SummaryBorrelia burgdorferi , a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia . This complementation also restored the growth and host adaptation of lp25 -B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia .

VlsE is an outer surface lipoprotein of Borrelia burgdorferi that undergoes antigenic variation through an elaborate gene conversion mechanism and is thought to play a major role in the immune response to the Lyme disease borellia. The crystal structure of recombinant variant protein VlsE1 at 2.3-Å resolution reveals that the six variable regions form loop structures that constitute most of the membrane distal surface of VlsE, covering the predominantly ␣-helical, invariant regions of the protein. The surface localization of the variable amino acid segments appears to protect the conserved regions from interaction with antibodies and hence may contribute to immune evasion.Lyme disease is a multistage, tick-borne infection that is endemic to regions of the United States, Europe, and Asia (1). The causative bacteria are a family of closely related spirochetes, including Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii, and are transmitted from one mammal to another by Ixodes ticks. Lyme disease borrelia cause persistent infections and chronic neurologic, cardiovascular, and arthralgic manifestations that can last for months to years in humans and other mammals if not treated successfully, indicating that the spirochetes can effectively evade the host's immune defenses.The mechanisms of immune evasion are not well understood at this time but are thought to include at least one form of antigenic variation. The variable major protein (VMP)-like sequence (vls) 1 locus of B. burgdorferi (2) is a complex antigenic variation system that in many ways resembles the more thoroughly characterized variable major protein system of relapsing fever borrelia (3). The vls locus of B. burgdorferi B31 consists of the expression site vlsE and a contiguous set of 15 vls silent cassettes. The exact function of the 35-kDa surfaceexposed lipoprotein VlsE is unknown; however, it is thought that the vls system may play an important role in mammalian infection because loss of the encoding linear plasmid lp28-1 results in reduced infectivity (4, 5).

The presence of the linear plasmids lp25 and lp56 of Borrelia burgdorferi B31 was found to dramatically decrease the rate of transformation by electroporation with the shuttle vector pBSV2, an autonomously replicating plasmid that confers kanamycin resistance (P. E. Stewart, R. Thalken, J. L. Bono, and P. Rosa, Mol. Microbiol. 39:714-721, 2001). B. burgdorferi B31 clones had transformation efficiencies that were either low, intermediate, or high, and this phenotype correlated with the presence or absence of lp25 and lp56. Under the conditions utilized in this study, no transformants were detected in clones that contained both lp25 and lp56; the few kanamycin-resistant colonies isolated did not contain pBSV2, indicating that the resistance was due to mutation. Intermediate electroporation rates (10 to 200 colonies per g of DNA) were obtained with B31 clones that were either lp25 ؊ and lp56 ؉ or lp25 ؉ and lp56 ؊ . Clones in this group that initially contained lp25 lacked this plasmid in pBSV2 transformants, a finding consistent with selective transformation of lp25 ؊ variants. High transformation rates (>1,000 colonies per g of DNA) occurred in clones that lacked both lp25 and lp56. Sequence analysis indicated that lp25 and lp56 contain genes that may encode restriction and/or modification systems that could result in the low transformation rates obtained with strains containing these plasmids. The previously reported correlation between lp25 and infectivity in mice, coupled with the barrier lp25 presents to transformation, may explain the difficulty in obtaining virulent transformants of B. burgdorferi.Lyme disease is a multistage, systemic disease caused by members of the spirochete genus Borrelia and is transmitted to humans by Ixodes ticks. Borrelia burgdorferi is the principal causative agent of Lyme disease in the United States, whereas B. burgdorferi, B. afzelii, and B. garinii have each been shown to cause human disease in regions of Europe and Asia. The genome of B. burgdorferi B31 has been sequenced (7) and is composed of a linear chromosome, nine circular plasmids (cp) and 12 linear plasmids (lp). These plasmids range in size from 9 kb (cp9) to 56 kb (lp56). In vitro passage of organisms leads to spontaneous loss of plasmids. The loss of lp25 and lp28-1 correlates with reduced infectivity of B. burgdorferi B31 in needle-inoculated mice (11,17,27), indicating that factors important in the virulence of Lyme disease borrelia are encoded by these plasmids.Efforts to identify virulence-associated genes in B. burgdorferi have been hampered by the lack of efficient methods for genetic manipulation of this bacterium. In recent years, great strides have been made in the development of genetic tools. Initial success in transformation and genetic exchange in B. burgdorferi was achieved by the electroporation of DNA segments containing gyrB with point mutations that conferred resistance to coumermycin (20). Unfortunately, the high rate of recombination into the native gyrB site on the chromosome and the occurrence...

The loss of linear plasmid lp28-1, which contains the vls antigenic variation locus, is associated with reduced infectivity of Borrelia burgdorferi in immunocompetent mice. The recombinant shuttle vector pBBE22, which includes the virulence determinant BBE22 from lp25 and restores infectivity to readily transformable B. burgdorferi lacking lp25 and lp56, was used to determine the effect of trans expression of vlsE on virulence. Spirochetes lacking lp28-1 were complemented with the plasmid pBBE22:vlsE, containing both BBE22 and vlsE. VlsE protein produced by this construct was expressed and surface accessible in in vitro-cultured B. burgdorferi, as determined by surface proteolysis and immunoblot analysis. Clones lacking lp25 but containing lp28-1 and either pBBE22 or pBBE22:vlsE were reisolated consistently from immunocompetent mice 8 weeks after infection. In contrast, a clone lacking both lp25 and lp28-1 and complemented with pBBE22:vlsE was isolated from only a single tissue of one of six C3H/HeN mice 8 weeks postinfection. These results indicate that either an intact vls antigenic variation locus or another determinant on lp28-1 is required to restore complete infectivity. In addition, an isogenic clone that retained lp28-1 was complemented with the vlsE shuttle plasmid and was examined for vlsE sequence variation and infectivity. Sequence variation was not observed for the shuttle plasmid, indicating that the cis arrangement of vlsE and the vls silent cassettes in lp28-1 facilitate vlsE gene conversion. Lack of vlsE sequence variation on the shuttle plasmid thus did not result in clearance of the trans-complemented strain in immunocompetent mice under the conditions tested.Lyme borreliosis, the most prevalent vector-borne disease in the United States, is a chronic infection caused by Borrelia burgdorferi and other members of the genus Borrelia (6). Spirochetes are transmitted to mammalian hosts by Ixodes ticks, leading to the development of an annular rash called erythema migrans at the site of inoculation and progressing to a multisystemic infection with neurological, arthritic, and cardiac manifestations (45). As infection advances and Borrelia disseminate into deeper tissues in the host, a strong immune response is elicited towards the pathogen, including the development of Borrelia-specific antibodies (7,8,11,14,17). Though an active immune response develops early during infection, Borrelia is able to escape clearance and persist for months to years. Elucidation of the mechanisms of immune evasion may lead to a better understanding of the pathobiology of Lyme disease.The vls (Vmp-like sequence) locus of B. burgdorferi B31 is on the linear plasmid lp28-1, a plasmid associated with infectivity in the mouse model (26, 27, 42, 52). The vls locus consists of an expression site (vlsE) and 15 unexpressed (silent) vls cassettes. The silent cassettes have high homology to the central cassette region of vlsE. Within the cassettes, there are six variable regions interspersed between highly conserved regions (52). ...

Yersinia pestis causes an acute infection known as the plague. Conventional techniques to enumerate Y. pestis can be labor intensive and do not lend themselves to high throughput assays. In contrast, bioluminescent bioreporters produce light that can be detected using plate readers or optical imaging platforms to monitor bacterial populations as a function of luminescence. Here, we describe the development of two Y. pestis chromosomal-based luxCDABE bioreporters, LuxPtolC and LuxPcysZK. These bioreporters use constitutive promoters to drive expression of luxCDABE that allow for sensitive detection of bacteria via bioluminescence in vitro. Importantly, both bioreporters demonstrate a direct correlation between bacterial numbers and bioluminescence, which allows for bioluminescence to be used to compare bacterial numbers. We demonstrate the use of these bioreporters to test antimicrobial inhibitors (LuxPtolC) and monitor intracellular survival (LuxPtolC and LuxPcysZK) in vitro. Furthermore, we show that Y. pestis infection of the mouse model can be monitored using whole animal optical imaging in real time. Using optical imaging, we observed Y. pestis dissemination and differentiated between virulence phenotypes in live animals via bioluminescence. Finally, we demonstrate that whole animal optical imaging can identify unexpected colonization patterns in mutant-infected animals.