Tuberculosis, which has plagued humanity for thousands of years, is re- emerging as a serious threat to the public health of the United States. A crucial early event in infection is the ingestion of Mycobacterium tuberculosis (Mtb) by alveolar macrophages (AM0). Subsequently, an important struggle ensues, as the AM0 seek to kill and degrade the Mtb, while the organisms strive to survive in this hostile environment. Our proposal seeks to characterize mechanisms that contribute significantly to the outcome of this initial interaction. Our novel multifaceted biochemical and molecular biological approach focuses on two fundamental questions concerning the initial responses of AM0 to ingested Mtb. These are: 1. What Mtb cell wall breakdown products result from the AM0 chemical assault on Mtb? and 2. How do these cell wall catabolites trigger cytokine production in the AM0? We plan to answer these questions by: 1. Characterizing the products resulting from catabolic breakdown of mycobacterial cell walls, using biosynthetically radiolabeled cell walls and AM0 cell lines. We will employ techniques of subcellular fractionation, chemical and chromatographic purification, and structural analysis.
We aim to accomplish three objectives: A. Identify lipoarabinomannan (LAM) or related structures that result from AM0 breakdown of Mtb. B. Identify arabinogalactan and peptidoglycan catabolites. C. Identify differences between AM0 catabolites from virulent and avirulent Mtb strains. These studies will test the hypothesis that AM0 catabolism of Mtb produces substances that affect host defenses. 2. Determining the mechanism(s) by which LAM-like molecules and other catabolites induce gene expression for cytokines and NO synthase in AM0. To test the hypothesis that a virulent strain of Mtb suppresses host cell responses, relative to an avirulent strain, we will A. Characterize the pattern of expression for NO synthase and a set of cytokines likely to be important in mourning a successful AM0 response to Mtb: tumor necrosis factor (TNF) and interleukins 1, 6, and 8 (IL-1, IL-6, IL-8). We will first compare the ability of whole Mtb and isolated cell wall catabolites to induce NO synthase and cytokine mRNA production in AM0, then determine if altered steady-state mRNA levels result from changes in rates of transcription, mRNA turnover, or both. B. We will characterize the intracellular signaling pathways activated by Mtb cell walls and catabolic fragments by identifying components of these pathways that are activated by interaction with mycobacterial cell wall products. We will also examine the nuclear regulatory proteins that are known to control gene transcription of these cytokines (e.g. NF-kappaB, NFIL-6). Since the AM0 response sets the stage for the final outcome -- successful clearance of organisms or development of disease -- understanding this initial interaction is crucial.
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