Virulence determinants are factors which play major pathogenic roles in microbial infections. While there is very little information available on the existence of virulence factors in the periodontopathic organisms disease they almost certainly do exist. The present study concerns one of these organisms, Actinobacillus actinomycetemcornitans. Detailed pathogenic mechanisms of A. actinomycetemcornitans infections are not known, but the organism produces a variety of potential virulence factors including a leukotoxin capable of killing human polymorphonuclear leukocytes and macrophages in vitro. If the toxin has similar effects on PMNs and MNs in the human gingival crevice it could represent a "strategy" for A. acrinomycetemcornitans survival and virulence because it could impair both innate and adaptive host defense system. We are presently studying the isolated and purified the leukotoxin from A. acrinomycetemcomitans and have cloned the toxin genes into E. coli. The primary objective of the present application is continue building upon past progress in understanding the relationship of the structure of the leukotoxin to its function at the molecular level The project has four major areas of investigation: 1) To determine critical region(s) of the repeat domain of the Actinobacillus actinomycetemcomitans leukotoxin which determine its specificity for human target cells; 2) To identify the domain(s) on the leukotoxin molecule that form the ion-conducting channel; 3) To investigate the conformations and underlying structure of both the aqueous and membrane active forms of the leukotoxin; and 4) To define target cell molecules required for leukotoxin specificity and activity. Identifying and understanding the role of virulence factors in periodontal disease will represent a major advance in our understanding of mechanisms of tissue destruction in the periodontium. As a sidelight the proposed studies may also provide information on the basic biology of the macrophage/granulocyte. In this regard an intriguing problem is the reason for the high cell selectivity exhibited by the A. actinomycetemcornitans toxin which appears to be achieved in the absence of a classic receptor mediated mechanism. Fundamental insights into the pathobiology of the Actinobacillus leukotoxin will bring us closer to a molecular definition of its role in human periodontal infections. In addition, detailed studies on the action of the Actinobacillus leukotoxin will contribute to a better understanding of the biology of this interesting family of membrane-active bacterial cytolysins.