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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI023807-14
Application #
6373094
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Voulgaropoulou, Frosso
Project Start
1988-04-01
Project End
2004-08-31
Budget Start
2001-09-01
Budget End
2004-08-31
Support Year
14
Fiscal Year
2001
Total Cost
$372,554
Indirect Cost
Name
Columbia University (N.Y.)
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
167204994
City
New York
State
NY
Country
United States
Zip Code
10032
McCabe, Denise; Cukierman, Tali; Gabay, Joelle E (2002) Basic residues in azurocidin/HBP contribute to both heparin binding and antimicrobial activity. J Biol Chem 277:27477-88
Parish, C A; Jiang, H; Tokiwa, Y et al. (2001) Broad-spectrum antimicrobial activity of hemoglobin. Bioorg Med Chem 9:377-82
Almeida, R P; Vanet, A; Witko-Sarsat, V et al. (1996) Azurocidin, a natural antibiotic from human neutrophils: expression, antimicrobial activity, and secretion. Protein Expr Purif 7:355-66
Witko-Sarsat, V; Halbwachs-Mecarelli, L; Almeida, R P et al. (1996) Characterization of a recombinant proteinase 3, the autoantigen in Wegener's granulomatosis and its reactivity with anti-neutrophil cytoplasmic autoantibodies. FEBS Lett 382:130-6
Henshaw, T J; Malone, C C; Gabay, J E et al. (1994) Elevations of neutrophil proteinase 3 in serum of patients with Wegener's granulomatosis and polyarteritis nodosa. Arthritis Rheum 37:104-12
Gabay, J E (1994) Antimicrobial proteins with homology to serine proteases. Ciba Found Symp 186:237-47;discussion 247-9
Newman, S L; Gootee, L; Gabay, J E (1993) Human neutrophil-mediated fungistasis against Histoplasma capsulatum. Localization of fungistatic activity to the azurophil granules. J Clin Invest 92:624-31
Bini, P; Gabay, J E; Teitel, A et al. (1992) Antineutrophil cytoplasmic autoantibodies in Wegener's granulomatosis recognize conformational epitope(s) on proteinase 3. J Immunol 149:1409-15
Almeida, R P; Melchior, M; Campanelli, D et al. (1991) Complementary DNA sequence of human neutrophil azurocidin, an antibiotic with extensive homology to serine proteases. Biochem Biophys Res Commun 177:688-95
Campanelli, D; Melchior, M; Fu, Y et al. (1990) Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils. J Exp Med 172:1709-15

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