Isolation and characterization of different forms of Bordetella pertussis adenylate cyclase

This thesis describes the different forms of AC produced by B. pertussis, B. parapertussis, B. bronchiseptica and those expressed in E. coli. The work was done with both crude and purified preparations of AC and also with whole cells of B. pertussis. Immunoblot analysis with various antisera, including anti-AC monoclonal, monospecific, and polyclonal antibodies, revealed the production of two different forms of AC with different electrophoretic mobilities i.e., 210 and 200 kDa as the major cell-associated forms in B. pertussis and B. bronchiseptica. In 3 B. parapertussis and E. coli, the predominant form was 210 kDa. These forms may represent the toxic and non-toxic forms observed by Rogel et al. (1989). Production of the holotoxic AC was not observed in culture supernates under the growth conditions used. Only low MW forms of 45-50 kDa were observed and these forms also appeared to be produced by proteolysis of AC during storage and other experimental manipulations. The breakdown was partially prevented by using a protease inhibitor. The enzymic activity of AC was found to be unstable in purified and crude forms of AC in the absence of urea. Thus, urea seemed to be beneficial in protecting loss of activity of AC during storage. A noteworthy observation of the present study was that the different Bordetellae, including B. pertussis Tn5-insertion mutants deficient in individual virulence factors, and a recombinant B. pertussis strain, produced different amounts of AC enzymic and toxic activities under the same set of condition which indicate that the production varied from strain to strain . The highest yields of enzymically-active and toxic AC were obtained from recombinant strain B. pertussis BP348 (pRMBl) and from B. pertussis Tn5-insertion mutant BP357 (deficient in a functional PT gene). On the other hand, B. pertussis Tohama and Tn5 mutant BP353 showed the lowest AC enzymic and toxic activities of the B. pertussis strains examined. B. bronchiseptica produced an intermediate level of AC enzymic and toxic activities compared to those of the B. pertussis strains. Enzymically and biologically-active AC was obtained from the E. coli constructs. However, the toxic activity was approximately half than that of native AC. E. coli strains harbouring the cloned B. pertussis cyaA, B, D genes together with C genes either from P. vulgaris, E. coli, OTB. pertussis on separate plasmids, showed production of CyaA (AC), as assessed by immunoblotting and the Salomon assay,and its activation by C genes, as assessed by inhibition of chemiluminescence. Various attempts were made to develop more convenient, economical, and sensitive methods for detection of AC and determination of its enzymic and toxic activities. An alternative method for detection of AC by calmodulin-affinity probing of a Western blot was found to be reproducible and suitable for use when specific anti-AC antibodies were not available. This is the first report of such a use for calmodulin. A microplate assay was developed for detection of AC enzymic activity and this proved economical, reproducible, and convenient for preliminary screening of AC-producing strains and as a semi-quantitative assay. The method has some advantages over conventional assays, such as the Salomon assay and the Binding protein assay, in that it does not require radioisotope and it is rapid. Further development of this assay as a diagnostic test for pertussis may be possible. A comparative study was made of different assays for determination of toxic effects of AC on a variety of target cells. These included : haemolysis of sheep and rabbit RBCs, CHO cell elongation, arborization of BHK cells, inhibition of the nitroblue tetrazolium reduction capacity of rabbit neutrophils, and inhibition of the chemiluminescence response of neutrophils to various stimuli. The study indicated that CL inhibition by AC toxin was a sensitive assay and could detect ACT in crude preparations at the level of 1mug protein /10