Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden
Correspondence
Sven Bergström
sven.bergstrom{at}molbiol.umu.se
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ABSTRACT |
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INTRODUCTION |
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In this study we investigated the heterogeneity of the p13 gene from all species causing Lyme borreliosis. Significantly, the most heterogeneous, surface-exposed region of P13 appeared to be the natural epitope. Furthermore, we combined epitope mapping with computer-based predictions to determine the membrane-spanning architecture of P13 in the outer membrane. The presence of the other paralogous genes of family 48 in a number of different Borrelia strains was also examined, and transcription of p13 and three of its paralogues (bba01, bbi31, bbh41) was investigated both under normal culture conditions and during infection in mice. Expression of the paralogue BBA01 in Lyme disease Borrelia was also investigated in vitro.
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METHODS |
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Identification and sequencing of p13 and its paralogous genes from different Borrelia species.
Genes of interest were amplified by PCR using Taq polymerase (Roche) under the following conditions: 6 cycles at 94 °C for 30 s, 40 °C for 1 min, 72 °C for 1 min followed by 35 cycles at 94 °C for 30 s, 50 °C for 1 min, 72 °C for 1 min.
Primers Y12 and Y18 (Noppa et al., 2001) were used for amplification of the p13 gene. For amplification of bba01, bbi31 and bbh41, the primer combinations of A01-M2 with A01-M2Rev, I31-M2 with I31-M2Rev, and H41-M2 with H41-M2Rev were used respectively (Table 2
). Every PCR reaction was repeated twice to ensure the validity of the results. For sequencing, the p13 and bba01 PCR products were cloned into pGEM-T Easy vector (Promega) and maintained in DH5
. Plasmids were prepared using the Qiaprep Spin miniprep kit (Qiagen) and sequencing was performed using the BigDye Kit (Perkin Elmer) and an Applied Biosystems ABI 377 Sequencer. The sequences were analysed using GCG [Wisconsin Package Version 9.1, Genetics Computer Group (GCG)] software and alignment of the consensus sequences was obtained using the BioEdit program (Tom Hall, Department of Microbiology, North Carolina State University).
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Mouse infection.
B. burgdorferi strain B31 was used for mouse infections. Prior to infection, the presence of the query genes in B31 was confirmed by PCR. Four-week-old C3H/HeN mice (Bomholt Gård Breeding, Denmark) were subcutaneously injected with 105 spirochaetes in 0·1 ml culture medium. The number of bacteria was determined microscopically in a PetroffHausser chamber. Negative controls were injected with 0·1 ml BSK-II medium. Three weeks post-infection, the mice were anaesthetized by injection of a mixture of Dormicum (Roche), Hypnorm (Janssen Pharmaceutica) and water (1 : 1 : 2), and killed by cardiac puncture. Ears, heart and bladder were aseptically removed and one ear and heart were immediately frozen in liquid nitrogen. The other ear and bladder were added to BSK-II medium supplemented with sulfamethoxazole (1·25 µl ml-1) and phosphomycin (4 µl ml-1) for detection of Borrelia spirochaetes.
Isolation of RNA and RT-PCR.
All reagents were prepared with diethylpyrocarbonate (DEPC)-treated water. Total RNA was isolated from in vitro-cultured Borrelia using the Ultraspec-II RNA isolation system (Biotex Laboratories) or Trizol reagent (Invitrogen) and from mouse heart with Trizol reagent essentially according to the manufacturer's instructions. Aliquots (2 µg) of each RNA preparation were then treated with 3 units of RNase-free DNaseI (Roche) to remove contaminating DNA.
Both 2-step and 1-step RT-PCR were used in this study. First-strand cDNA reactions were primed with 5 pmol gene-specific primer (Table 2) and synthesized using the AMV reverse transcriptase (Roche). For gene-specific PCR amplification from cDNA, 5 µl of first-strand cDNA reaction was amplified in a total volume of 50 µl, containing 20 nM of each primer, 200 µM of each dNTP and 0·5 units of Taq DNA polymerase. For 1-step RT-PCR from the 1 µg of RNA, the Superscript One-Step RT-PCR with Platinum Taq kit (Invitrogen) was used following the manufacturer's instructions. In each RT-PCR, two negative controls were included: sterile water to verify the purity of the reagents, and RNA from uninfected mice to confirm the reaction specificity and the absence of cross-contamination during sample processing. Using DNA extracted from B. burgdorferi cultures we monitored amplification efficiency. Two additional positive controls were included: primers specific for the mouse housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) (Pinto do et al., 1998
) were used to monitor RT-PCR efficiency on RNA preparations from organs, and the flagella-specific flaB primers FB1 and FB2 (Noppa et al., 2001
) were used for Borrelia RNA detection. For all 1-step RT-PCR reactions the absence of DNA contamination was verified by PCR. Ten microlitres of each RT-PCR product was analysed on 1 % TBE-agarose gel stained with ethidium bromide (5 µg ml-1).
Protein electrophoresis, immunoblotting and antibodies.
Proteins were separated in Tricine 1020 % polyacrylamide gradient gels or in Bistris 412 % polyacrylamide gradient NuPage gels by using the Novex XCell Sure Lock electrophoresis cell (Invitrogen). Total Borrelia proteins were prepared by growing cells to stationary phase, harvesting by centrifugation at 8000 g at 4 °C, and washing twice in phosphate-buffered saline (PBS). The outer-membrane proteins (B-fraction) of B. burgdorferi strain B31 were prepared as described elsewhere (Magnarelli et al., 1989). For immunoblotting, proteins were transferred to a PVDF membrane (PALL Corp.) and probed with antibodies. The polyclonal antibody recognizing P13 is described elsewhere (Noppa et al., 2001
). For production of serum against the BBA01 protein, a synthetic peptide with the sequence EKGIESFNKYDKEKC (amino acids 2841 plus a terminal cysteine) was used (obtained from Innovagen). The peptide fragment was conjugated to a keyhole limpet haemocyanin (KLH) carrier via its terminal cysteine with maleimide cross-linker (Agrisera). The synthetic peptide was used for immunizing a rabbit and an immune serum was obtained (Agrisera). The monoclonal antibody (mAb) 15G6, recognizing P13, was previously described (Sadziene et al., 1995
). Bound antibodies were detected using peroxidase-conjugated anti-rabbit or anti-mouse antibodies (DAKO) and enhanced chemiluminescence (ECL) reagents according to the manufacturer's instructions (Amersham Pharmacia Biotech).
Overexpression of different P13 fragments.
Three fragments (A, B and C) covering the sequence of mature P13 were obtained using the primer pairs P13M1F with Y54MR (A), Y55MF with Y56MR (B), and Y57MF with P13M1R (C). All primer sequences are given in Table 2; the restriction sites included were: Acc65I for primers P13M1R, Y54MR and Y56MR, BspHI for primers Y55MF and Y57MF, and NcoI for primer P13M1F. The PCR conditions used were as follows: 5 cycles at 94 °C for 30 s, 45 °C for 1 min, 72 °C for 1 min and 35 cycles at 94 °C for 30 s, 55 °C for 1 min and 72 °C for 1 min. The PCR products were further purified using High Pure PCR Product Purification Kit (Roche) columns; fragments B and C were digested with Acc65I and BspHI restriction enzymes, whereas fragment A was digested with Acc65I and NcoI restriction enzymes (New England Biolabs). Thereafter, the PCR products were cloned in pETM-20 vector (EMBL, Heidelberg, Germany) digested with Acc65I and BspHI and transformed into E. coli TOP10. Colonies were screened for the correct inserts and plasmids containing the three different fragments of the p13 gene (A, B and C) were purified using the Qiaprep plasmid miniprep kit (Qiagen) and transformed into Rosetta (DE3)pLysS cells for protein expression. After 3 h induction, the cultures were harvested by centrifugation at 8000 g for 5 min, resuspended in 200 µl PBS, and the protein concentration was estimated using Bio-Rad Protein Assay kit. Protein samples (10 µg) were then analysed on 412 % Bistris acrylamide gels and stained with Coomassie G-250. Epitope mapping was performed using Western blotting and mAb 15G6.
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RESULTS |
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Given the widespread distribution of the p13 allele within Lyme disease Borrelia species and strains, the heterogeneity among the P13 protein was investigated. Amino acid sequence comparison of P13 revealed that it is considerably homogeneous and that most sequence heterogeneity is found outside the predicted transmembrane regions, especially between the transmembrane regions III and IV (Fig. 1). This observation is consistent with the probable surface exposure of these regions of P13, as is considered to be true also for the corresponding parts of the paralogue proteins within family 48 (Noppa et al., 2001
). Additionally, the most sequence heterogeneity of P13 is found between species. P13 of the B. garinii strains was found to be the least well conserved, as expected, since this genospecies is the most heterogeneous of all Lyme disease Borrelia spp. (Baranton et al., 1998
). We also analysed the sequence heterogeneity of the bba01 from different Lyme disease Borrelia strains by sequencing an internal region (located at nucleotide position 60313) of the gene. The bba01 gene was very homogeneous, with only minor heterogeneity between species (data not shown).
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DISCUSSION |
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The loss of plasmids during in vitro cultivation is a well-known phenomenon for Borrelia spirochaetes (Barbour, 1989; Busch et al., 1997
; Grimm et al., 2003
; Labandeira-Rey & Skare, 2001
; McDowell et al., 2001
; Norris et al., 1995
), although this is restricted to plasmids harbouring genes whose products are important for infectivity but not in vitro growth (Labandeira-Rey & Skare, 2001
; Purser & Norris, 2000
). Our results showed that paralogue bba01, situated on the 54 kb linear plasmid (lp54), is not lost during in vitro passage. Moreover, bba01 is also the only paralogue detected in all Lyme disease Borrelia species. These data correlate with earlier studies and indicate that genes situated on plasmid lp54 are not needed for infectivity, but probably play a role during other stages of the Borrelia life cycle (Grimm et al., 2003
; Labandeira-Rey & Skare, 2001
; Purser & Norris, 2000
). On the other hand, we could show that plasmids lp28-3 and lp28-4 are lost during in vitro cultivation. This indicates that genes situated on these plasmids are not needed for growth of Borrelia in vitro, but could play a role in infectivity and survival of Borrelia in vivo. However, independent studies indicated that B. burgdorferi plasmids lp54 and lp28-3 are not lost during cultivation in vitro (Grimm et al., 2003
) and are always present in clones examined after murine infection (Purser & Norris, 2000
). In addition, B. burgdorferi B31 lacking lp28-4 were modestly attenuated in a mouse infection model and carried an important antigen (VraA) for their infectivity in rabbits (Labandeira-Rey et al., 2001
; Labandeira-Rey & Skare, 2001
). This correlates with our finding that the bbi31 gene situated on this plasmid is transcribed in mice, while neither bba01 nor bbh41 was (Table 5
). Taken together, however, these data contradict another study in which it was concluded that lp28-4 is not needed during infection of mice (Purser & Norris, 2000
). Perhaps the different model systems used for the investigation of plasmid stability could explain these controversies. Nevertheless, the results also show that Borrelia is a complicated organism requiring a distinct plasmid content during different stages of its life cycle.
Since almost all plasmids (with exception of lp54) harbouring the investigated paralogous genes of family 48 are lost during cultivation, they are probably not needed for growth of B. burgdorferi in vitro, but could be needed for their pathogenicity. To investigate the possible function(s) of the paralogue proteins of family 48, we therefore analysed expression of p13 and its paralogues in laboratory culture and during murine infection. Our results showed that the p13 and bbi31 are transcribed in both conditions, while all the other paralogous genes are only transcribed during in vitro culture and are below the detectable level in mice (Tables 4 and 5). Thus, our RT-PCR results indicate that the P13 and BBI31 proteins could be needed during the infection process and may constitute important virulence factors of Borrelia.
Our sequencing data for the bba01 gene from B. burgdorferi strains B31, N40 and Sh2-82 have revealed sequence homology with two earlier published B. burgdorferi N40 genes, p11 and p5, located in an operon on lp54 (Feng et al., 1996). The analysis of the genome sequence of B. burgdorferi B31 showed that the p11 and p5 genes were absent; instead the bba01 gene was defined (Fraser et al., 1997
). In the study by Feng et al. (1996)
a deletion of one nucleotide in the gene sequence had apparently led to the frameshift and a stop codon. Our sequencing data of the bba01 gene from B. burgdorferi strains B31, N40 and Sh2-82 and the published B. burgdorferi B31 genome sequence have revealed that there is no frameshift in that particular region in any of these strains (data not shown). Therefore, the results presented in that study were confused by a single nucleotide deletion and are not due to a difference between the two B. burgdorferi strains B31 and N40.
The immune serum against the BBA01 paralogue was used to analyse BBA01 synthesis during in vitro culture of Lyme disease Borrelia (Fig. 4). Expression could not be detected in high-passage strain B31, although the plasmid and the gene are still present (Table 1
). Interestingly, we could detect BBA01 in another high-passage strain, B31-A. This indicates that BBA01 synthesis is downregulated in continuously cultured B. burgdorferi B31, but not in B31-A. This could be explained by different plasmid content or maybe loss of some regulator for the BBA01 protein expression in B. burgdorferi B31 during in vitro propagation. This also supports the idea that P13 paralogues are not needed for growth of Borrelia in vitro but could be important during infection or survival in vivo. Surprisingly, we could not detect BBA01 in outer-membrane preparations of low-passage B31, although we expected that it could be an integral outer-membrane protein like P13 (Noppa et al., 2001
). This might suggest different subcellular localizations of BBA01 and P13 or that BBA01 is rapidly degraded during preparation of the outer-membrane proteins. We also observed that the highest levels of BBA01 are found in B. afzelii strain ACAI, perhaps reflecting an important role for this protein in this Lyme-disease-species. However, we can not rule out that the epitope of BBA01 from ACAI is recognized by the polyclonal antiserum more efficiently. Finally, we have shown that the BBA01 paralogue is expressed in all three Lyme-disease-causing species. Interestingly, BBA01 synthesis is upregulated in the P13 knockout compared to the wild-type (Fig. 4
, lanes 5 and 6), which suggests that BBA01 could possibly compensate for the function of P13. The results also showed that polyclonal antibodies raised against the synthetic peptide of BBA01 are specific, such that the region used to synthesize the peptide could be the epitope of BBA01. Further studies are needed to elucidate this possibility.
It has been postulated that B. burgdorferi has evolved many plasmids and paralogous gene families to be capable of adaptation to different environments (Ojaimi et al., 2003). Within this theme, we have shown that all investigated paralogues of family 48 can be expressed, but under different conditions, some of which are difficult to mimic in vitro. We also present a model of the P13 architecture in the outer membrane of B. burgdorferi, although more studies on the structure of this protein are required. Taking the results together, we have shown that the paralogue family 48 has at least two members (P13 and BBA01) that are synthesized during laboratory cultivation. Conversely, p13 and bbi01 are transcribed in mice and could therefore be important during establishment of infection in animals. In this context, we believe that altered expression of the various paralogues is important during adaptation to different environments. Upregulation of the BBA01 protein in the p13 knockout strain indicates that paralogues not only have sequence homology, but also could exhibit some functional redundancy. Since Borrelia has no advanced metabolic capacity and obtains all the essential nutrients from the haemolymph of ticks or the blood of mammals, an efficient and selective uptake mechanism, such as porins, is required. Earlier, we established that P13 is a channel-forming protein (Östberg et al., 2002
). It is also evident that BBA01 is expressed in vitro and is apparently processed in a similar manner to P13. In addition, the gene is present in all strains of Lyme disease Borrelia investigated. Therefore, we propose that the BBA01 paralogue could also be a channel-forming protein, needed at certain stages of the Borrelia life cycle. Further studies are needed to investigate this possibility. Moreover, expression of the paralogues in different environments, in both ticks and different animals, may give a better understanding of the biology of Borrelia spirochaetes and the pathogenesis during Lyme disease infection.
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ACKNOWLEDGEMENTS |
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We thank Laila Noppa for helpful discussions, Matthew Francis for carefully reading the manuscript, Alan G. Barbour for providing mAbs 15G6 and B. burgdorferi strain N40 and Sara af Bjerkén for the help with RT-PCR.
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Received 22 August 2003;
revised 28 October 2003;
accepted 4 December 2003.
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