Histatins constitute a distinct family of low molecular weight, histidine-rich, cationic salivary proteins which exhibit a broad range of antimicrobial activities. Unlike several other cationic antimicrobial peptides which act in local environments and in close proximity to their site of synthesis, histatins are secreted by both parotid and submandibular glands and are subsequently transported to the oral cavity protecting oral, pharyngeal and esophageal tissues. The long term objective of this project is to understand how histatins, representing an important part of the oral innate host defense system, protect against the multiple potentially adverse effects of microorganisms entering and residing in the oral cavity. The mechanism by which histatins kill Candida albicans, a pathogenic yeast, has not been fully elucidated. It has been shown that histatins, by virtue of their weakly amphipathic nature and reluctance to form helical structures in hydrophobic environments, do not form pore structures in cell membranes. Since histatins are taken up only by metabolically active cells, target mitochondria, inhibit cellular respiration and form reactive oxygen species (ROS), it is likely that the candidacidal activity of the histatins is related to the deleterious effects of ROS on cellular membranes. Aim 1 focuses on the mechanism of action of histatins by identification of: a) the site of inhibition within the respiratory chain, b) the mechanism of ROS formation triggered by histatins and c) ROS induced destabilization of cell membranes by the characterization of nucleotides and proteins/peptides released into the extracellular environment. Another unique feature of histatins is their effect against bacterial virulence factors such as host tissue destroying enzymes and bacterial toxins. Aim 2 is to characterize these "second generation type antibiotic" effects of histatins by investigating the inhibition of several bacterial enzymes, such as the gingipains from Porphyromonas gingivalis, host-derived proteases such as metalloproteinases and the process of neutralization of the leukotoxin released from the periodontal pathogen Actinobacillus actinomycetemcomitans. Aim 3 will assess the structure/function relationships between histatins and their antimicrobial activity using recombinant technologies to construct artificial histatins containing naturally occurring sequences of functional importance, and to generate hybrid molecules exhibiting bi-functional activities. Aim 4 is planned to investigate the functional consequences of the propensity of histatins to form heterotypic complexes employing the molecular approach of the yeast two-hybrid system and the direct biochemical characterization of complexes formed in saliva. Aim 5 will establish the in vivo relationship between histatin levels in whole saliva and the oral microbial profile using the DNA-DNA checkerboard assay providing quantitative information on C. albicans and 80 species of oral bacteria ranging from harmless commensal organisms to periodontal pathogens.