate that strain B31 mlp genes encode a diverse
array of lipoproteins which may participate in early infection
processes in the mammalian host.
Lyme disease caused by the bacterium
Borrelia burgdorferi is the most prevalent arthropod-borne
disease in the United States (47). Humans acquire the
infection when the organism is transmitted by the bite of infected
Ixodes ticks. Subsequent tissue invasion results in diverse
clinical manifestations such as erythema migrans, flu-like symptoms,
and neurologic, musculoskeletal, and cardiac problems (4, 21, 33,
36, 37, 46).
Most B. burgdorferi outer surface proteins are lipoproteins
(29). B. burgdorferi strain B31-M1 has 21 extrachromosomal elements, which may carry up to 91 lipoprotein-encoding genes (9). The synthesis of several
outer surface lipoproteins increases when B. burgdorferi
cultures are shifted from 23 to 35°C (5, 34, 42, 45).
Temperature-shifted cultures are presumed to mimic the warming that
occurs when the tick attaches to the mammal and feeds. Several outer
surface lipoproteins synthesized by B. burgdorferi grown at
35°C are recognized by sera from infected animals (34, 42), indicating that the mammal is exposed to these proteins during infection or transmission. Antigens expressed early in infection
have potential serodiagnostic or vaccine utility.
A lipoprotein-encoding family of seven genes designated 2.9 LP located on 30- and 18-kb supercoiled plasmids was
originally discovered and characterized in B. burgdorferi
strain 297 (28). Recently, three new members of this gene
family were characterized in strain 297 and renamed mlp (for
"multicopy lipoprotein genes") (49). Mlp homologues also
are made by Borrelia hermsii (41) and
Borrelia afzelii (44). The mlp genes
in B. burgdorferi strain 297 can be assigned to categories
on the basis of molecular size, protein sequence, and serologic
reactivity (28). Two distinct categories of noncoding
DNA sequences located immediately upstream of the ribosomal binding
site of the mlp genes have been identified (28,
49).
B. burgdorferi strain B31-M1 may contain a combination of
nine related 32-kb circular plasmids (designated cp32-1 through cp32-9)
and a related linear plasmid (designated lp56) that contains an
integrated cp32 plasmid (9, 10, 53, 54). Analysis of the
seven cp32 plasmids and lp56 plasmid from the strain B31-M1 sequenced
genome identified three families of loci named erp, orfC/orf3, and mlp. Two of these gene families
(erp and mlp) encode lipoproteins, whereas the
third (orfC/orf3) is believed to participate in plasmid
partitioning (40).
Several lines of evidence suggest that members of the Mlp family
participate in host-pathogen interactions. First, Akins et al.
(2) demonstrated that one mlp operon in strain
297 was expressed only in vivo in dialysis chambers and not at 23, 34, or 37°C following a temperature shift. Second, Yang et al.
(49) analyzed three other mlp genes in strain 297 and discovered that their expression increased when the cultures were
shifted from 23 to 37°C and that they were antigenic in infected
mice. Third, Mlps are lipoproteins, molecules that constitute a
significant fraction of the spirochete outer surface and induce
immunological responses in the host (16, 22, 24, 48).
Fourth, a recent investigation has discovered that Mlp homologs in
B. hermsii are antigenic in relapsing-fever patients
(41).
Taken together, these observations suggest that Mlps are important
molecules that may participate in the pathogenesis of human Lyme
disease. The goal of the present study was to investigate molecular
variation, expression, and antigenicity of nine mlps identified in the genome of B. burgdorferi strain B31.
Bacterial strains.
B. burgdorferi strain B31 was
originally isolated from an infected Ixodes scapularis tick
collected on Shelter Island, N.Y. (7). This strain has been
established in the laboratory by means of an infectious cycle between
I. scapularis and mice (34). Clone B31-4A
was derived from a single colony of infectious B31 plated on solid
Barbour-Stoenner-Kelly (BSK) and retains mouse infectivity (10,
19). Clone B31-e1 was derived from a single colony of a
high-passage, noninfectious culture of B31 (10).
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Expression and Immunological Analysis of the
Plasmid-Borne mlp Genes of Borrelia burgdorferi
Strain B31
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Cloning the B31 mlp genes.
Total genomic
DNA from strains B31-e1 and B31-4A was purified from 500 ml of
mid-log-phase BSK-H broth cultures with a DNA extraction kit
(Stratagene, San Diego, Calif.). DNA was quantitated by UV
spectroscopy, and 0.1 µg of DNA was used for PCRs. B31 mlp genes were given an alphabetical designation related to the cp32 plasmid (9, 10) on which they are found. The mlp
gene names in relation to their respective plasmid location are
designated as follows: mlpA, cp32-1; mlpB,
cp32-2; mlpC, cp32-3; mlpD, cp32-4; mlpF, cp32-6; mlpG, cp32-7; mlpH,
cp32-8; mlpI, cp32-9; and mlpJ, lp56. The
sequences for the mlpB and mlpE genes were not
available from The Institute for Genomic Research (TIGR) website
(www.tigr.org) because the plasmids encoding these genes (cp32-2 and
cp32-5) were not present in the sequenced B31-MI strain (9).
For the mlpB gene, Expand Long Template PCR (Boehringer
Mannheim, Indianapolis, Ind.) was performed, as recommended by the
manufacturer, on B31-e1 genomic DNA with primers designated
ErpC-prime and BB-2 (Table 1). A 12-kb
fragment was amplified, and the region containing the mlpB
gene was sequenced directly with primers ErpC-prime, Cp-2, ErpC-Lp2,
and BB-2 (Table 1).
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)EtBr spin columns (Supelco, Bellefonte, Pa.) as
specified by the manufacturer. Fragments were then quantitated by UV
spectroscopy, ligated with the pCR2.1 vector, and transformed into
INV
F' cells as specified by the manufacturer (Invitrogen). Recombinants were selected, and the plasmids were purified and sequenced. The resulting plasmids were designated pCR-MlpA, pCR-MlpB, pCR-MlpC, pCR-MlpD, pCR-MlpF, pCR-MlpG, pCR-MlpH, pCR-MlpI, and pCR-MlpJ.
DNA sequencing.
PCR fragments were sequenced by the
following method. PCR products that were free of contaminating bands as
assessed by agarose gel electrophoresis were purified and concentrated
with a Centricon 100 concentrator (Millipore, Bedford, Mass.). PCR
products with additional contaminating bands were purified in a
different manner. The fragment of interest was excised from the gel and
purified with a Supelco ()EtBr spin column. DNA was quantitated by UV spectroscopy and diluted for automated DNA sequencing with an Applied
Biosystems Inc. model 373 Stretch automated DNA sequencer and ABI PRISM
dye terminator ready-reaction cycle-sequencing kit (PE Biosystems,
Foster City, Calif.). DNA plasmids were purified with a Qiagen Midi kit
(Qiagen, Valencia, Calif.).
DNA sequence analysis.
Nucleotide and deduced amino acid
sequences were analyzed with MacVector version 6.5.1 (Oxford Molecular,
Beaverton, Oreg.) and compared to reference sequences obtained from the
TIGR website. Sequence alignments, phylogenetic tree construction, and
calculation of protein sequence similarity values were performed with
DNASTAR (Lasergene, Madison, Wis.). Protein sequence identity values
were calculated by pairwise alignment of protein sequences in GAP
(Genetics Computer Group, Madison, Wis.). Sequence and primer extension data were further analyzed with the 
70 consensus search
program MACTARGSEARCH (25). Detection and analysis of
recombination between mlp alleles were performed by previously described statistical methods (38).
Bendability/curvature propensity plots were calculated with the bend.it
server, with the DNase I-based trinucleotide bendability parameters
described by Brukner et al. (6) and the consensus
bendability scale (12).
Northern blots.
Total RNA was extracted from B. burgdorferi B31-4A and B31-e1 cells grown at 23°C or after the
temperature shift to 35°C, with Ultraspec isolation solution
(Biotecx, Houston, Tex.). The RNA was denatured with glyoxal and
dimethyl sulfoxide, separated by agarose gel electrophoresis in 10 mM
sodium phosphate buffer (pH 7.0) (32), and transferred to
nylon membranes (Micron Separations, Westborough, Mass.). Probes
specific for each of the B31 mlp genes and the
flaB (flagellin) gene were generated by PCR from pCR2.1 recombinant plasmids with the cognate gene. Probe specificities were
first established by searching probe DNA sequences against the B31-M1
genome on the TIGR website. The oligonucleotides used to generate these
PCR fragment probes are listed in Table 1. The flaB-specific
probe was generated as previously described (39). DNA
fragments generated by PCR were gel purified by using QIAEX II kit
(QIAGEN) and radiolabeled with [-32P]dATP (NEN
Research Products, Du Pont, Boston, Mass.) by random priming.
Unincorporated radiolabel was removed by column chromatography. To
check for probe specificity, purified DNAs from the pCR2.1 recombinant
clones were digested with EcoRI for 2 h at 37°C to linearize the DNA and 1 µg of the DNA was transferred to
nitrocellulose with a vacuum dot-blot apparatus (Bio-Rad, Hercules,
Calif.). Dot blot filters were hybridized overnight at 65°C in 2×
Denhardt's solution-6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M
sodium citrate)-1 mM EDTA-100 µg of sonicated salmon sperm DNA per
ml in a rotating hybridization oven (Robbin's Scientific Corp.,
Sunnyvale, Calif.). Following hybridization, the nitrocellulose filters
were washed twice for 15 min at 65°C in 2× SSC containing 0.1%
sodium dodecyl sulfate (SDS) and then four times for 15 min each at
room temperature in 0.1× SSC containing 0.1% SDS. The filters then
were subjected to autoradiography at 70°C for 4 to 48 h.
Primer extension analysis. RNA for primer extension analysis was extracted from B. burgdorferi B31-4A or B31-e1 grown at 23°C or after the shift to 35°C by using Ultraspec isolation solution (Biotecx). The RNA was treated with DNase (Promega, Madison, Wis.), dissolved in sterile deionized water treated with 0.1% diethylpyrocarbonate, and quantitated by UV spectroscopy.
mlpA and mlpC were chosen for study on the basis of the uniqueness of their upstream and coding sequences for primer specificity. The DNA sequences of mlpA and mlpF are identical for the first 124 bases upstream and 275 bases downstream of the start codons. This high level of identity precluded the design of a primer that would differentiate between transcripts for these two genes. Therefore, a primer (designated mlpA/F-PE) that would bind equally well to mlpA or mlpF transcripts was made. Base differences within the regulatory and coding regions for mlpC, mlpD, mlpG, mlpH, mlpI, and mlpJ allowed the design of a primer specific for mlpC transcript expression, and this primer was designated mlpC-PE (Table 1). Alignment of the mlpA/F-PE and mlpC-PE primers against the B31-M1 total genome sequence established that these primers were specific for their targets. Extension reactions were performed with the avian myeloblastosis virus reverse transcriptase primer extension system (Promega). The reaction products were loaded onto a 6% or 8% acrylamide sequencing gel. A 1,093-bp DNA fragment containing the start codon and upstream sequence for the mlpA gene was made with primers designated mlpAprom-5' and mlpAprom-3' (Table 1) and cloned into the pCR2.1 vector (Invitrogen). The plasmid DNA (designated pmlpA-prom) was purified and used as a sequencing marker. For primer extension analysis of the mlpC gene, a 1,174-bp DNA fragment containing the mlpC ATG start codon and upstream sequence was made with primers designated mlpCprom-5' and mlpCprom-3' (Table 1). This fragment was cloned into the pCR2.1 vector (designated pmlpC-prom) and used as a sequencing marker for mlpC primer extension analysis. All sequencing reactions were performed with the Amplicycle sequencing kit (PE Biosystems).Recombinant Mlp proteins and Western blotting. Genes encoding Mlp proteins were amplified with PCR primers that would result in a XhoI site and a BamHI site for restriction digestion and cloning in frame with an amino-terminal histidine tag in the pET-15b expression vector (Novagen, Madison, Wis.). The same 5' primer (designated MlpA/C/F/H-His5') was used for the mlpA, mlpC, mlpF, and mlpH genes (Table 1). For mlpB, mlpD, mlpG, mlpI, and mlpJ, unique 5' primers were designated MlpB-His5', MlpD-His5', MlpG-His5', MlpI-His5', and MlpJ-His5', respectively (Table 1). The same 3' primer, designated All(-G)His3' (Table 1), was used with all of the above-mentioned 5' primers. Unique primers designated MlpG-His5' and MlpG-His3' were used for cloning and expression of the mlpG gene. The 5' fusion primers begin with the codon for the first amino acid immediately downstream of the lipidated cysteine, and the 3' primers correspond to the sequence in the pCR2.1 vector. The Mlp-His fusion proteins were terminated by the encoded Borrelia mlp termination sequence in each construct.
All PCRs were performed in a total volume of 100 µl under mineral oil with the appropriate primer set and the appropriate pCR2.1 recombinant DNA as the template. The PCR conditions used were 25 cycles of 94°C for 1 min, 50°C for 30 s, and 72°C for 2 min followed by one cycle of 7 min at 72°C. The pET15b vector (Novagen) was digested with XhoI and BamHI enzymes, purified with ()EtBr
spin columns, and ligated to the fusion PCR fragments. Ligation
reaction mixtures were transformed into BLR(DE3) competent cells
(Novagen). Recombinants were induced with
isopropyl-
-D-thiogalactopyranoside (IPTG) and the
proteins encoded by mlpA, mlpC, mlpD,
mlpF, mlpG, mlpH, mlpI, and
mlpJ were identified in supernatant fractions by
SDS-polyacrylamide gel electrophoresis analysis. These proteins were
purified as recommended by the manufacturer (Novagen). The MlpB fusion
protein was present in the cell pellet fraction, and attempts to purify
the protein under denaturing conditions were unsuccessful.
Purified proteins were quantitated with the Bio-Rad protein assay kit,
and 2 µg of each protein was loaded onto 15% polyacrylamide gels and
subjected to SDS-polyacrylamide gel electrophoresis. The gels were
stained with Coomassie blue or transferred to nitrocellulose. Mouse
serum collected 4 weeks (two mice) or 8 weeks (three mice) after
infection of the mouse, via a tick bite, with a low-passage, nonclonal
population of B31, was provided by Tom Schwan, Rocky Mountain
Laboratories (39). The individual 4- and 8-week mouse sera
were pooled separately prior to immunoblotting of nitrocellulose membranes. Control sera were collected from two mice not infected with
B. burgdorferi. Eleven human sera were collected from Lyme disease patients and provided by Tom Schwan. The diagnosis of Lyme
disease was based on p39 immunoreactivity and clinical presentation consistent with Lyme disease (35). In addition, sera
collected from five noninfected humans, with no previous exposure to
B. burgdorferi, were used in immunoblotting experiments.
Western blotting was performed as previously described with slight
modifications (39). Briefly, primary incubations were performed for 1 h with mouse or human sera at a 1:100 dilution. A
rabbit anti-mouse or anti-human horseradish peroxidase-conjugated secondary antibody at 1:20,000 dilution was incubated with the membrane
for 1 h in phosphate-buffered saline-Tween. Membranes were
developed with the ECL kit as specified by the manufacturer (Amersham,
Piscataway, N.J.).
Nucleotide sequence accession number. The sequence for the B31 mlpB gene has been deposited in GenBank under accession number AF245449.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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mlp genes in strain B31. B. burgdorferi strain B31 can contain nine different cp32 plasmids and a related linear plasmid designated lp56. The B. burgdorferi strain recently sequenced (B31-M1) contains seven cp32 mlp genes (cp32-2 and cp32-5 are absent) and one lp56 mlp gene. We used DNA purified from a B31 passage variant known to contain cp32-2 to amplify a large fragment that extended from the erpC gene to the intergenic region located 3' of the mlp gene on cp32-2. Sequence analysis of this PCR fragment indicated that a mlp gene homologue (designated mlpB) was present. Our attempt to clone the mlp gene located on the cp32-5 plasmid with a similar strategy was unsuccessful.
Alignment of the Mlp protein sequences encoded by mlp genes in strains 297 and B31 identified a relatively conserved consensus signal peptidase II cleavage site for all Mlps (Fig. 1). Mlp proteins have been previously assigned to two categories or classes on the basis of sequence homology, reactivity with polyclonal antisera, and molecular weight (28, 49). Phylogenetic tree construction with the amino acid sequences of 9 strain B31 Mlps and 10 strain 297 Mlps confirmed that two distinct sequence families exist and that mlpB encodes a class I protein (Fig. 2). Identity values ranged from 22.1 to 43.1% for comparison of Mlp class I proteins with class II proteins within or between the two strains. Within the two classes, the identity values were 68.7 to 86.4% for class I proteins and 65.8 to 88.6% for class II proteins. The phylogenetic analysis also found that divergent or unique Mlp proteins exist between the two strains (Fig. 2). For example, only four of nine strain B31 Mlp proteins were related to strain 297 Mlp proteins (Mlp-2 and MlpC, 81.1% identity; Mlp-4 and MlpJ, 88.6% identity; Mlp-8 and MlpH, 81.4% identity; and Mlp-3 and MlpG, 74.4% identity). Hence, there are no closely related Mlp homologs (>95%) in strains B31 and 297.
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Northern analysis of mlp gene expression at tick (23°C) and mammalian (35°C) temperatures. The expression of several B. burgdorferi outer surface lipoproteins increases when cultures are shifted from 23 to 35°C (34, 42). We investigated if the mlp genes of B31 were differentially expressed at temperatures normally associated with the tick (23°C) or mammal (35°C) (42). Oligonucleotide primers were designed that amplify unique regions of the B31 mlp genes. The resulting PCR products varied in size from 84 to 160 bp for mlpA through mlpJ. Each probe was confirmed to be specific by being tested against the full-length cloned mlp genes digested with EcoRI.
When the PCR products were probed against B. burgdorferi B31-4A RNA under stringent hybridization conditions, it was found that full-length mlp gene transcripts were not present at 23°C (Fig. 3). In striking contrast, predicted full-length transcripts were identified when the cultures were shifted from 23 to 35°C (Fig. 3). The RNA transcripts smaller than the predicted full-length transcript observed for mlpA, mlpC, mlpF, and mlpH (Fig. 3) may be caused by functional inactivation, a process involving endonucleolytic cleavage or degradation of the 5' terminus of the transcript (18). The intensities of RNA transcripts for mlpA, mlpC, mlpF, and mlpH were considerably greater than those observed for mlpD, mlpG, mlpI, and mlpJ. Variance in transcript intensity could be due, in part, to differences in mlp promoter sequences. For example, mlpA and mlpF, which contain nearly identical upstream sequences, had a high level of band intensity, whereas mlpC, mlpD, mlpG, mlpH, mlpI, and mlpJ, with a different upstream sequence, had comparatively lower band intensity.
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Primer extension analysis of mlpA/F and mlpC. Primer extension analysis was performed to identify the mRNA initiation sites and promoter regions responsible for transcript initiation. The DNA sequence of mlpC, mlpD, mlpG, mlpH, mlpI, and mlpJ in strain B31 differs completely from that of the analogous region of mlpA and mlpF beginning 6 bases upstream of the ATG start codon. The high homology between the coding and noncoding regions of mlpA and mlpF precluded the design of a primer which would bind specifically to one gene but not the other. Therefore, the primer used (designated mlpA/F-PE) had the potential to bind to both mlpA or mlpF transcripts. Primer extension experiments were performed with RNA isolated from temperature-shifted strain B31-e1, a passage variant known to contain cp32-1 (mlpA present) but not cp32-6 (mlpF absent). No product was detected in primer extension experiments (data not shown). Northern blot analysis with the mlpA-specific probe also failed to detect an mRNA transcript for the mlpA gene in RNA from temperature-shifted strain B31-e1 (data not shown). PCR amplification and sequence analysis with the mlpA/F-5' and mlpA-3' primers confirmed the presence of the mlpA gene in DNA from strain B31-e1.
Primer extension analysis of RNA obtained from temperature-shifted strain B31-4A with the mlpA/F-PE primer produced a single, dominant extension product (Fig. 4A). This product is most probably the result of extension from mlpA and mlpF transcripts known to be present in this preparation (Fig. 3). The calculated point of initiation is 95 bases from the 3' end of the primer sequence and 56 bases upstream of the ATG start codon for mlpA and mlpF. No additional extension products were detected during brief or prolonged runs of the gel or subsequent repeated experiments (data not shown). The faint bands observed immediately above and below the primary initiation site (Fig. 4A) may represent initiation just before and after the adenine 56 bases upstream of the ATG start codon for mlpA and mlpF during transcription initiation, a phenomenon reported in studies of ospC transcription initiation (23).
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70 promoter with a similarity score of 56.8% (Fig. 4C).
Moreover, further analysis of this promoter indicated the presence of
an Escherichia coli S (RpoS) consensus
sequence (11) in the 10 region (Fig. 4C). Consistent with
this observation, a curvature-propensity plot calculated with DNase
I-based trinucleotide parameters (6, 12) identified an
upstream sequence capable of adopting a curved conformation. We note
that E. coli S promoters usually are located
immediately downstream of DNA capable of adopting a curved conformation
(11).
A 70 consensus promoter sequence with a homology score
of 55% was found immediately upstream of the first initiation site for mlpC (Fig. 4D). Alignment of the regulatory regions of
mlpC, mlpD, mlpG, mlpH,
mlpI, and mlpJ and the first and second
initiation sites is shown in Fig. 4D. Surprisingly, no consensus
sequence with a homology score greater than 45%, the cutoff value for
functional 70 promoter sequences in E. coli,
was identified upstream of the second initiation site (25).
In addition, no S consensus sequence or curved DNA was
identified upstream of the first or second initiation site.
Immunoblot analysis of the B31 Mlp proteins with sera
from infected mice and Lyme disease patients.
Identification of
temperature-induced mRNA transcripts corresponding to the B31
mlp genes led us to test if the protein products were
antigenic in mammalian hosts. Each Mlp protein was expressed as a
His-tagged fusion protein and purified to homogeneity (Fig. 5A). MlpB was present in insoluble form
in the E. coli pellet fraction and was intractable to
purification (Fig. 5A).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Sequence analysis. Phylogenetic analysis of the B31 cp32 mlp genes compared with two other physically linked heterogeneous gene families established that the sequence diversity present in the mlp genes is unique and is unrelated to location or linkage to variant sequences. Caimano et al. (8) concluded that the importance of recombination as a mechanism for generating sequence diversity at the B31 mlp loci was less apparent than their results obtained for the strain 297 mlp genes, presumably because of the lower level of sequence diversity seen among the strain B31 mlp alleles. However, our focused analysis of polymorphic sites within each of the seven class II mlp alleles demonstrated that these genes, and presumably the class I mlp genes, have been diversified by recombination events. Comparative analysis of the mlp genes present in strains 297 and B31 (including the newly discovered mlpB gene) demonstrated that only four mlp genes were relatively homologous. Taken together, the data suggest that recombination events contribute to the diversity of the mlp genes and suggest that the rate of generating new allelic variants may be relatively high. Although the host processes that select new Mlp variants are unknown, immune avoidance or functional selection processes may participate.
mlp Northern blot analysis. We discovered that upregulation of the mlp genes in B. burgdorferi strain B31 occurs when cultures are shifted from 23 to 35°C, a result in agreement with data obtained from analysis of three newly identified mlp genes in B. burgdorferi strain 297 (49). We note that unlike the Northern blot results reported for the erp genes (40), no mlp-specific transcripts were detected in bacteria grown at 23°C. Hence, our results add the strain B31 mlp gene family to other Borrelia lipoprotein genes that are differentially upregulated by growth at 35°C, a temperature characteristic of many mammalian hosts, including humans (2, 34, 42).
Primer extension analysis of mlp expression.
Primer extension analysis identified the transcriptional initiation
sites of the mlp transcripts produced during temperature shift conditions. These experiments identified 70
promoter elements that may participate in the expression of
mlpA, mlpF, and mlpC. Promoters with
70-like homology in B. burgdorferi have been
described previously for the genes encoding variable, lipidated
proteins such as OspA, OspC, OspD, and OspE (20, 23, 26) and
flagellin (13). We note that primer extension studies with
the mlp genes of strain 297 were not performed due to high
sequence identity between genes and the lack of defined cp32 passage
variants for strain 297 (28).
70 can be recognized in
vitro by S, a sigma factor involved in stationary-growth
gene expression (43). Our discovery of a consensus
S sequence (CTATCT) in the 10 region of the promoter
region of mlpA and mlpF, together with
identification of a region of potential curved DNA upstream, suggests
that these genes may be regulated by S. In E. coli, the presence of a 35 region in a S promoter
generally favors recognition by 70 (11).
However, direct evidence of molecular interaction between the
mlpA and mlpF promoters and
S and/or 70 was not sought in the
present study.
The promoter region identified for the second transcription initiation
site located upstream of the mlpC gene was not homologous to
70 or S sequences, suggesting a distinct
molecular mechanism of mRNA initiation. We discovered that this cryptic
promoter is no longer active in strain B31-e1 (which lacks cp32
plasmids and some other plasmids and is noninfectious
[10]), a result mimicking that seen for the promoter
element of the mlpA gene. It is probable that cis
and trans factors responsible for expression of
mlpA and mlpC are not present, are present at
greatly reduced levels, or are not active in B31-e1. Altered gene
expression has been described in high-passage B31 variants (17,
30).
Among the B31 mlpC, mlpD, mlpG,
mlpH, mlpI, and mlpJ genes,
mlpC and mlpH had the highest level of expression
by Northern analysis. These two genes also had identical sequences in
the spacer region and the 10 region of the promoter identified for the first upstream initiation site. In contrast, mlpJ had
the lowest level of expression and the greatest divergence in sequence of this promoter region. It is likely that the number and location of
minor base changes in this promoter region affect the level of
expression of these genes in vitro. Minor base changes in promoter sequences that lead to altered promoter activity are well described in
many organisms.
Antigenicity of purified Mlp recombinant proteins. We discovered that mice made anti-Mlp antibodies within the first 4 weeks of experimental infection. This antibody reactivity was not Mlp class specific, because Mlps from both classes reacted similarly with the mouse sera. In addition, there was no simple relationship between the Mlp proteins that were immunoreactive and the upstream regulatory region of their structural genes. Importantly, similar results were obtained with sera from the Lyme disease patients. The mlp complement of the organisms that infected these Lyme disease patients is not known. It is possible that some of the structural features of the Mlp proteins from B31 may be conserved among different B. burgdorferi strains or isolates. In addition, we do not know if these Mlp-reactive antibodies were produced early or late in these human infections, although the mouse data favor early production. We note that our data are consistent with results obtained from recent work on B. hermsii Mlp homologs, also shown to be antigenic in infected humans (41).
Potential pathogenesis roles have been attributed to protein families such as Vls, BlyAB, and the Opps on the basis of functional assays or sequence homologies (5, 14, 50-52). Putative functions have yet to be assigned to the Erp, 39-kDa, and Mlp proteins and numerous other paralog proteins of B. burgdorferi. The identification of multiple mlp alleles in strain B31, together with evidence of intragenic recombination, suggests that more alleles will arise in a strain by recombination. In addition, other B. burgdorferi strains will undoubtedly have diversity in mlp sequence. The extensive diversity of mlp genes within and between strains could constrain the serodiagnostic and vaccine utility of these proteins unless broadly conserved epitopes are identified.Conclusions. Our data and those contributed by others (2, 8, 28, 44, 49) suggest that Mlp proteins may play an important, albeit undefined role in B. burgdorferi transmission, establishment of infection, or immune evasion. Analysis of early expression of the mlp genes in the tick during the transmission cycle and their expression within different host tissues may provide important new insights into Lyme disease pathogenesis.
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ACKNOWLEDGMENTS |
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We thank Thomas S. Whittam for help in analysis of the B31 mlp alleles, Tom Schwan for providing sera from Lyme disease patients and infected mice, and Gary Hettrick for help with the graphics. J. M. Musser edited the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th St., Hamilton, MT 59840. Phone: (406) 363-9271. Fax: (406) 363-9204. E-mail: sporcella{at}nih.gov.
Editor: D. L. Burns
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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|---|
| 1. | Abeles, A. L., K. M. Snyder, and D. K. Chattoraj. 1984. P1 plasmid replication: replicon structure. J. Mol. Biol. 173:307-324[CrossRef][Medline]. |
| 2. | Akins, D. R., K. W. Bourell, M. J. Caimano, M. V. Norgard, and J. D. Radolf. 1998. A new animal model for studying Lyme disease spirochetes in a mammalian host-adapted state. J. Clin. Investig. 101:2240-2250[Medline]. |
| 3. |
Barbour, A. G.,
C. J. Carter,
V. Bundoc, and J. Hinnebusch.
1996.
The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes.
J. Bacteriol.
178:6635-6639 |
| 4. | Barthold, S. W., D. H. Persing, A. L. Armstrong, and R. A. Peeples. 1991. Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice. Am. J. Pathol. 139:263-273[Abstract]. |
| 5. |
Bono, J. L.,
K. Tilly,
B. Stevenson,
D. Hogan, and P. Rosa.
1998.
Oligopeptide permease in Borrelia burgdorferi: putative peptide-binding components encoded by both chromosomal and plasmid loci.
Microbiology
144:1033-1044 |
| 6. | Brukner, I., R. Sanchez, D. Suck, and S. Pongor. 1995. Trinucleotide models for DNA bending propensity: comparison of models based on DNaseI digestion and nucleosome packaging data. J. Biomol. Struct. Dyn. 13:309-317[Medline]. |
| 7. |
Burgdorfer, W.,
A. G. Barbour,
S. F. Hayes,
J. L. Benach,
E. Grunwaldt, and J. P. Davis.
1982.
Lyme disease a tick-borne spirochetosis?
Science
216:1317-1319 |
| 8. |
Caimano, M. J.,
X. Yang,
T. G. Popova,
M. L. Clawson,
D. R. Akins,
M. V. Norgard, and J. D. Radolf.
2000.
Molecular and evolutionary characterization of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297.
Infect. Immun.
68:1574-1586 |
| 9. | Casjens, S., N. Palmer, R. van Vugt, W. M. Huang, B. Stevenson, P. Rosa, R. Lathigra, G. Sutton, J. Peterson, R. J. Dodson, D. Haft, E. Hickey, M. Gwinn, O. White, and C. Fraser. 2000. A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi. Mol. Microbiol. 35:490-516[CrossRef][Medline]. |
| 10. |
Casjens, S.,
R. van Vugt,
K. Tilly,
P. A. Rosa, and B. Stevenson.
1997.
Homology throughout the multiple 32-kilobase circular plasmids present in Lyme disease spirochetes.
J. Bacteriol.
179:217-227 |
| 11. |
Espinosa-Urgel, M.,
C. Chamizo, and A. Tormo.
1996.
A consensus sequence for S-dependent promoters.
Mol. Microbiol.
21:657-659[CrossRef][Medline].
|
| 12. | Gabrielian, A., and S. Pongor. 1996. Correlation of intrinsic DNA curvature with DNA property periodicity. FEBS Lett. 393:65-68[CrossRef][Medline]. |
| 13. |
Ge, Y.,
I. G. Old,
I. Saint Girons, and N. W. Charon.
1997.
The flgK motility operon of Borrelia burgdorferi is initiated by a 70-like promoter.
Microbiology
143:1681-1690 |
| 14. | Guina, T., and D. B. Oliver. 1997. Cloning and analysis of a Borrelia burgdorferi membrane-interactive protein exhibiting haemolytic activity. Mol. Microbiol. 24:1201-1213[CrossRef][Medline]. |
| 15. |
Higgins, D. G.,
A. J. Bleasby, and R. Fuchs.
1992.
CLUSTAL V: improved software for multiple sequence alignment.
CABIOS
8:189-191 |
| 16. |
Hirschfeld, M.,
C. J. Kirschning,
R. Schwandner,
H. Wesche,
J. H. Weis,
R. M. Wooten, and J. J. Weis.
1999.
Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by toll-like receptor 2.
J. Immunol.
163:2382-2386 |
| 17. |
Jonsson, M., and S. Bergstrom.
1995.
Transcriptional and translational regulation of the expression of the major outer surface proteins in Lyme disease Borrelia strains.
Microbiology
141:1321-1329 |
| 18. | King, T. C., and D. Schlessinger. 1990. Processing of mRNA transcripts, p. 703-718. In F. C. Neidhardt, J. L. Ingram, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. |
| 19. |
Kurtti, T. J.,
U. G. Munderloh,
R. C. Johnson, and G. G. Ahlstrand.
1987.
Colony formation and morphology in Borrelia burgdorferi.
J. Clin. Microbiol.
25:2054-2058 |
| 20. |
Lam, T. T.,
T.-P. K. Nguyen,
R. R. Montgomery,
F. S. Kantor,
E. Fikrig, and R. A. Flavell.
1994.
Outer surface proteins E and F of Borrelia burgdorferi, the agent of Lyme disease.
Infect. Immun.
62:290-298 |
| 21. | Logigian, E. L., R. F. Kaplan, and A. C. Steere. 1990. Chronic neurologic manifestations of Lyme disease. N. Engl. J. Med. 323:1438-1444[Abstract]. |
| 22. |
Ma, Y.,
K. P. Seiler,
K. F. Tai,
L. Yang,
M. Woods, and J. J. Weis.
1994.
Outer surface lipoproteins of Borrelia burgdorferi stimulate nitric oxide production by the cytokine-inducible pathway.
Infect. Immun.
62:3663-3671 |
| 23. |
Marconi, R. T.,
D. S. Samuels, and C. F. Garon.
1993.
Transcriptional analyses and mapping of the ospC gene in Lyme disease spirochetes.
J. Bacteriol.
175:926-932 |
| 24. | Marietta, E. V., J. J. Weis, and J. H. Weis. 1997. CD28 expression by mouse mast cells is modulated by lipopolysaccharide and outer surface protein A lipoprotein from Borrelia burgdorferi. J. Immunol. 159:2840-2848[Abstract]. |
| 25. | Mulligan, M. E., D. K. Hawley, R. Entriken, and W. R. McClure. 1984. Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res. 12:789-800. |
| 26. |
Norris, S. J.,
C. J. Carter,
J. K. Howell, and A. G. Barbour.
1992.
Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein.
Infect. Immun.
60:4662-4672 |
| 27. | Ogura, T., and S. Hiraga. 1983. Partition mechanism of F plasmid: two plasmid gene-encoded products and a cis-acting region are involved in partition. Cell 32:351-360[CrossRef][Medline]. |
| 28. |
Porcella, S. F.,
T. G. Popova,
D. R. Akins,
M. Li,
J. D. Radolf, and M. V. Norgard.
1996.
Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family.
J. Bacteriol.
178:3293-3307 |
| 29. | Rosa, P. A. 1997. Microbiology of Borrelia burgdorferi. Semin. Neurol. 17:5-10[Medline]. |
| 30. |
Sadziene, A.,
B. Wilske,
M. S. Ferdows, and A. G. Barbour.
1993.
The cryptic ospC gene of Borrelia burgdorferi B31 is located on a circular plasmid.
Infect. Immun.
61:2192-2195 |
| 31. | Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425[Abstract]. |
| 32. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 33. | Schutzer, S. E. 1992. Lyme disease: molecular and immunological approaches, vol. 6. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 34. |
Schwan, T. G.,
J. Piesman,
W. T. Golde,
M. C. Dolan, and P. A. Rosa.
1995.
Induction of an outer surface protein on Borrelia burgdorferi during tick feeding.
Proc. Natl. Acad. Sci. USA
92:2909-2913 |
| 35. |
Simpson, W. J.,
M. E. Schrumpf, and T. G. Schwan.
1990.
Reactivity of human Lyme borreliosis sera with a 39-kilodalton antigen specific to Borrelia burgdorferi.
J. Clin. Microbiol.
28:1329-1337 |
| 36. | Steere, A. C. 1989. Lyme disease. N. Engl. J. Med. 321:586-596[Abstract]. |
| 37. | Steere, A. C., W. P. Batsford, M. Weinberg, J. Alexander, H. J. Berger, S. Wolfson, and S. E. Malawista. 1980. Lyme carditis: cardiac abnormalities of Lyme disease. Ann. Intern. Med. 93:8-16. |
| 38. | Stephens, J. C. 1985. Statistical methods of DNA sequence a analysis: detection of intragenic recombination or gene conversion. Mol. Biol. Evol. 2:539-556[Abstract]. |
| 39. |
Stevenson, B.,
J. L. Bono,
T. G. Schwan, and P. Rosa.
1998.
Borrelia burgdorferi Erp proteins are immunogenic in mammals infected by tick-bite and their synthesis is inducible in cultured bacteria.
Infect. Immun.
66:2648-2654 |
| 40. |
Stevenson, B.,
S. Casjens, and P. Rosa.
1998.
Evidence of past recombination events among the genes encoding the Erp antigens of Borrelia burgdorferi.
Microbiology
144:1869-1879 |
| 41. | Stevenson, B., S. F. Porcella, K. L. Oie, C. A. Fitzpatrick, S. J. Raffel, L. Lubke, M. E. Schrumpf, and T. G. Schwan. 2000. The relapsing fever spirochete Borrelia hermsii contains multiple antigen-encoding circular plasmids Homologous to the cp32 plasmids of Lyme disease spirochetes. Infect. Immun. 7:3900-3908. |
| 42. | Stevenson, B., T. G. Schwan, and P. A. Rosa. 1995. Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi. Infect. Immun. 63:4535-4539[Abstract]. |
| 43. |
Tanaka, K.,
Y. Takayanagi,
N. Fujita,
A. Ishihama, and H. Takahashi.
1993.
Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli.
Proc. Natl. Acad. Sci. USA
90:3511-3515 |
| 44. |
Theisen, M.
1996.
Molecular cloning and characterization of nlpH, encoding a novel, surface-exposed, polymorphic, plasmid-encoded 33-kilodalton lipoprotein of Borrelia afzelii.
J. Bacteriol.
178:6435-6442 |
| 45. | Tilly, K., S. Casjens, B. Stevenson, J. L. Bono, D. S. Samuels, D. Hogan, and P. Rosa. 1997. The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene. Mol. Microbiol. 25:361-373[CrossRef][Medline]. |
| 46. | Van der Linde, M. R. 1991. Lyme carditis: clinical characteristics of 105 cases. Scand. J. Infect. Dis. Suppl. 77:81-84[Medline]. |
| 47. | Walker, D. H. 1998. Tick-transmitted infectious diseases in the United States. Annu. Rev. Public Health 19:237-269[CrossRef][Medline]. |
| 48. |
Wooten, R. M.,
T. B. Morrison,
J. H. Weis,
S. D. Wright,
R. Thieringer, and J. J. Weis.
1998.
The role of CD14 in signaling mediated by outer membrane lipoproteins of Borrelia burgdorferi.
J. Immunol.
160:5485-5492 |
| 49. |
Yang, X.,
T. G. Popova,
K. E. Hagman,
S. K. Wikel,
G. B. Schoeler,
M. J. Caimano,
J. D. Radolf, and M. V. Norgard.
1999.
Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family.
Infect. Immun.
67:6008-6018 |
| 50. | Zhang, J.-R., J. M. Hardham, A. G. Barbour, and S. J. Norris. 1997. Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes. Cell 89:275-285[CrossRef][Medline]. |
| 51. |
Zhang, J.-R., and S. J. Norris.
1998.
Genetic variation of the Borrelia burgdorferi gene vlsE involves cassette-specific, segmental gene conversion.
Infect. Immun.
66:3698-3704 |
| 52. |
Zhang, J.-R., and S. J. Norris.
1998.
Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi.
Infect. Immun.
66:3689-3697 |
| 53. | Zückert, W. R., E. Filipuzzi-Jenny, J. Meister-Turner, M. Stålhammar-Carlemalm, and J. Meyer. 1994. Repeated DNA sequences on circular and linear plasmids of Borrelia burgdorferi sensu lato, p. 253-260. In J. S. Axford, and D. H. E. Rees (ed.), Lyme borreliosis. Plenum Press, New York, N.Y. |
| 54. |
Zückert, W. R., and J. Meyer.
1996.
Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element.
J. Bacteriol.
178:2287-2298 |
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