A System for Site-Specific Genetic Manipulation of the Relapsing Fever Spirochete Borrelia hermsii - Bacterial Pathogenesis: Methods and Protocols - page 66

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A System for Site-Specific Genetic Manipulation
of the Relapsing Fever Spirochete Borrelia hermsii
James M. Battisti, Sandra J. Raffel, and Tom G. Schwan
Summary
The lack of a system for genetic manipulation has hindered studies on the molecular
pathogenesis of relapsing fever Borrelia . The focus of this chapter is to describe selectable
markers, manipulation strategies, and methods to electro-transform and clone wild-type
infectious Borrelia hermsii . Preliminary studies suggest that the v ariable t ick p rotein (Vtp)
of B. hermsii is involved in tick-to-mammal transmission. To address this hypothesis, we
have developed a system for genetic manipulation and have constructed clones of a Vtp
mutant and an isogenic reconstituted strain. The methods described here are applicable
for the inactivation of other loci in B. hermsii and should be adaptable for other species
of relapsing fever spirochetes.
Key Words: Borrelia hermsii ; tick-borne relapsing fever; genetic manipulation;
transformation; mutagenesis; variable tick protein (Vtp).
1. Introduction
The most rigorous method to assess the contribution of a specific gene
product in the virulence of a particular pathogen is by testing molecular Koch's
postulates (1) , wherein isogenic strains (wild type, mutant, reconstituted mutant)
are constructed and compared in experimental animals. Relapsing fever is a
recurrent febrile illness caused by spirochetes in the genus Borrelia . Tick-borne
relapsing fever (TBRF) in the USA is caused primarily by Borrelia hermsii and
Borrelia turicatae , which are maintained in enzootic cycles between mammals
and Ornithodoros ticks (2) . Outbreaks of TBRF are generally localized and
sporadic. Louse-borne relapsing fever (LBRF) is caused by Borrelia recurrentis ,