The purpose of this research is to investigate the role of alpha-defensins in innate immunity of the rhesus macaque (Macaca mulatta). The goals of the proposed studies are to define the determinants of primate alpha-defensin bactericidal activity, their mechanisms of action, and to characterize mechanisms that regulate myeloid (RMADs) and Paneth cell (REDs) alpha-defensin biosynthesis at the transcriptional and posttranslational levels. We propose to quantitate the expression of RED and RMAD mRNAs and peptides in small bowel mucosa, neutrophils and NK cells, characterize and compare their mechanisms of action and the structural basis of that activity, investigate mechanisms of proRED and proRMAD activation in Paneth cells and myeloid cells, and identify cis-acting structural components of RMAD and RED genes that regulate the specificity of their transcription.
In Aim #1, the microbicidal activities of REDs 1-6 will be tested against varied bacterial species, and membrane disruptive activities of natural and mutant REDs and RMADs will be characterized and quantitated. The hypothesis that mutagenesis alters peptide structure will be tested by determining NMR structures of native and mutant RED4 and RMAD4 peptides in solution and in the presence of rapidly-tumbling phospholipid bicelles to test the hypothesis that specific residue positions interact with phospholipid bilayers.
In Aim #2, we will test the hypothesis that alpha-defensin precursor processing is mediated by lineage-specific proteolytic events by measuring the extent of RED and RMAD precursor processing in intact monkey small intestine and in rhesus neutrophils, NK cells, and monocytes. We will identify the respective processing enzymes, determine whether precursor proteolysis is coincident with activation of bactericidal activity, and determine the effect of mutations at conserved alpha-defensin positions on peptide folding, disulfide bond formation, activation, and membrane binding and disruption. Thus, in Aim #3, the hypotheses that primate alpha-defensin gene expression is regulated by lineage-specific, cis-acting elements will be tested by quantitating the expression and distribution of individual RED and RMAD mRNAs in enteric and myeloid tissues and by identifying determinants of lineage-specific alpha-defensin gene transcription. Potential lineage-specific gene regulatory regions will be assayed by functional analyses of RED and RMAD promoter constructs and chimeric minigenes in cultured monocytes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI059346-02
Application #
7250290
Study Section
Special Emphasis Panel (ZRG1-III-F (01))
Program Officer
Rothermel, Annette L
Project Start
2006-07-01
Project End
2011-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
2
Fiscal Year
2007
Total Cost
$357,814
Indirect Cost
Name
University of California Irvine
Department
Pathology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Tai, Kenneth P; Kamdar, Karishma; Yamaki, Jason et al. (2015) Microbicidal effects of ?- and ?-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Innate Immun 21:17-29
Mastroianni, Jennifer R; Lu, Wuyuan; Selsted, Michael E et al. (2014) Differential Susceptibility of Bacteria to Mouse Paneth Cell ?-Defensins under Anaerobic Conditions. Antibiotics (Basel) 3:493-508
Tai, Kenneth P; Le, Valerie V; Selsted, Michael E et al. (2014) Hydrophobic determinants of ?-defensin bactericidal activity. Infect Immun 82:2195-202
Wilmes, Miriam; Stockem, Marina; Bierbaum, Gabriele et al. (2014) Killing of staphylococci by ?-defensins involves membrane impairment and activation of autolytic enzymes. Antibiotics (Basel) 3:617-31
Patil, Amar A; Ouellette, Andre J; Lu, Wuyuan et al. (2013) Rattusin, an intestinal ?-defensin-related peptide in rats with a unique cysteine spacing pattern and salt-insensitive antibacterial activities. Antimicrob Agents Chemother 57:1823-31
Mastroianni, Jennifer R; Costales, Jessica K; Zaksheske, Jennifer et al. (2012) Alternative luminal activation mechanisms for paneth cell ?-defensins. J Biol Chem 287:11205-12
Burkhardt, Amanda M; Tai, Kenneth P; Flores-Guiterrez, Juan P et al. (2012) CXCL17 is a mucosal chemokine elevated in idiopathic pulmonary fibrosis that exhibits broad antimicrobial activity. J Immunol 188:6399-406
Andersson, Håkan S; Figueredo, Sharel M; Haugaard-Kedström, Linda M et al. (2012) The ?-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance. Amino Acids 43:1471-83
Schaal, Justin B; Tran, Dat; Tran, Patti et al. (2012) Rhesus macaque theta defensins suppress inflammatory cytokines and enhance survival in mouse models of bacteremic sepsis. PLoS One 7:e51337
Schmidt, Nathan W; Tai, Kenneth P; Kamdar, Karishma et al. (2012) Arginine in ?-defensins: differential effects on bactericidal activity correspond to geometry of membrane curvature generation and peptide-lipid phase behavior. J Biol Chem 287:21866-72

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