RESUMO
Borrelia 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.
Assuntos
Borrelia burgdorferi/enzimologia , Borrelia burgdorferi/patogenicidade , Nicotinamidase/metabolismo , Plasmídeos/genética , Animais , Borrelia burgdorferi/genética , Teste de Complementação Genética , Inflamação/metabolismo , Articulações/microbiologia , Articulações/patologia , Doença de Lyme/metabolismo , Membranas Artificiais , Camundongos , Camundongos Endogâmicos C3H , Camundongos SCID , Mutação , Nicotinamidase/genética , Plasmídeos/metabolismo , Ratos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMO
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 micro 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 micro 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.
