Mycobacterium avium and Mycobacterium tuberculosis are intracellular pathogens that infect mononuclear phagocytes and survive within tissue macrophages. Tuberculosis is responsible for millions of deaths annually, and M. avium is the most common pathogen causing systemic disease in AIDS patients. Both bacteria survive inside membrane-bound vacuoles in macrophages. The intracellular localization of these organisms suggest that they are well adapted for survival in the host and that they probably can obtain sufficient amounts of nutrients inside the host cells. The presence or absence of elements, such as iron, in the environment is known to influence gene expression in bacteria. It has been assumed that macrophage vacuoles are deficient in iron, as a host defense mechanism against infection. Improving our understanding about the environment within the mycobacterial vacuole and how mycobacteria respond to the conditions present in the vacuoles will provide new insights into pathogenic mechanisms. We propose to: (1) Determine the presence and total concentration as well as the available concentration of important elements such as iron, potassium, phosphorus, sulphur, calcium, magnesium, manganese, copper and zinc in M. avium vacuole in macrophages and dentritic cells compared to M. smegmatis vacuoles by using two methods: (a) hard X-ray microscopy, and (b) bacterial reporte systems. (2a) Establishing the role of the environment within mycobacterial vacuoles on the expression of mycobacterial putative virulence determinants; (b) determine the importance of these determinants in virulence. This is the first time that we will be able to obtain data on the concentrations of single elements in mycobacterial phagosomes. We believe that the proposed studies will generate important information about the phagosome environment and the mycobacterial response to it.
