The role of Iron Availability in the Pathogenesis of Actinobacillus actinomycetemcomitans. The purpose of the project is to study the role of iron availability on the pathogenesis of Actinobacillus actinomycetemcomitans (A.a.). Little is known about the iron transport systems of anaerobic bacteria, although it is clear that the acquisition of iron from the host is required in order to sustain growth. The identification of an iron-repressible outer membrane protein of ~70 kilodaltons (kD) that is immunogenic in humans may represent a vaccine candidate for the prevention and treatment of A.a. infections. The specific aims of the study are to identify potential in vivo iron sources utilized by A.a to help in targeting iron transport systems. In addition, the role of the 70kD iron-repressible protein in iron uptake will be studied. To achieve these goals, an iron-limited growth medium will be developed in order to study iron utilization with a limited number of variables. We are currently employing 8-hydroxyquinoline extraction of complex media and cation exchange chromatography of defined media to produce an medium with ~100nM iron that will also support the growth of A.a. The gene for the 70kD protein will be cloned an sequenced to determine if it is similar to other know iron transport systems. An oligonucleotide probe derived form the N-terminal amino acid sequence of the 70kD protein was utilized to screen a ~gt11 library of A.a. genomic DNA to obtain potential clones. In addition, monospecific antisera generated against the 70kD antigen was also used for screening the library. Positive clones are currently being characterized to definitively demonstrate that we possess the gene for the 70kD protein. Once the wild-type gene has been identified, it will be subcloned into an appropriate vector and utilized to generate mutations in the wild- type gene in order to characterize the function of the protein in iron uptake. Keys Words: Actinobacillus, Iron, Outer Membrance Proteins, Iron Transport