The role of extrachromosomal elements in virulence has been an area of intense research in B. burgdorferi, as well as in other pathogenic bacteria. In B. burgdorferi, several plasmids that are required to complete the infectious cycle have been identified. We have developed a B. burgdorferi shuttle vector, pBSV38G, using the genes putatively responsible for lp38 autonomous replication and here show that the shuttle vector autonomously replicates, is incompatible with lp38, and effectively displaces lp38 from low-passage-number infectious B. burgdorferi isolates. We have analyzed these lp38-deficient clones in vitro for phenotypic changes associated with the loss of this plasmid, as well as in vivo to determine the role of this plasmid in the infectious cycle.
In vitro, we were unable to detect differences in growth rate, protein profile, or production of antigenic proteins between strains lacking lp38 and their parental strains. Furthermore, our data indicate that both the B31-A3 and B31-S9 strains lacking lp38 are fully infectious in a mouse model by needle inoculation and that loss of lp38 does not affect the ability of spirochetes to disseminate from the inoculation site to distant tissues. Additionally, spirochetes lacking lp38 are readily acquired from the skin of infected mice by feeding I. scapularis larvae, survive through the molt, and can establish infection when transmitted by feeding nymphs. Our use of naturally infected ticks as a route of transmission also suggests that strains lacking lp38 are competent to establish infection at a low dose.
We find these results surprising for several reasons. The low sequence divergence of lp38 among strains suggests that these genes have been conserved for a functional purpose (61
). In vivo
, several lp38 genes are upregulated when B. burgdorferi
resides in the skin of a mouse (31
). Many of these genes are also differentially regulated in response to conditions mimicking those found in vivo
, suggesting a possible role in survival within the tick vector or mammalian host (10
). Some of these genes, including the hypothetical lipoprotein genes bbj08
and components of a putative ABC transport system composed of bbj26
, represent possible virulence determinants. We were, however, unable to detect differences in the antigenic protein profiles of wild-type strains from those lacking lp38. This suggests that either the upregulated lp38 genes do not produce sufficient protein to elicit an immune response in vivo
, the proteins are not antigenic, or these conditions do not result in a protein expression profile by in vitro
-grown spirochetes that adequately reflects what occurs in the tick or mammalian host environments. Although we could detect OspD in wild-type strains but not in strains lacking lp38, OspD does not elicit an immune response in infected mice and therefore is not detected by our assay (55
). Furthermore, our results do not support the preliminary findings of Botkin et al. (9
), in whose study a clone lacking lp38 exhibited a significant decrease in mouse infectivity. Our data clearly demonstrate that all of the genes on lp38 located outside the region required for autonomous replication are dispensable for B. burgdorferi
to complete its infectious cycle.
These results raise interesting questions regarding lp38. If this plasmid is dispensable for completion of the B. burgdorferi
infectious cycle, why has it been retained in nature? What role do lp38-borne genes play in the B. burgdorferi
life cycle? This plasmid has been shown to be retained in B. burgdorferi
after 25 passages at both 25°C and 35°C (24
), and in a study analyzing the plasmid contents of 19 clonal isolates, lp38 was present in all of the strains tested (43
). In a recent study analyzing 44 low-passage-number B. burgdorferi
clones and 4,464 B. burgdorferi
transformants, all 44 clones retained lp38 and only 1.1% of the transformants had lost lp38 (38
). Human clinical and field isolates that lack lp38 have been identified, although it is unclear if these isolates originally carried lp38 and lost it during the course of isolation. Complicating this genotypic heterogeneity is the inability of some of these isolates to establish infection when reintroduced in a mouse model (65
). This highlights the utility of selectively displacing an individual plasmid from a defined genetic background in order to link a phenotype with a specific genetic element.
We have examined a selected set of variables, including growth rate, protein and antigenic profile, the ability to infect a mouse by both artificial and natural routes of infection, and the ability to survive within a tick. We were unable to find a significant phenotype under any of the conditions tested. However, BSK medium used to grow B. burgdorferi is complex and may mask differences that would be detectable under more stringent growth conditions, perhaps in a more minimal medium. It is also possible that in a natural host, such as Peromyscus, the retention of lp38 might be required to establish and maintain infection. In addition, neither seroreactivity nor reisolation of spirochetes from various tissues adequately gauges the pathogenicity of a strain; they gauge only whether the spirochetes have elicited an immune response from the host and are proficient at disseminating to distant tissues. It is possible that within the ecology of the natural infectious cycle, which includes different host species, mixed infections, and varying physical environments, conditions under which spirochetes carrying lp38 have a selective advantage may exist. However, within the context of this experimental mouse-tick infectious cycle, we conclude that all the genes on lp38 can be added to the growing list of conserved and regulated yet expendable elements of the B. burgdorferi genome.