Mycobacterium tuberculosis is the largest single infectious disease killer in the world today. Its penetrance of the human population is due its ability to cause persistent infections in the human host. Work from our laboratory and several other labs have reported enhanced levels of isocitrate lyase expression intracellular or dormant bacilli. Isocitrate lyase is the first enzyme of the glyoxylate shunt pathway which exploits acetate or long chain fatty acids as an alternate carbon source. ICL competes with isocitrate dehydrogenase to divert the flux of carbon away from the TCA cycle and into the alternative pathway of carbon acquisition. We propose to examine the role of the glyoxylate shunt and its potential as a drug target. These are the specific goals of this project. 1. Identification of the environmental stimuli responsible for up- regulation of ICL expression. We will examine the culture conditions required to induce maximal expression of ICL. In addition we will use the ICF:GFP construct that we have generated to probe the intracellular environment to determine what effect changes in immune status (and the vacuolar environment( have on ICL expression. 2. The role of anaplerotic metabolism in sustaining a latent infection. We shall use both the ICL:GFP construct and antibodies against ICL and malate synthase to probe the expression of these enzymes in murine infections. This work will be undertaken in collaboration with Dr's McKinney and Jacobs who will be investigating infections in different KO mice with different mycobacterial mutants. 3. Identification of compounds inhibitory to ICL and malate synthase activities and their development as anti mycobacterial agents. We will use the recombinant enzymes to screen drug libraries at GlaxoWellcome. Structural information generated by Dr. Sacchettini will be used to modify and improve inhibitors which will be evaluated in different culture and infection models.
