Defense Date

2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Dr. Richard T. Marconi

Abstract

Borrelia hermsii, the primary etiological agent of tick-borne relapsing fever in North America, binds the complement regulatory protein factor H (FH) as a means of evading opsonophagocytosis and the alternative pathway of complement. The sequence of the gene encoding the FH-binding protein has been determined. The protein is unique to B. hermsii and has been designated FhbA. Analyses of B. hermsii isolates revealed that FhbA is expressed by 24 of the 25 isolates tested. fhbA was demonstrated to be a single genetic locus through pulsed-field gel electrophoresis, restriction digest, and hybridization analyses, and all isolates possessing fhbA carry it on a 200 kb linear plasmid, whereas isolates that lack fhbA instead carry a 170 kb linear plasmid.To investigate the molecular basis of the interaction between FhbA and FH, truncated FhbA proteins were generated and tested for FH binding. Binding required both N- and C-terminal domains indicating conformational determinants are needed for FH binding. Further mutagenesis established that two C-terminal coiled-coil domains and a loop are involved in the interaction with FH. Potential variation in FhbA among isolates was analyzed by DNA sequence analysis. Two FhbA types, FhbAl and FhbA2, were delineated. Both FhbA types share a conserved C terminus containing the coiled-coil structures involved in binding FH. Additionally, it was demonstrated through whole-cell adsorption and ALBI assays that B. hermsii also binds FHL-1. To assess the specificity of the immune response to FhbA, recombinant FhbAl and FhbA2 were screened with serum from infected mice and humans. FhbA was found to be expressed and antigenic during infection. To localize the epitopes of FhbAl and FhbA2, truncations were screened with infection serum. The epitopes were determined to be conformational. Lastly, type-specific PCR primers were generated to implement rapid differentiation of strains bearing fhbA1 versus fhbA2.Together, these analyses indicate that FH/FHL-1 binding is a prevalent virulence mechanism allowing complement evasion by B. hermsii and provide insight into the antigenic structure of FhbA. Additionally, the data can be applied to the future development of species-specific diagnostic tools and will advance the studies on the epidemiology of relapsing fever in North America.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008

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