Kimberly Weiss, Ph.D.

Dissertation research performed under the direction of David H. Walker

ABSTRACT
My dissertation project was designed to extend our knowledge of the biology of the spotted fever group rickettsiae. Members of the genus Rickettsia are obligate intracellular bacteria and are difficult to grow in large quantities. Using recombinant DNA technology, an immunoreactive rickettsial antigen was identified, its expressed structural gene was cloned in Escherichia coli, and the expressed protein was purified. Genomic R. conorii DNA libraries were constructed in the expression vector lambda-gt11. Antigen-producing constructs were identified by plaque lifts using polyclonal antibodies to R. conorii. One construct, designated lambda4-7, encodes a beta-galactosidase fusion protein that migrates at 210 kDa on SDS-PAGE gels. Monospecific antibodies to the recombinant protein recognize a R. conorii (Malish 7 strain) antigen of 120 kDa. DNA sequencing of the insert confirmed that this construct was too small to encode the entire gene. Two other overlapping constructs were isolated and the entire gene was identified and sequenced. An open reading frame of 3,068 base pairs with a calculated molecular weight of 111.8 kDa was identified. Sequence data were analyzed using the computer program PC/Gene (IntelliGenetics, Mountain View, CA), which allow predictions concerning the primary and secondary structural features of the protein. The DNA coding region shares no significant homology with other rickettsial antigen-genes (i.e., R. rickettsii 190 kDa, 120 kDa, and 17 kDa genes).  E. coli-produced recombinant antigens that encode the middle 67% portion of the 120 kDa protein were used in T-lymphocyte proliferation assays to ascertain if they contain T-lymphocyte epitopes. R. conorii-immune T lymphocytes proliferate eight-fold over controls in the presence of recombinant E. coli (lambda4-7) antigens. Lymph node cells from mice immunized with sonic lysates of recombinant E. coli respond to native R. conorii antigen with a stimulation index approximately 10-fold over controls. T lymphocytes from mice immunized with recombinant antigen are able to transfer local delayed-type hypersensitivity, a characteristic of T lymphocytes of the TH-1 type. These data confirm the importance of the 120 kDa antigen as a component in generation of an immune response to rickettsiae and possible cell populations that may be generated in response to infection.