Lyme spirochetes can be isolated from Ixodes
ticks in regions of endemicity, and several studies have shown that more than one strain often exists in individual ticks and that all of these strains are then transmitted to the mammalian hosts (33
). In the early days of the study of Lyme disease, this multistrain infection of ticks was not fully appreciated. Thus, mixed cultures were disseminated among laboratories, and when strain cloning became possible, different laboratories isolated different clones from the same culture, which were subsequently given the identical strain designation as the original culture, thus confusing the outcome of some research. We began to suspect that this was the scenario for the “N40” strain that has been studied extensively by various laboratories. Since our research primarily uses a clone, N40D10/E9, from the original N40 isolate, we decided to investigate whether we could differentiate various N40 cultures from one another using a series of tests, with the aim of using this information as a tool to reinterpret the results with “N40” that have been reported in the literature from a number of laboratories. The laboratory of Stephen Barthold cloned cN40 independently, and our results strongly indicate that N40D10/E9 and cN40 do indeed represent different strains cloned from the original N40 isolate. We suggest that the specific designations of various N40 clonal cultures, probably as defined here, be used in future studies to avoid confusion.
Using a combination of five techniques, PFGE, Southern hybridization, RST typing, PCR identification of the cN40 complete plasmid set, and sequence comparisons of ospC
genes or dbpA
genes, we compared four divergent “N40” cultures. These techniques have previously been employed to divide clinical and tick isolates of B. burgdorferi
into different categories (8
), but all these techniques have not been used together in a single study, until now. The PFGE display and Southern analysis of linear plasmids, PCR identification of plasmids present, and sequence analysis of the ospC
genes all showed substantial differences among the four cultures and indicated that our cN40 and N40B clones were derivatives of the same strain and N40C and N40D10/E9 were similar and represent two similar strains that are rather different from cN40 and N40B. The difference in PFGE stained linear plasmid patterns in A, the PCR identification of plasmids in , and the nearly 20% difference in ospC
gene sequences in Fig. S3 in the supplemental material demonstrate this most clearly.
PFGE followed by Southern hybridization of whole-cell DNA is a time-consuming and labor-intensive technique that can accurately determine the linear plasmid content of a B. burgdorferi
isolate (and circular plasmids, if combined with restriction digestion); however, it does not distinguish plasmids of very similar size, which is not uncommon in Borrelia
isolates. A PCR-based assay described by Norris and coworkers (80
) allows the determination of the plasmid content of strain B31 after growth or mutagenesis, and this has resulted in real progress in understanding the molecular pathogenesis of this strain (42
). In a recent study, the same group has described a high-throughput method using the Luminex multiplex technology to determine the plasmid content of this strain (64
). Unfortunately, these techniques are currently limited to the sequenced strain B31. In the course of the work described here, we devised a set of tested and confirmed PCR primers from the reported N40 (N40B) genome sequence that can detect all 17 of the N40 plasmids (; ; see Table S1 in the supplemental material). Poor amplification or a different-size PCR product likely indicates the differences in the sequence of that plasmid and/or partial loss of the plasmid from the culture. This primer set will also allow the careful study of the pathogenesis of this strain, which has not previously been possible due to the inability to determine whether all plasmids were present.
We also noticed during our studies of the N40 cultures that our primer set, which is heavily weighted toward virulence factor genes, amplified mostly fragments of the same size as those from the B31 and N40D10/E9 strains. This caused us to examine the plasmid sections that carry these genes in the other sequenced B. burgdorferi
). Although there appears to have been substantial genetic exchange among the linear plasmids in different isolates, we found that such exchange is especially prevalent near the ends of the plasmids. Thus, although exceptions do exist for specific virulence factors, especially if their genes lie near plasmid ends, as is the case with the especially variable B31 lp36 and lp28-1 plasmids and their homologs, these observations depict a limited genetic exchange in the middle region of the linear plasmids of B. burgdorferi
(S. Casjens, unpublished data). We therefore suggest that virulence factor genes will be particularly useful to determine the plasmid content of different strains and that such an analysis can also be used for differentiation of B. burgdorferi
new isolates and also for epidemiological analyses of the strains prevalent in the regions of endemicity of the United States and Europe.
Epidemiological studies often involve isolation and characterization of multiple B. burgdorferi
strains from regions of endemicity (58
). However, there is no simple test to determine whether a new isolate is indeed a very different strain or is similar to one of the strains previously isolated from the same region. In addition, there is a need for a method that can easily determine whether plasmids are lost during propagation or genetic manipulation of a particular B. burgdorferi
strain. An infectious strain appears to be much more likely to have retained the important virulence factor genes on related plasmids rather than in random locations. Therefore, we suggest that in the beginning of a new strain characterization, PFGE of the endogenous plasmids could be conducted, especially if the clonal isolate will be used for the genetic studies later on. PCR-amplified products of virulence factors encoding genes located in each plasmid of B31 (or perhaps the new strain) can then be used as probes in Southern hybridization of the PFGE blots. Such a characterization will then help develop a PCR-based assay that can be used to determine the plasmid content of this strain in the follow-up studies. Some characteristics and virulence mechanisms are restricted to particular strains of any pathogen, and although such studies for Lyme disease have been severely limited in the past, B. burgdorferi
studies involving multiple strains will be required to provide a complete picture of Lyme disease pathogenesis and will help determine the complete repertoire of the virulence factors contributing to this disease.
As a proof of this principle, we examined several known virulence factor-encoding genes as probes for Southern hybridization of the blot of the PFGE of the B. burgdorferi
N40 and B31 strains (). In addition, examination of a new potentially important metabolic enzyme, MtnN, indicates that mtnN
is located on a linear plasmid of 28 kb (probably lp28-4) in all tested strains. This is similar to the finding that the locations of important virulence factor-encoding genes, such as dbpA-dbpB
, and ospC
, are conserved in particular genome segments, even though sizes may be somewhat different due to genetic rearrangements, especially on the outer edges of the linear plasmids. Gene duplication followed by rearrangement and mutations appear to have resulted in the evolution and antigenic variation of a number of B. burgdorferi
virulence factors and other proteins (4
). Often, duplicated genes that encode functionally redundant proteins that exhibit overlapping roles are present in the same operon, with each protein demonstrating altered specificities for host cell factors. Although mtnN
and its homologs do not follow this pattern, their location is still conserved, further supporting our premise regarding the relatively conserved location of virulence factor-encoding genes. Hence, we believe that the use of the approach described here can help any researcher in designing a PCR-based assay to examine new strains and to examine the previously unstudied role of bacterial variation in the molecular pathogenesis of Lyme disease. Finally, a comparative analysis of the protein profiles can be used to determine association between the levels of protein production with the functional activity of a molecule. This analysis could further distinguish cN40 and N40D10/E9. Such analyses will be useful to differentiate other strains in the future.
Summary and conclusions.
A comprehensive study of four B. burgdorferi N40 isolates that have been used in the study of Lyme disease pathogenesis allowed us to differentiate these isolates and establish that they are derivatives of two different strains. This information will help to clarify the confusion that is due to the use of two different strains that were thought to be the same but that are in fact very different, and it will allow this research field to avoid future confusion on this point. We also propose that the use of PFGE and PCR of virulence factor genes followed by Southern hybridization is a useful strategy to discriminate different strains of B. burgdorferi sensu stricto. A PCR-based assay can then help determine the plasmid content of the strains.