Abstract
A DNA microarray containing fragments of 137 Borrelia burgdorferi B31 putative lipoprotein genes was used to examine Lyme disease spirochetes. DNA from B. burgdorferi sensu stricto B31, 297, and N40; Borrelia garinii IP90; and Borrelia afzelii P/Gau was fluorescently labeled and hybridized to the microarray, demonstrating the degree to which the individual putative lipoprotein genes were conserved among the genospecies. These data show that a DNA microarray can globally examine the genes encoding B. burgdorferi lipoproteins.
Lyme disease is caused by genetically diverse spirochetes collectively termed Borrelia burgdorferi sensu lato. This complex includes several genospecies, of which at least three cause significant disease in humans: B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii (17, 23). All North American pathogenic strains that have been identified are B. burgdorferi sensu stricto. In Europe, all three genospecies are found, with B. garinii and B. afzelii being the most prevalent. Manifestations of Lyme disease vary geographically, depending in part on the predominant genospecies. Lymphocytoma, acrodermatitis chronica atrophicans, and encephalomyelitis are mainly found in Europe, whereas disseminated early infection and arthritis are more common in the United States (1, 22, 25). B. burgdorferi sensu stricto strain B31 has a genome consisting of a linear chromosome and 21 linear and circular plasmids, which carries approximately 1,700 open reading frames (ORFs) (4, 7). Approximately 150 ORFs encode putative lipoproteins, more than any other known bacterium (4, 7). Nineteen of these putative lipoproteins have homologues that have been already identified in other organisms (7). Some of the remaining lipoproteins have demonstrated roles in B. burgdorferi pathogenesis, including enabling the spirochetes to adhere to decorin (8, 21), fibronectin (5, 6, 18, 19), or the tick midgut (14, 15) or to evade the immune system (26) or complement (12).
Putative lipoprotein gene DNA microarray.
To efficiently study the putative lipoprotein genes among B. burgdorferi isolates, a lipoprotein DNA microarray was developed. B. burgdorferi B31 clone 5A11, which contains all 21 known plasmids (20), was grown to the stationary phase in Barbour-Stoenner-Kelly H medium at 33°C. The spirochetes were isolated, and DNA was purified by using the DNeasy Mini kit (Qiagen Inc., Valencia, Calif.). The genomic sequence of B. burgdorferi B31 was downloaded from The Institute for Genomic Research website (http://www.tigr.org), and 137 pairs of primers were designed to amplify a 150- to 500-bp internal fragment of each lipoprotein gene. As a control, flaB primers were also designed. Since some gene families share sequence identity, it is not possible to individually differentiate these genes by hybridization. For example, the rev family consists of three members (bbc10, bbm27, and bbp27) and two of them are almost identical (bbm27 and bbp27) (4, 7). Therefore, two pairs of primers were designed for this family. One pair was unique for bbc10 while the second pair was specific for both bbm27 and bbp27. Similarly, only two pairs of primers were used for the eight members of the mlp family. Each pair only amplified one of the eight ORFs. All PCR primer sequences are available by request. PCR products were assessed by agarose gel electrophoresis, and all showed the expected sizes. Amplified DNA was purified, dissolved to a concentration of 0.05 μg/μl in 50% dimethyl sulfoxide, and spotted in triplicate on CMT-GAPS aminosilane-coated slides (Corning Inc., Corning, N.Y.) by using a Virtek SDDC-2 Arrayer (Toronto, Canada). To cross-link DNA to the microarray, printed slides were treated with 200 mJ of UV. The array was then blocked in 25% formamide plus 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) plus 0.1% sodium dodecyl sulfate (SDS) plus 1% bovine serum albumin at 42°C for 45 min, washed with distilled water at room temperature, and spun dry at 500 × g for 3 min.
Probe DNA was purified from cultured B. burgdorferi sensu stricto B31, 297, and N40; B. garinii IP90; and B. afzelii P/Gau or from murine spleens (control). Aminoallyl dUTP (Molecular Probes, Inc., Eugene, Oreg.) was incorporated by nick translation, fluorescently labeled with the Alexa Fluor 546 kit (B31 DNA) or the Alexa Fluor 647 kit (mouse, 297, N40, IP90, and P/Gau DNA) from Molecular Probes, purified, and lyophilized. A pair of fluorescently labeled DNA probes (one was B31 and the other was mouse, 297, N40, IP90, or P/Gau DNA) plus 10 μg of blocking mouse DNA (MboI digested) were dissolved in 15 μl of hybridization buffer (25% formamide, 5× SSC, and 0.1% SDS) and denatured at 95°C for 5 min. The heated samples were centrifuged at 8,000 × g for 2 min and applied to a preblocked microarray. The slide was covered with a coverslip and incubated in a humid chamber at 42°C for 16 to 20 h. The hybridized slide was stringently washed in hybridization buffer for 3 min and then in 2× SSC plus 0.1% SDS for an additional 3 min at 42°C. The slide was transferred into 0.2× SSC plus 0.1% SDS for 3 min, into 0.2× SCC for 1 min, and into 100% ethanol for 30 s at room temperature and spun dry. The hybridized microarray was scanned with an Axon GenePix 4000A array scanner, and raw data were captured with GenePix 3.0 software (Axon Instruments, Foster City, Calif.).
Specificity and normalization of microarray hybridization.
To determine hybridization specificity, labeled murine (control) and B31 spirochetal DNAs were allowed to bind to the microarray. The ratios of 635-nm (murine DNA) and 532-nm (B31 DNA) emission values for each spot on the microarray were <0.2; therefore, readings below this value were considered to be insignificant. Normalization was achieved by adjusting both channels of laser intensity to reach a ratio of approximately 1 when the 635- and 532-nm emission values of flaB, a highly conserved B. burgdorferi gene, were used as calibration standards for each individual microarray. The degree of conservation was determined with DNA probes from B31 and one test strain (297, N40, IP90, or P/Gau) hybridized to each spot on the microarray and was calculated as a ratio of the 635- and 532-nm emission values. The values ranged from 0 to approximately 1.0. When the ratio obtained with two hybridized DNA probes was >0.8, 0.6 to 0.79, 0.4 to 0.59, 0.2 to 0.39, or <0.2, the gene was defined as highly conserved, conserved, less conserved, may exist, or may not exist, respectively. Three separate experiments demonstrated that the microarray data were highly reproducible.
Assessment of putative lipoprotein genes among B. burgdorferi genospecies.
There are 19 lipoproteins that have homologues whose functions have been identified in other organisms (7). All of these genes were highly conserved in the American strains (Table 1). Sixteen of the genes were highly conserved in the European strain IP90. Although only 10 of the 19 genes were highly conserved in B. afzelii P/Gau, this strain shared all of these 19 genes. These genes were generally more conserved in the respective strains than were their counterparts that are unique to B. burgdorferi, regardless of whether they were located on the chromosome or on a plasmid of the spirochetes.
TABLE 1.
Conservation of putative lipoprotein genes that have homologues in other organisms
| Gene | Potential function | Conservation in straina:
|
|||
|---|---|---|---|---|---|
|
B. burgdorferi
|
B. garinii IP90 | B. afzelii P/Gau | |||
| 297 | N40 | ||||
| bb0088 | GTP-binding protein | HC | HC | HC | HC |
| bb0098 | DNA mismatch repair protein | HC | HC | HC | LC |
| bb0100 | Glutamate racemase | HC | HC | HC | HC |
| bb0122 | Translation elongation factor TS | HC | HC | HC | HC |
| bb0141 | Membrane fusion protein | HC | HC | HC | HC |
| bb0300 | Cell division protein | HC | HC | HC | HC |
| bb0341 | Glu-tRNA amidotransferase, subunit B | HC | HC | HC | C |
| bb0372 | Glutamyl-tRNA synthetase | HC | HC | C | C |
| bb0387 | Ribosomal protein S12 | HC | HC | HC | C |
| bb0489 | Ribosomal protein L24 | HC | HC | HC | HC |
| bb0536 | Zinc protease | HC | HC | HC | HC |
| bb0620 | β-Glucosidase | HC | HC | HC | C |
| bb0621 | 4-Methyl-5(β-hydroxyethyl)- thiazole monophosphate biosynthesis protein | HC | HC | LC | C |
| bb0652 | Protein export membrane protein | HC | HC | HC | HC |
| bb0687 | Phosphomevalonate kinase | HC | HC | C | C |
| bb0785 | Stage V sporulation protein G | HC | HC | HC | HC |
| bb0828 | DNA topoisomerase | HC | HC | HC | HC |
| bba34 | Oligopeptide ABC transporter | HC | HC | HC | M |
| bbb16 | Oligopeptide ABC transporter | HC | HC | HC | LC |
HC, highly conserved; C, conserved; LC, less conserved; M, may exist.
Of the 137 putative lipoprotein genes investigated in this study, 62 are located on the chromosome. Like B31, American strains 297 and N40 and European isolate IP90 had all 62 of these genes. These genes were highly conserved in the American strains except for two genes, bb0424 and bb0844, in strain N40 (Tables 1 and 2). Most of the 62 genes were also highly conserved in European strain IP90. The second European strain, P/Gau, might lack 3 of the 62 genes (bb0224, bb0321, and bb0424). Interestingly, these were 3 of the 5 least conserved genes (bb0224, bb0321, bb0424, bb0806, and bb0844) in strain IP90. The fourth least conserved gene was bb0844, which also was the least conserved in strain N40 (Table 2).
TABLE 2.
Conservation of putative chromosomal lipoprotein genes
| Gene | Conservation in straina:
|
|||
|---|---|---|---|---|
|
B. burgdorferi
|
B. garinii IP90 | B. afzelii P/Gau | ||
| 297 | N40 | |||
| bb0003 | HC | HC | HC | C |
| bb0028 | HC | HC | HC | C |
| bb0038 | HC | HC | HC | LC |
| bb0070 | HC | HC | HC | HC |
| bb0071 | HC | HC | HC | C |
| bb0142 | HC | HC | HC | HC |
| bb0155 | HC | HC | HC | C |
| bb0185 | HC | HC | HC | HC |
| bb0213 | HC | HC | C | LC |
| bb0224 | HC | HC | LC | N |
| bb0227 | HC | HC | HC | C |
| bb0298 | HC | HC | HC | HC |
| bb0309 | HC | HC | HC | HC |
| bb0321 | HC | HC | LC | N |
| bb0324 | HC | HC | C | C |
| bb0352 | HC | HC | HC | LC |
| bb0365 | HC | HC | C | C |
| bb0382 | HC | HC | HC | C |
| bb0383 | HC | HC | C | HC |
| bb0384 | HC | HC | HC | C |
| bb0385 | HC | HC | HC | HC |
| bb0398 | HC | HC | C | M |
| bb0424 | HC | C | M | N |
| bb0458 | HC | HC | HC | C |
| bb0464 | HC | HC | HC | HC |
| bb0475 | HC | HC | C | C |
| bb0524 | HC | HC | HC | C |
| bb0542 | HC | HC | C | LC |
| bb0553 | HC | HC | HC | HC |
| bb0606 | HC | HC | C | HC |
| bb0628 | HC | HC | HC | HC |
| bb0644 | HC | HC | HC | HC |
| bb0664 | HC | HC | HC | LC |
| bb0689 | HC | HC | HC | C |
| bb0696 | HC | HC | HC | HC |
| bb0740 | HC | HC | HC | LC |
| bb0758 | HC | HC | C | C |
| bb0760 | HC | HC | HC | HC |
| bb0806 | HC | HC | LC | LC |
| bb0813 | HC | HC | C | C |
| bb0815 | HC | HC | HC | LC |
| bb0823 | HC | HC | HC | HC |
| bb0832 | HC | HC | C | LC |
| bb0840 | HC | HC | HC | LC |
| bb0844 | HC | M | LC | M |
HC, highly conserved; C, conserved; LC, less conserved; M, may exist; N, may not exist.
Seventy-five putative lipoprotein genes, located on B. burgdorferi B31 plasmids, were also included in this study. Strain 297 had all of the genes, while N40 and IP90 lacked 4 (bbc10, bbf20, bbh18, and bbh32) and 2 (bbc10 and bbh32) of these 75 genes, respectively (Tables 1 and 3). Most of these genes were highly conserved in strains 297 and N40 but less conserved in IP90. B. afzelii P/Gau shared approximately 30 of these 75 genes, and most of them were not conserved.
TABLE 3.
Conservation of putative plasmid lipoprotein genes
| Gene | Conservation in straina:
|
Gene | Conservation in straina:
|
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
B. burgdorferi
|
B. garinii IP90 | B. afzelii P/Gau |
B. burgdorferi
|
B. garinii IP90 | B. afzelii P/Gau | |||||
| 297 | N40 | 297 | N40 | |||||||
| bba04 | HC | HC | LC | M | ||||||
| bb05 | HC | HC | C | M | ||||||
| bb07 | HC | HC | HC | M | ||||||
| bb14 | HC | HC | HC | LC | ||||||
| bb15 | HC | HC | C | M | ||||||
| bb16 | HC | HC | LC | N | ||||||
| bb24 | HC | HC | M | N | ||||||
| bb25 | HC | HC | LC | N | ||||||
| bb33 | HC | HC | LC | M | ||||||
| bb36 | HC | HC | C | M | ||||||
| bb57 | HC | HC | C | M | ||||||
| bb59 | HC | HC | LC | N | ||||||
| bb60 | HC | HC | LC | M | ||||||
| bb62 | HC | HC | LC | N | ||||||
| bb64 | HC | HC | LC | N | ||||||
| bb65 | HC | HC | LC | M | ||||||
| bb66 | HC | HC | LC | N | ||||||
| bb73 | HC | HC | M | N | ||||||
| bbb08 | HC | HC | C | M | ||||||
| bbb09 | HC | HC | HC | LC | ||||||
| bbb14 | HC | HC | C | M | ||||||
| bbb19 | LC | LC | C | M | ||||||
| bbb27 | HC | HC | C | M | ||||||
| bbc10 | LC | N | N | N | ||||||
| bbd10 | HC | HC | HC | LC | ||||||
| bbd24 | HC | C | C | M | ||||||
| bbe06 | HC | C | LC | N | ||||||
| bbe08 | LC | HC | LC | N | ||||||
| bbe09 | HC | HC | LC | N | ||||||
| bbe28 | M | HC | M | N | ||||||
| bbe31 | HC | HC | M | N | ||||||
| bbf20 | HC | N | HC | M | ||||||
| bbf32 | M | M | C | M | ||||||
| bbg-02 | HC | HC | M | N | ||||||
| bbg25 | LC | HC | HC | M | ||||||
| bbh01 | HC | M | M | N | ||||||
| bbh18 | HC | N | M | N | ||||||
| bbh32 | HC | N | N | N | ||||||
| bbi16 | HC | HC | M | N | ||||||
| bbi28 | HC | HC | M | N | ||||||
| bbi29 | HC | HC | LC | N | ||||||
| bbi32 | HC | HC | LC | N | ||||||
| bbi34 | HC | HC | M | N | ||||||
| bbi42 | HC | C | M | M | ||||||
| bbj01 | HC | HC | LC | M | ||||||
| bbj09 | M | C | HC | N | ||||||
| bbj34 | LC | C | LC | N | ||||||
| bbj36 | M | C | HC | N | ||||||
| bbj51 | M | C | M | N | ||||||
| bbk04 | HC | LC | C | C | ||||||
| bbk07 | HC | HC | M | N | ||||||
| bbk12 | HC | HC | M | N | ||||||
| bbk19 | HC | C | LC | N | ||||||
| bbk32 | HC | HC | HC | N | ||||||
| bbk47 | M | HC | M | N | ||||||
| bbk49 | M | C | M | N | ||||||
| bbk53 | HC | LC | LC | M | ||||||
| bbl39 | C | C | C | N | ||||||
| bbl40 | HC | LC | M | N | ||||||
| bbm27 | LC | HC | M | N | ||||||
| bbm38 | LC | LC | C | N | ||||||
| bbn38 | LC | C | LC | N | ||||||
| bbn39 | HC | C | M | N | ||||||
| bbo39 | HC | HC | LC | M | ||||||
| bbo40 | C | C | M | N | ||||||
| bbp38 | HC | HC | HC | N | ||||||
| bbq03 | HC | LC | C | C | ||||||
| bbq05 | C | M | M | N | ||||||
| bbq35 | HC | LC | M | N | ||||||
| bbq47 | M | LC | M | N | ||||||
| bbr42 | HC | HC | LC | M | ||||||
| bbs30 | HC | HC | LC | N | ||||||
| bbs41 | LC | LC | C | M | ||||||
HC, highly conserved; C, conserved; LC, less conserved; M, may exist; N, may not exist.
B. burgdorferi B31 contains 21 linear and circular plasmids, 19 of which carry at least one putative lipoprotein gene, including plasmids A (lp54), B (cp26), C (cp9), D (lp17), E (lp25), F (lp28-1), G (lp28-2), H (lp28-3), I (lp28-4), J (lp38), K (lp36), L (cp32-8), M (cp32-6), N (cp32-9), O (cp32-7), P (cp32-1), Q (lp56), R (cp32-4), and S (cp32-3) (4, 7). Linear plasmids T (lp5) and U (lp21) do not carry any suspected lipoprotein genes. The DNA microarrays contained gene fragments amplified from these 19 lipoprotein gene-carrying plasmids. Examination of bbc10, the only suspected lipoprotein gene on cp9, would suggest that N40 and IP90 may lack this plasmid (Table 3). Lack of conservation of this gene in strains N40 and IP90 or loss of the cp9 plasmid during in vitro cultivation might, however, also explain the low ratios of bound DNA probes of these two strains (3). In contrast, lp54 contains 19 lipoprotein genes. Therefore, it is more likely that the detection or absence of these genes in the DNA microarray would reflect the presence or lack of this plasmid. Palmer and colleagues studied the distribution of linear plasmids with a set of different markers and suggested that strains N40, IP90, and P/Gau lack lp56 (16). In B. burgdorferi B31, this plasmid contains 4 lipoprotein genes, bbq03, bbq05, bbq35, and bbq47 (4, 7), all of which can be detected in both N40 and IP90 with our microarray (Table 3). Plasmid gene arrangements among spirochetes could potentially account for these differences. For example, vlsE is carried by lp28-1 in strain B31 (26) but may be located on a different size plasmid in other isolates (13, 24). When vlsE was used as the marker for lp28-1, our studies indicated that both IP90 and P/Gau contained this plasmid, in contrast to Palmer and colleagues' result that both isolates lack lp28-1 (16). Therefore, the complete sequencing of individual plasmids is the only valid method of determining which lipoprotein genes are present on specific plasmids.
The B. burgdorferi B31 genome has 17 chromosomal lipoproteins with homologues whose functions have been defined in other organisms (7), and our data suggest that these genes were very conserved in the spirochetes tested. In addition, bba15 (ospA, which facilitates tick attachment) (14, 15), bbb19 (ospC) (2), and bbf32 (vslE, involved in immune evasion) (26) may play important roles in the B. burgdorferi enzootic life cycle and are generally detectable in all of the genospecies. Plasmid loss might partially explain why the extrachromosomal genes were less conserved and few plasmid genes were detected in isolate P/Gau. However, these data on the chromosomal genes should not be affected by this phenomenon. In fact, our studies strongly indicated that most of the chromosomal lipoprotein genes were significantly less conserved in B. afzelii P/Gau than other strains, including the European isolate IP90. This may provide a reasonable explanation for the observations reported by other researchers that antigenic proteins are highly divergent among European isolates (9-11).
We have developed a DNA microarray to examine 137 lipoprotein genes of five B. burgdorferi isolates. Our results suggest that the DNA microarray can be used globally to examine the putative lipoprotein genes among B. burgdorferi isolates, including the three major B. burgdorferi sensu lato genospecies, B. burgdorferi sensu stricto, B. garinii, and B. afzelii. The B. burgdorferi lipoprotein gene microarray should prove useful in the genetic studies of geographic collections of B. burgdorferi, the examination of B. burgdorferi lipoprotein gene expression under different in vitro conditions, and the expression of B. burgdorferi lipoproteins throughout the B. burgdorferi life cycle in the arthropod vector and the mammalian host.
Acknowledgments
We thank Steven Norris (University of Texas, Houston) for providing clonal isolate B31 5A11.
This study was supported by grants from the National Institutes of Health and the American Heart Association. E.F. is the recipient of a Burroughs Wellcome Clinical Scientist Award in Translational Research.
Editor: D. L. Burns
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