Recent evidence suggests that cationic peptides isolated from human polymorphonuclear leucocyte (PMN) granules may play an important role in oxygen independent inhibition of bacteria. PMNs are major components of the acute inflammatory process associated with periodontal disease, yet little information exists on PMN-derived, oxygen-independent antibacterial systems and periodontal plaque bacteria. Knowledge in this area may be important to our understanding of PMN-bacterial interactions. A major investigative problem, however, involves the lengthy chromatographic techniques which give only low yields and poor resolution of the peptides of interest. The purposes of this study are 1) to develop suitable HPLC methodologies for the separation and isolation of cationic peptides from human PMN graunules of sufficient quantity and quality to perform detailed bacteriologic studies and 2) to examine the antibacterial activity of isolated cationic peptides on specific periodontopathic bacteria. The HPLC methodology proposed includes combining gel-permeation, ion-exchange and hydrophobic-interaction chromatography. It is anticipated that the techniques proposed will not only improve resolution of the peptide fractions but will decrease preparation times by up to 70%. Peptides will be assayed for bactericidal activity against several plaque bacteria including Actinobacillus actinomycetemcomitans, and Bacteroides gingivalis. The anticipated long-term objectives, following completion of the proposed project will be: 1) to define in more detail the interactions of PMNs and plaque bacteria, 2) to identify the metabolic and physiologic targets of cationic peptide action, 3) to investigate the feasibility of producing low molecular weight cationic peptides by molecular cloning techniques for possible use as topical antibacterial agents, and 4) development of HPLC techniques suitable for isolating cationic peptides from a variety of sources including saliva.
