Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, MS 415 Chandler Medical Center, Lexington, KY 40536-0298, USA
Correspondence
Brian Stevenson
bstev0{at}uky.edu
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ABSTRACT |
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INTRODUCTION |
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Among the many borrelial plasmids is the cp32 family, a group of closely related elements that are generally circular plasmids of about 32 kb in size (Stevenson et al., 2001). All examined Lyme disease spirochaetes contain numerous different members of this plasmid family. The cp32 plasmids are largely identical to each other and vary significantly at only a few loci, one of which is the erp locus. The encoded Erp proteins are surface-exposed lipoproteins that are produced during mammalian infection and bind to complement regulatory factor H, apparently contributing to the resistance of these bacteria to host complement-mediated killing (Alitalo et al., 2002
; Hellwage et al., 2001
; Kraiczy et al., 2001a
; Kurtenbach et al., 2002a
; Stevenson et al., 2002
). One of the first erp loci to be identified, the pG gene of B. burgdorferi strain ZS7, contains an unrelated downstream gene named bapA (Wallich et al., 1995
). The erp genes of two other B. burgdorferi strains have since been extensively studied, and both strains B31 and 297 contain an erp locus that includes a bapA gene (Akins et al., 1995
; Stevenson et al., 1996
). Northern blot analysis indicated that the bapA gene of strain B31 is cotranscribed with the adjacent erp gene (Stevenson et al., 1998a
). This genetic pairing suggests that BapA may also perform an important function for B. burgdorferi. While that role remains unknown, previous immunological evidence indicates that BapA functions during mammalian infection (Bauer et al., 2001
).
Some B. burgdorferi also contain a gene related to bapA, named eppA; these two genes together constitute B. burgdorferi gene paralogue family 95 (Casjens et al., 2000). Strain B31 is known to carry two different eppA alleles, eppA1 and eppA2, each on a different 9 kb circular plasmid, cp9-1 and cp9-2, respectively (Champion et al., 1994
; Miller et al., 2000a
). Sequence analysis of cp9-1 indicated that it probably evolved from a cp32 plasmid through a series of deletion and rearrangement mutations (Casjens et al., 2000
). The only other previously described eppA gene is that of strain N40, which is likewise located on a 9 kb circular plasmid (Stewart et al., 2001
). An earlier study found that infected laboratory rabbits and at least one tested human Lyme disease patient produced antibodies that recognized recombinant strain B31 EppA1 protein (Champion et al., 1994
), indicating that both members of protein paralogue family 95 are expressed by B. burgdorferi during mammalian infection.
Prior to the present study, only a very limited number of B. burgdorferi strains had been examined for either bapA or eppA, so it was unknown how widely spread these genes are among Lyme disease bacteria, or how well conserved are their sequences. Likewise, it was unknown how frequently human Lyme disease patients produce antibodies directed against BapA or EppA. Since serological diagnosis of Lyme disease is often complicated by amino acid differences among antigenic proteins, or by lack of antigenicity by proteins synthesized during human infection, such knowledge would be useful for the development of improved serodiagnostic tests. In addition, the juxtaposition of bapA genes in operons with genes encoding factor H-binding Erp proteins raises questions as to the origin of bapA. To answer these questions, we studied serum reactivity of numerous Lyme disease patients, and examined the genetics of a broad spectrum of spirochaetes isolated from human patients and wild animals around the globe.
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METHODS |
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Southern blot analysis.
B. burgdorferi were grown to late exponential phase (approx. 107 to 108 bacteria ml-1). Plasmid DNAs were purified using Qiagen mini-prep kits. Aliquots (300 ng) of total plasmid DNAs, either uncut or digested with EcoRI, were separated by agarose gel electrophoresis and transferred to nylon membranes.
DNA probes derived from the strain B31 bapA and eppA1 genes were generated by PCR amplification of B31-MI or B31-RML genomic DNA, utilizing oligonucleotide primer pairs EA-C plus EA-D and BA-3 plus BA-4, respectively (Table 2). An aliquot of each PCR reaction was examined for amplicon purity by agarose gel electrophoresis and staining with ethidium bromide. PCR products were purified through Centricon-100 concentrators (Ambion) (Stevenson et al., 1996
), and labelled with [32P]dATP by random priming (ICN). Each labelled probe was individually incubated with the nylon membranes overnight at 45 °C in 6x SSC (where 20x SSC is 3 M NaCl, 0·3 M sodium citrate), 0·1 % SDS and 0·5 % nonfat dry milk. Membranes were washed four times for 15 min each at room temperature in 2x SSC, 0·1 % SDS (low-stringency wash) then exposed to a phosphoscreen or Kodak X-OMAT film overnight. Phosphoscreen blots were analysed utilizing a STORM phosphoimager (Molecular Dynamics). Blots were next washed further at 55 °C in 0·2x SSC, 0·1 % SDS (high-stringency wash), then exposed and analysed as above. For reuse, blots were then stripped of hybridized probe by successive washes in boiling water. Successful stripping was confirmed by overnight exposure of the membrane to X-ray film.
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Recombinant proteins and polyclonal antibodies.
Polyhistidine-tagged fusion proteins were produced for use in immunoblot analyses. B. burgdorferi B31 bapA, eppA1 and eppA2 genes were PCR amplified and cloned into pUni/V5-His-TOPO (Invitrogen), and polyhistidine-tagged fusions were produced according to the manufacturer's instructions. Recombinant proteins were then purified over nickel affinity columns (Novagen) and analysed for purity by SDS-PAGE with Coomassie brilliant blue staining. Antisera were raised to recombinant EppA1 and BapA proteins in New Zealand White rabbits (El-Hage et al., 2001). Polyclonal antibodies were then affinity purified using the appropriate recombinant protein immobilized on a nylon membrane (Hefty et al., 2001
).
Western blot analysis.
Aliquots (50 µg) of each polyhistidine-tagged fusion protein were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Each membrane was blocked overnight at 4 °C in Tris-buffered saline-Tween 20 (TBS-T) (20 mM Tris/HCl, pH 7·5, 150 mM NaCl, 0·05 % Tween 20) containing 5 % (w/v) non-fat dry milk. Patient serum samples were each diluted 1 : 200 in TBS-T and incubated with membranes in a mini-PROTEAN II multiscreen apparatus (Bio-Rad). Membranes were then washed extensively with TBS-T and incubated with protein Ahorseradish peroxidase conjugate (Amersham) in TBS-T. Bound antibodies were detected by enhanced chemiluminescence (Amersham).
Human sera.
Pre-existing human serum samples were obtained and used in strict accordance with policies and guidelines established by the US Public Health Service and the University of Kentucky Office of Research Integrity. Patient serum samples designated NY1 through NY20 (Table 3), taken from confirmed Lyme disease patients, were provided by Gary Wormser (New York Medical College, Valhalla, NY, USA) and the time interval between onset of symptoms and serum collection was approximately 30 days (Miller et al., 2000b
). Patient serum samples designated 90-2631 through 96-1103 (Table 3
) were provided by Martin Schriefer (Centers for Disease Control and Prevention, Fort Collins, CO, USA). The time interval between onset of symptoms and collection of these sera varied and has previously been reported (Miller et al., 2000b
). Dr Schriefer also provided six control serum samples collected from healthy people residing in parts of the USA where Lyme disease is non-endemic (Table 3
) (Miller et al., 2000b
). Lyme disease serum samples designated NY62 through NY137 (Table 3
) were collected from patients residing on Long Island, NY (Simpson et al., 1990
; Stevenson et al., 1998a
). The interval between onset of symptoms and sera collection is unknown for this last set of samples (Simpson et al., 1990
; Stevenson et al., 1998a
).
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RESULTS AND DISCUSSION |
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To assess the degree of diversity among BapA proteins, 28 of the bapA amplicons were randomly selected for sequencing. Comparison of the predicted proteins indicated a tremendous degree of conservation among BapA proteins, with identities ranging from 92 to 100 % (Fig. 1A, Fig. 2
). All analysed bapA genes are predicted to yield proteins having molecular masses of approximately 20 kDa. Most BapA proteins are predicted to have slightly basic pI values of approximately 7·8, although some proteins, such as that of strain 297, have acidic pI values of approximately 5·6. The bapA genes of strains 91-1226 and BL203, isolated from Lyme disease patients in Maryland and New York, respectively, were identical to the genes of the New York strain B31 and German strain ZS7. Genes from several other geographically dispersed B. burgdorferi isolates were also identical. For example, identical bapA genes are carried by strains 297 and Sh-2-82, and seven additional strains isolated from patients living in New York and Wisconsin, and from ticks captured in New York and California. With only two exceptions, all the BapA proteins contained an arginine-glycine-aspartate (RGD) motif at amino acids 6668 (Fig. 1
). Virulence-associated proteins of many other pathogenic bacteria contain such RGD motifs, which are critical for integrin binding that facilitates attachment of bacteria to eukaryotic host tissues (Hynes, 1992
).
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bapA genetic recombination events
Analysed strains of B. burgdorferi contain anywhere between six and ten different erp loci, but, at most, only one locus per strain contains an adjacent bapA gene (this work; Akins et al., 1999; Casjens et al., 2000
). It is therefore most reasonable to assume that an ancestral bapA gene recombined into a region immediately 3' of one or more erp locus at some point in the past. We examined several bapA loci for evidence of this postulated recombination event, and used physical linkage with distinctive plasmid markers to ascertain the genetic stability of bapA.
The examined bapA genes all contain a well-conserved 55 bp direct repeat sequence on either side of the gene (Fig. 3). Direct repeats are a hallmark of transposable elements, due to staggered strand-cutting during integration events (Shapiro, 1983
). We note that the bapA gene does not bear significant homology to any known transposon, nor is it found elsewhere in the B. burgdorferi genome, as might be expected for a transposable element. However, consistent with the possibility that the ancestral bapA integrated into a cp32 via a transposition-like event, a single copy of the flanking sequence is found 3' of some B. burgdorferi erp genes. For example, the strain N40 ospF gene lies adjacent to a nearly perfect copy of the bapA flanking sequence, while a somewhat divergent version is located 3' of the strain B31 erpK gene. Other known erp loci contain either an extremely divergent version or an unrelated sequence (data not shown). No similar sequences are located adjacent to any known eppA gene. The regions 5' of the bapA genes of several strains were examined, and all were found to contain a single erp gene. Comparison of these erp genes with those of the well-characterized strains B31, 297 and N40 indicates that all bapA-linked erp genes are rather closely related and constitute a distinct clade (Fig. 4
). These genetic similarities suggest that the current bapA loci all arose from a single insertion event into a region 3' of just one ancestral erp gene.
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B. burgdorferi eppA genes
Bacteria were also examined for the second type of gene in paralogue family 95, eppA. Southern blot analysis using a probe derived from the strain B31 eppA1 gene demonstrated hybridization with 63 % of the strains under low-stringency wash conditions, and 35 % under high-stringency conditions (Table 1). These blots indicated that all detected eppA genes were located on DNAs having electrophoretic mobilities comparable to 9 kb circular plasmids (data not shown). Next, PCR was performed using oligonucleotide primers complementary to sequences found in the strain B31 eppA1 and eppA2 genes (Table 2
), yielding an eppA amplicon from 28 of 52 tested strains. An eppA amplicon could not be obtained from the uncloned B356 parental isolate, or from four out of the five B356 clones examined, but a gene was obtained for clone B356-4. Including those strains already known to contain an eppA gene, 38 of 54 (70 %) analysed B. burgdorferi isolates were demonstrated by either Southern blotting or PCR to contain an eppA gene, and 65 % contain both types of paralogue family 95 genes.
Nineteen eppA PCR products were chosen at random and sequenced. Alignment of the predicted EppA proteins indicated a great deal of both identity and diversity among the examined isolates, with amino acid sequence identities that ranged from 54 to 100 % (Fig. 1B, Fig. 2)
. Most predicted EppA proteins have molecular masses of approximately 21 kDa and basic pI values ranging between 8·6 and 9·7. Curiously, the EppA proteins of strains 114a and 119a are predicted to have sizes similar to all other analysed EppA proteins, yet have very acidic pI values of approximately 4·9. There was considerable evidence of past insertion or deletion mutations having occurred within these genes, although high degrees of stability were also observed. The eppA gene of strain 91-1226, isolated from a human patient in Maryland, is identical to the eppA1 allele of strain B31. The EppA proteins of the New York strains N40 and B356-4 are predicted to be identical to the B31 EppA2 protein. The eppA genes of the New York patient isolates 114a and 119a exhibited a very high degree of divergence from other genes, but were identical to each other. Two bacteria isolated from ticks collected in California, strains CA17 and CA19, contain eppA genes identical to that of strain B348, which was isolated from a human in New York. Only one of the eppA genes is predicted to encode a protein with the RGD motif found in most BapA proteins (see above), although all contain a variation on that amino acid sequence (Fig. 1
).
As noted above, all eppA genes appeared to be located on approximately 9 kb circular plasmids. The complete sequences of three related 9 kb plasmids from three different B. burgdorferi strains have been determined, and found to exhibit limited sequence conservation even in homologous loci (Dunn et al., 1994; Fraser et al., 1997
; Stewart et al., 2001
). These variations are probably reflective of the tortuous evolutionary paths these plasmids have followed in their descent from cp32 plasmids (Casjens et al., 2000
). Due to an inability to identify informative, conserved sequences in the previously sequenced plasmids that would be suitable for PCR analysis, we did not attempt to characterize DNAs adjacent to the newly identified eppA genes.
The widespread variability in EppA sequence suggests that it is under some sort of selective pressure. EppA is proposed to be an integral outer-membrane protein (Champion et al., 1994) and may therefore be exposed to the host immune system. However, we note that the examined bacteria were isolated from a wide variety of hosts, including humans, mice, birds, and ticks that feed on a wide variety of vertebrates. It has been proposed that amino acid sequence differences of some proteins between strains influence the efficiency of bacteria for infection of different host species (Stevenson et al., 2001
). As one example, sequence differences among Erp proteins alter their ability to bind complement factor H, which is thought to contribute to the host range of Lyme disease borreliae (Kraiczy et al., 2001b
; Kurtenbach et al., 2002a
, b
; Stevenson et al., 2002
). Perhaps EppA proteins also interact with host proteins that differ in sequence between diverse hosts in nature? Continued studies of this bacterial protein and elucidation of its function(s) will resolve this question.
Although significant diversity was noted among the eppA genes of many strains, there were also several examples of identity between strains. The eppA genes of Wisconsin isolate 95-0024 and New York isolate 127b are identical, as are those of the New York isolate B348-2 and California strains CA17, CA19 and CA22. It has been proposed that B. burgdorferi is largely a clonal organism (Dykhuizen & Baranton, 2001; Qiu et al., 2002
), so the occurrence of similar sequences in bacteria from widely separated geographical regions may be a reflection of that characteristic. However, genetic exchange between bacteria may also be responsible for the widespread nature of certain alleles. For example, strains B31 and N40 have identical eppA genes but differ at many other examined loci (our unpublished results; Liang et al., 2002
; Roberts et al., 1998
; Stevenson & Barthold, 1994
). These data argue against a hypothesis that only very small fragments of DNA can be transferred between B. burgdorferi (Dykhuizen & Baranton, 2001
). Further comparisons of strains possessing identical eppA and other loci will continue to test of the validity of that hypothesis.
Analysis of infected human sera
Serum samples obtained from 49 Lyme disease patients and six healthy humans were assayed for the presence of antibodies that recognized recombinant B31 BapA, EppA1, and EppA2 proteins (Table 3). Six out of 49 (12 %) Lyme disease patient sera contained antibodies that recognized the B31 BapA protein, confirming that B. burgdorferi produces this protein during mammalian infection. Of the 16 strains isolated from patients who also provided serum samples, 14 contained a bapA gene, yet only one of these patients produced detectable levels of antibodies against BapA. As described above, all of those infectious isolates encoded BapA proteins sharing over 95 % amino acid identities with the B31 protein. These results indicate that the failure of some patients to produce antibodies that recognize the B31 BapA fusion was not because they were infected by bacteria that lacked a gene similar to that of strain B31.
Eighteen out of 49 (37 %) patient serum samples contained antibodies that recognized the EppA1 fusion protein, and 17 out of 49 (35 %) contained antibodies that bound the EppA2 fusion protein. A positive correlation was noted between the production of EppA-binding antibodies and the presence of an eppA gene in the infecting bacteria: of the bacteria isolated from patients who also provided serum samples, eight strains contained eppA genes and seven of those patients were seropositive. Ten patients produced antibodies that recognized both EppA1 and EppA2, indicating that these two proteins contain some similar epitopes. However, some regions of the two B31 proteins studied are antigenically distinct, since several patients produced antibodies that recognized only one recombinant protein. This raises the possibility that many of the serologically negative Lyme disease patients actually produced antibodies to the EppA proteins of their infecting organisms, which contained epitopes that differ from those of the strain B31 EppA1 and EppA2 proteins. The diversity of eppA sequences among the strains analysed in this study suggests the possibility of an even wider degree of variation throughout nature, some of which is too great to have been detected by our PCR primers or Southern blot probes.
Conclusions
A large majority of Lyme disease spirochaetes carry a member of gene paralogue family 95, with most bacteria examined containing both a bapA and an eppA gene. These high proportions strongly suggest that the ability to produce BapA and/or EppA confers a selective advantage to B. burgdorferi in nature. A high degree of sequence conservation was noted among bapA genes, although it was also evident that DNAs adjacent to these genes have undergone significant deletion and replacement mutations. The plasmid location of bapA genes is also quite variable, with analysed loci being mapped to five different cp32 plasmid groups. The stability of the bapA gene in the face of such extensive genetic rearrangements suggests that the encoded protein performs a function that does not permit much structural variation. On the other hand, mutations have run riot through the eppA locus. Extensive variation is also evident throughout the 9 kb circular plasmids that carry eppA genes, and these two phenomena may be linked. Finally, the highly conserved BapA does not appear to be highly antigenic, while the extremely variable EppA was much more antigenic. The reasons for these genetic and antigenic variations are presently unknown, and indicate a need for continued studies of these intriguing genes and their proteins to determine their roles in B. burgdorferi biology and the pathogenesis of Lyme disease.
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ACKNOWLEDGEMENTS |
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We thank Sherwood Casjens, Russell Johnson, Martin Schriefer, Tom Schwan, Ira Schwartz and Gary Wormser for providing B. burgdorferi strains and Lyme disease patient serum samples; and Kelly Babb, Nazira El-Hage, Melissa Hines, Julie Stewart and Natalie Mickelsen for technical assistance and thoughtful discussions.
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Received 11 November 2002;
revised 14 January 2003;
accepted 23 January 2003.