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.
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