The broad aim of the project is to characterize oxidative microbicidal mechanisms of human neutrophils (PMN), especially as they relate to the azurophil granule enzyme myeloperoxidase (MPO). In this renewal proposal, the emphasis has been shifted from investigations of disrupted microbial energy metabolism to a focus on inhibition of microbial DNA replication. The basis for the change is a series of experiments using MPO, as well as other cell free oxidative (xanthine oxidase plus acetaldehyde) and nonoxidative (gentamicin) microbicidal systems, that demonstrate, only for the MPO system, a close correlation among loss of viability, cessation of DNA synthesis, and inhibition of microbial DNA-membrane interactions. The significance of the last effect relates to findings that an interaction between the microbial membrane and the chromosomal origin of replication, oriC, is essential for initiation of chromosomal DNA replication. The proposal's central hypothesis is that MPO-derived oxidants somehow interfere with chromosomal replication initiation, presumably through a membrane effect, and render the bacterium non-viable. Preliminary experiments suggest that intact PMNs inhibit microbial DNA synthesis in a fashion similar to the cell-free MPO system. The PMN effect requires a functional MPO system.
Specific aims are:
Aim l) To complete studies of PMN-mediated inhibition of DNA synthesis and to determine whether metabolic reconstitution of PMNs that have defective MPO systems (chronic granulomatous disease, MPO deficiency) permits these PMN to inhibit microbial DNA synthesis more normally.
Aim 2) To determine whether the inhibition of microbial DNA synthesis is indeed related to chromosomal replication initiation. E. coli mutants with a lethal, temperature sensitive, defect in replication initiation (dnaA[ts]) will be compared to MPO-treated normal cells with respect to replication of chromosomal, phage, and plasmid DNAs which have different dependencies on oriC for replication initiation.
Aim 3) To determine which elements of replication initiation are altered by MPO-derived oxidants. Principal candidates are membrane phospholipids and the dnaA initiator protein, product of the dnaA gene. Candidate structures will be extracted from MPO-treated organisms and tested for functional integrity in a cell-free assay for oriC- dependent DNA synthesis.
Aim 4) To relate knowledge obtained with the cell-free MPO system to an assessment of MPO function in intact PMNs. The anticipated benefit of the project is an improved understanding of PMN- mediated host defense against bacterial infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
7R37AI025606-08
Application #
2063026
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1988-03-01
Project End
1998-02-28
Budget Start
1994-09-01
Budget End
1995-02-28
Support Year
8
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Washington
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Rosen, Henry; Klebanoff, Seymour J; Wang, Yi et al. (2009) Methionine oxidation contributes to bacterial killing by the myeloperoxidase system of neutrophils. Proc Natl Acad Sci U S A 106:18686-91
Rosen, Henry; Crowley, Jan R; Heinecke, Jay W (2002) Human neutrophils use the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate but not nitrate bacterial proteins during phagocytosis. J Biol Chem 277:30463-8
Staudinger, Benjamin J; Oberdoerster, Michele A; Lewis, Patrick J et al. (2002) mRNA expression profiles for Escherichia coli ingested by normal and phagocyte oxidase-deficient human neutrophils. J Clin Invest 110:1151-63
Rosen, H; Michel, B R; vanDevanter, D R et al. (1998) Differential effects of myeloperoxidase-derived oxidants on Escherichia coli DNA replication. Infect Immun 66:2655-9
Kuhl, S J; Rosen, H (1998) Nitric oxide and septic shock. From bench to bedside. West J Med 168:176-81
Rosen, H; Michel, B R (1997) Redundant contribution of myeloperoxidase-dependent systems to neutrophil-mediated killing of Escherichia coli. Infect Immun 65:4173-8
Rosen, H (1997) Superantigens. Int J Dermatol 36:14-6
Durden, D L; Kim, H M; Calore, B et al. (1995) The Fc gamma RI receptor signals through the activation of hck and MAP kinase. J Immunol 154:4039-47
Rakita, R M; Michel, B R; Rosen, H (1994) Inactivation of Escherichia coli penicillin-binding proteins by human neutrophils. Infect Immun 62:162-5
Durden, D L; Rosen, H; Cooper, J A (1994) Serine/threonine phosphorylation of the gamma-subunit after activation of the high-affinity Fc receptor for immunoglobulin G. Biochem J 299 ( Pt 2):569-77

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