Innate immunity is important for controlling pathogens independently of humoral and cell mediated responses. Polymorphonuclear leukocytes generate lethal reactive oxygen intermediates and contain antibiotic polypeptides that can kill ingested pathogens independently of O2. The long term goals are to understand the contribution and the mechanism of action of oxygen independent antibiotic proteins found in PMN to both the killing of pathogens and to inflammation. The proposed studies will focus on azurocidin, a protein that is homologous to a family of serine proteases found in PMN. Azurocidin lacks proteolytic activity, but is highly bacteriocidal. In addition, azurocidin is a chemoattractant for monocytes and T cells. It is proposed to generate mutations that alter a highly cationic region in azurocidin that may be responsible for the electrostatic interaction with LPS, and a second region known to bind serine protease inhibitors. i) It will be determined if the mutations affect ligand binding and antibacterial and chemotaxis functions. ii) Linker-scanning mutagenesis will be used to introduce random mutations in the azurocidin coding region. iii) The mutant proteins will be tested for changes in antibacterial, ligand binding and chemoattractant activity. It will be determined if ligand binding has an effect on antibacterial and chemoattractant activity. iv) It is proposed to express the recombinant tagged azurocidin in macrophages and v) to measure the survival of Salmonella typhimurium in these macrophages. vi) Bacterial genetics will be used to determine if there are bacterial functions which are required for azurocidin to kill target bacteria.
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