Our preliminary studies (unpublished) indicate that the structure of capsular polysaccharide (CPS) of the pathogenic bacterium Klebsiella pneumoniae (K. pn) Type 2, as well as quality of this antigen probably have importance in its virulence. Two mutant strains, 84-1 and 84-5, were derived from our wildtype K. pn strain UC6 to study relationships of structure vs virulence, and to develop a genetic system for this organism. Thus, the aims of this project are to: (i) relate virulence to specific antigenic differences in CPS of the three strains; and (ii) to accomplish basic analysis and prepare for long-range studies on the genetic make-up of UC6.
The aims on virulence relations will be accomplished using UC6, 85-5, and 84-1 in in vivo and in vitro studies. The strain phenotypes vary based upon mouse virulence (UC6=less than 100; 84-5 about 71,000, and 84-1 greater than 10/7 bacteria), capsule size, and colony character. Mouse protection with CPS from each strain and challenge assays using homologous and heterologous bacteria will show antigen relatedness based on protection. Also, precipitin bands formed in crossed immunoelectrophesis with intermediate gel will allow direct observation of such relatedness because unique epitopes in any of the 3 antigens should lead to unique bands by this method. Monoclonal antibodies developed against purified CPS will provide epitode probes. Those important in immunity will be assessed by passive protection mice. Both parameters will be important in locating those epitopes considered important for chemical analysis. The latter will be done to identify epitope monosaccharide components, structure, and molecular size. The information generated here will be essential in furthering genetic analysis of K. pn, and area of scant study. For this project, preliminary genetic mapping of wild type UC6 genome, especially directed toward mechanisms of capsule formation, will be done. F- and Hfr strains transformed with a plasmid rendering them Lambda phage sensitive have already been developed. Furthermore, a DNA library from UC6 has been introduced into Escherichia coli using Lambda and cosmid vectors. Transformation techniques will also be used. The data derived from this study could have important implications concerning K. pn and the future direction of its study. Aside from the knowledge concerning its basic virulence and genetic mechanisms, it holds potential for development of useful pure cell products, including a vaccine.
