Borrelia burgdorferi, the cauative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi contains abundant linear and circular plasmids and there is growing evidence that these plasmids carry genes critical for adaptation. A major obstacle to studying the functions of specific plasmids and gene products in the infectious cycle of B. burgdorferi has been an inability to genetically manipulate pathogenic strains. To address this issue, we have developed conditions for site-directed mutagenesis in B31-MI, the infectious B. burgdorferi strain whose genome was recently sequenced. We inactivated several plasmid and chromosomal genes in B31-MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31-MI based on colony phenotype, growth rate, plasmid content and protein composition. This high degree of clonal polymorphism within B31-MI complicates the assessment of mutants and indicates that it is not an appropriate strain background for genetic studies of infectious B. burgdorferi. In order to establish a well-defined isogenic clone for targeted mutagenesis studies, we derived a wild type clone from B31-MI that was infectious for mice and transformable. Our studies also demonstrate that the instability of the B. burgdorferi genome mandates careful monitoring of plasmid content of derived mutants and complementation of inactivated genes in order to perform genetic studies with this pathogen. We have inactivated several plasmid-encoded genes in an infectious clone and demonstrated that these clones and the complemented controls have retained all plasmids. Thus we have finally succeeded in genetically manipulating a pathogenic B. burgdorferi clone. This allows comparisons of mutant and isogenic wild type clones in order to identify the functions of specific gene products. In collaboration with a consortium of extramural investigators, we have developed an array of all B. burgdorferi genes and are using this array to compare global patterns of gene expression under different growth conditions and between mutant and wild type clones. We have established an experimental infectious cycle in which we are assessing the competence of mutant clones lacking specific genes or plasmids for infectivity in both the tick vector and mammalian host, and for transmission between them. We are further characterizing the functions of specific B. burgdorferi gene products through biochemical analyses of isogenic mutant and wild type clones.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000917-01
Application #
6669981
Study Section
(LHBP)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Tilly, Kit; Bestor, Aaron; Dulebohn, Daniel P et al. (2009) OspC-independent infection and dissemination by host-adapted Borrelia burgdorferi. Infect Immun 77:2672-82
Sarkar, Amit; Tilly, Kit; Stewart, Philip et al. (2009) Borrelia burgdorferi resistance to a major skin antimicrobial peptide is independent of outer surface lipoprotein content. Antimicrob Agents Chemother 53:4490-4
Lawrence, Kevin A; Jewett, Mollie W; Rosa, Patricia A et al. (2009) Borrelia burgdorferi bb0426 encodes a 2'-deoxyribosyltransferase that plays a central role in purine salvage. Mol Microbiol 72:1517-29
Jewett, Mollie W; Lawrence, Kevin A; Bestor, Aaron et al. (2009) GuaA and GuaB are essential for Borrelia burgdorferi survival in the tick-mouse infection cycle. J Bacteriol 191:6231-41
Stewart, Philip E; Bestor, Aaron; Cullen, Jonah N et al. (2008) A tightly regulated surface protein of Borrelia burgdorferi is not essential to the mouse-tick infectious cycle. Infect Immun 76:1970-8
Tilly, Kit; Rosa, Patricia A; Stewart, Philip E (2008) Biology of infection with Borrelia burgdorferi. Infect Dis Clin North Am 22:217-34, v
Yang, Yu; Stewart, Philip E; Shi, Xiaoguang et al. (2008) Development of a transposon mutagenesis system in the oral spirochete Treponema denticola. Appl Environ Microbiol 74:6461-4
Jewett, Mollie W; Lawrence, Kevin; Bestor, Aaron C et al. (2007) The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi. Mol Microbiol 64:1358-74
Jewett, Mollie W; Byram, Rebecca; Bestor, Aaron et al. (2007) Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi. Mol Microbiol 66:975-90
Botkin, Douglas J; Abbott, April N; Stewart, Philip E et al. (2006) Identification of potential virulence determinants by Himar1 transposition of infectious Borrelia burgdorferi B31. Infect Immun 74:6690-9

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