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Infection and Immunity, December 2006, p. 6690-6699, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.00993-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Potential Virulence Determinants by Himar1 Transposition of Infectious Borrelia burgdorferi B31{triangledown}

Douglas J. Botkin,1,2,3 April N. Abbott,1,3 Philip E. Stewart,4 Patricia A. Rosa,4 Hiroki Kawabata,5 Haruo Watanabe,5 and Steven J. Norris1,2,3*

Graduate School of Biomedical Sciences,1 Program in Microbiology and Molecular Genetics,2 Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030,3 Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840,4 Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan5

Received 22 June 2006/ Returned for modification 8 August 2006/ Accepted 20 September 2006

Lyme disease Borrelia organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1 transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferi is feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia.

* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77225-0708. Phone: (713) 500-5338. Fax: (713) 500-0730. E-mail: steven.j.norris{at}uth.tmc.edu.

{triangledown} Published ahead of print on 2 October 2006.

Editor: V. J. DiRita

Infection and Immunity, December 2006, p. 6690-6699, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.00993-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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