1. The host/pathogen interplay in tuberculosis and HIV/TB co- infection. dgr8@cornell.edu M. tuberculosis (red) cholesterol (green), and fluorescent dextran (magenta) in macrophages

2. 1. The interplay between the immune environment and bacterial stress responses. 1. The contribution of host-derived stresses to the activity of anti-TB drugs 2. The impact of HIV on the host/pathogen balance and is significance to active TB. The Host Immune Environment 3. Bacteria in Medium (+ cytochalasin D) Bacteria on macrophages (+ cytochalasin D) Changes in gene expression induced by adherence Bacteria on macrophages (no cytochalasin D) Changes in gene expression induced by phagocytosis How does Mycobacterium detect and respond to the intracellular environment? Kyle Rohde Rohde, K, Abramovitch, R, and Russell, D.G. (2007) Mycobacterium tuberculosis invasion of macrophages: Linking bacterial gene expression to environmental cues. Cell, Host and Microbe. 2: 352-364 4. A. Macrophage bound (+ cytochalasin D) B. 2 hours post internalization A. Microarray comparison between Mtb in BMMO medium (+ cyto D) and Mtb bound to the surface of macrophages in the presence of cytochalasin D for 30 minutes. B. Microarray comparison between Mtb in BMMO medium and Mtb bound to BMMO and incubated for 2 hours in the absence of cyto D. The bacteria show a transcriptional response on phagocytosis but not on binding to macrophages. upregulated downregulated Transcriptional response 2 hrs post-infection 5. Production of ROIs Acidification of the phagosome Production of RNIs Acquisition of lysosomal hydrolases, ie. proteinases lipases glucosidases nucleotidases Nutrient limitation Intracellular cues that Mtb may sense and utilize as its GPS system. Physiological imperatives vs off-target responses 6. Mycobacterium tuberculosis Reporter Strains environmentally- responsive promoter mCherry gfp constitutively-active promoter hspX: Hypoxia and NO rv2390c: [Cl-] and low pH Tan, S., Sukumar, N., Abramovitch, R.B., Parish, T., and Russell, D.G. (2013) Mycobacterium tuberculosis senses chloride and pH as indicators of the immune status of its host cell. PLoS Pathogens. 9 (4), e1003282 Shumin Tan Neelima Sukamar 7. Erdman rv2390c::GFP a Sensor for phagosome maturation IgG-coated reporter beads in phagosomes ( senses [Cl-] ( senses pH] rpfD::gfp - reporter Mtb in defined buffers As pH goes down, [Cl-] goes up. Both increase activity of the 2390c promoter activity. Bis(carboxylpropyl) biacridinium 8. Erdman rv2390c::GFP smyc::mCherry infection 35d WT mice IFN-/- mice smyc::mCherryrpfD::GFP phalloidinDAPI iNOS 9. Erdman rv2390c::GFP smyc::mCherry infection - WT vs. IFN-/- 14 days 28 days 35 days Time post-infection GFP/m3 p