Neutrophils (PMN) and macrophages provide host defense against infection by ingesting and killing microorganisms. These essential functions depend on the creation of a toxic intracellular environment created by the fusion of lysosomes with the organism containing phagosome. One of the goals of this project is to better define the events which regulate this fusion process. Changes in Ca++ concentration and pH have been intimately linked to PMN and macrophages activation and are a major focus of the proposed studies. We have identified a new Ca++ pumping enzyme in human PMN lysosomes which functions at physiologic Ca++, ATP, and H+ concentrations and have proposed that this Ca++ pump plays a major role in activating phagocytes. Using isotopic and fluorescent methods, we will characterize the regulation of calcium uptake by PMN lysosomes and the release of calcium from these organelles. These studies will provide information on normal control mechanisms for triggering phagocytes and will form the basis of a strategy for isolating this Ca++ -ATPase. A combination of affinity and standard chromatographic methods are proposed. Failure to create a phagolysosomal environment sufficiently toxic to kill pathogenic microorganisms results in morbid infectious diseases. Intrinsic cell defects are one class of this failure and the Chediak-Higashi syndrome is a prominent example. The hallmark of this human disorder is giant, abnormal lysosomes which fuse defectively with the phagosome. This results in recurrent, often lethal, infections. While the fundamental defect is unknown, we have recently described an abnormal Ca++ pump in CHS PMN lysosomes. Using human and beige mouse PMN and macrophages lysosomes, we will further examine the nature of this pump and determine whether this represents the first example of an abnormal ion translocating pump which is the proximate cause of human disease. Organisms, including Yersinia, Legionella, and Mycobacteria which survive and multiply within phagolysosomes, cause morbid infectious diseases. Using a model of Yersinia pestis organisms which have been genetically engineered to report the Ca++ and pH conditions in their environment, we have begun to define the ionic constitution of the phagolysosome. Our goal is to identify characteristics of the organism-containing phagolysosome which can be mainipulated as part of a new antimicrobial strategy. We will genetically engineer additional mutants of Y. pestis and use these probes to examine whether modifying the ionic environment alters the virulence of the pathogen.
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