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David Russell Thaler Lecture
- 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 v’s 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 IFNγ-/- mice smyc’::mCherryrpfD’::GFP phalloidinDAPI iNOS
- 9. Erdman rv2390c’::GFP smyc’::mCherry infection - WT vs. IFNγ-/- 14 days 28 days 35 days Time post-infection GFP/µm3 p<0.0001 p<0.0001 p<0.0001 IFNγ-/- mice infection WT mice infection
- 10. Nitric Oxide is a major activator of DosR as shown by the reporter strain Erdman hspX’::GFP smyc’::mCherry GFP expression scored as a function of bacterial volume by Volocity (Perkin Elmer) 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
- 11. Erdman hspX’::GFP smyc’::mCherry WT infection 28d WT mice IFNγ-/- mice smyc’::mCherryhspX’::GFP phalloidinDAPI
- 12. Erdman hspX’::GFP smyc’::mCherry in mice GFP expression scored as a function of bacterial volume by Volocity (Perkin Elmer)
- 13. RelativeGFPexpression 7 14 21 28 35 42 rv2390c’::GFP hspX’:: GFP INNATE IMMUNE RESPONSES ADAPTIVE IMMUNE RESPONSES Kinetics of Expression of Reporter Strains in Mice Days post infection
- 14. Harvest lungs 14,28,42,56 days post-challenge 4 weeks Intranasal challenge with reporter strains 103 CFU in 25μl droplet Vaccination and Challenge: Procedure Intraperitoneal administration of 5 X 105 heat killed M.tuberculosis or sterile PBS WT, IFNγ-/-, NOS2 -/-
- 15. 10000 100000 1000000 10000000 100000000 7 14 21 28 42 56 TotalLungCFUS Days post infection Vaccinated PBS Kinetics of Colonization of Vaccinated vs Control C57BL/6 mice
- 16. 28 dpi Vaccinated Control 56 dpi Lung Pathology in C57BL/6 mice: H&E Stain
- 17. Vaccinated Day 28 Day 14 DAPI hspX’::GFP smyc’::mCherry phalloidin PBS Erdman hspX’::GFP smyc’::mCherry in C57BL/6 mice
- 18. Quantitation of hspX’::GFP in C57BL/6 mice Higher hspX’::GFP expression as early as 14 dpi in vaccinated mice
- 19. DAPI hspX’::GFP smyc’::mCherry phalloidin iNOS Erdman hspX’::GFP, smyc’::mCherry in C57BL/6 mice: iNOS Staining VaccinatedPBS Day 28 Day 14
- 20. Quantitation of hspX’::GFP in iNOS + and iNOS – lung regions D14 pi PBS Vac iNOS - Vac iNOS + p < 0.0001 N.S.
- 21. WT - PBS WT - Vaccinated Erdman rv2390c’::GFP,smyc’::mCherry-WT Day 14 DAPI rv2390c’::GFP smyc’::mCherry phalloidin
- 22. PBS Vac Day 14 PBS Vac Day 28 PBS Vac Day 42 PBS Vac Day 56 Quantitation of rv2390c’::GFP in C57BL/6 mice Lower rv2390c’::GFP expression in vaccinated mice p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001
- 23. Erdman hspX’::GFP smyc’::mCherry WT - PBS WT - vac Day 14 DAPI hspX’::GFP smyc’::mCherry phalloidin Day 28 IFNγ-/- - vacIFNγ-/- - PBS NOS2-/- - vac
- 24. Quantitation of Erdman hspX’::GFP smyc’::mCherry
- 25. M. smegmatis expressing ssB::GFP movie by Bree Aldridge, Tufts U. Real-time Replication Reporter
- 26. The % Foci-Positive Bacilli is inversely proportional to the Immune Response. Number of foci-positive Mtb scored at 14 days post challenge infection
- 27. PBS Vac INH PBS Vac RIF Drug Susceptibility Assay: D14 pi p < 0.0001 N.S. Increased INH resistance early on in vaccinated mice
- 28. What do the reporter strains tell us? 1. DosR reporter: Induction correlates well with the acquired immune response and the presence of IFN-g and iNOS. Expression is accelerated by vaccination. 2. Drug sensitivity: Sensitivity to INH in Mtb from 14d infection is higher in naive than vaccinated mice implying that these bacteria are more actively replicating. 3. ssB::GFP reporter: Scoring of replication forks indicates that replication correlates inversely with the development of the acquired immune response and is dependent on the presence of IFN-g. 3. Chloride reporter: induced early but trends down as the acquired immune response develops. Is not induced in immune incompetent mice so early neutrophil activity? Inverse correlation with granuloma establishment?
- 29. + drug Yancheng Liu Can we identify the host contribution to drug-induced death in the host?
- 30. Bacteria exposed to drugs inside cells Bacteria exposed to drugs in liquid culture Bacteria exposed to INH ETH PYR RIF at concentrations that induce 1/2 log death at 48 hrs (2-5 X MIC) Bacterial RNA isolated at 4hr and 24 hr time periods post-exposure to each drug and analyzed by microarray Transcriptional profile of M. tuberculosis exposed to a panel of 4 different drugs The role of environmental pressure(s) in drug-mediated killing of Mtb.
- 31. 1.0E+05 1.0E+06 1.0E+07 0 1 2 3 4 5 6 7 8 9 10 CFU/ml DMSO INH RIF PZA EMB DPI Bacterial CFUs following treatment of intracellular Mtb with front-line drugs. Macrophages were infected 5 days previously, treated with drug for 4 and 24 hrs, then isolated and processed for RNA purification. DMSO INH (0.2 mg/ml) RIF (0.4 mg/ml) PZA (200 mg/ml) EMB (12 mg/ml) Impact of Drug Pressure on Survival of Intracellular Mtb. Drug addition RNA isolation
- 32. 196 248128 Induced by INH in broth Induced by INH in mØ Expression ratio in broth ExpressionratioinmØ fadE23 fadE24 fbpC accD6 rv1772 iniA iniC iniB efpA kasA kasB rv1592c fabD acpM ahpC rv0448c rv0449c mpt70 mpt83 MT2941 rv2874 rv3290c Macrophage-specific response Focusing on INH: One can resolve both drug-specific responses (common to MØ and broth) and MØ-specific-responses observed only in the MØ
- 33. In vitro stimulus Genes overlapped with mφ- specific INH induced list (146 genes) Genes overlapped by random chanceh Acid stress (pH=5.5)a (291 genes) 35 8 Nutrient starvationb (440 genes) 37 12 Oxidative stress (H2O2)c (319 genes) 24 9 NO/CO/hypoxia(Dos regulon)d (50 genes) 16 1 Dos-independent hypoxiae (290 genes) 14 8 SDS-mediated membrane damagef (211 genes) 30 6 Triton-mediated membrane damageg (73 genes) 20 2 Table 1. Comparison of mφ-specific INH induced genes to in vitro stimuli induced genes. The MØ-specific responses are focused primarily on known host-cell mediated defense stresses.
- 34. Commonly induced genes Commonly repressed genes 4-Way Venn Diagram of Genes induced by 4 frontline drugs in Mtb in MØ
- 35. Table 2. Comparison of mφ-specific commonly induced genes to in vitro stimuli induced genes. In vitro stimulus Genes overlapped with mφ- specific commonly induced list (108 genes) Genes overlapped by random chanceh Acid stress (pH=5.5)a (291 genes) 28 6 Nutrient starvationb (440 genes) 27 9 Oxidative stress (H2O2)c (319 genes) 23 7 NO/CO/hypoxia(Dos regulon)d (50 genes) 18 1 Dos-independent hypoxiae (290 genes) 8 6 SDS-mediated membrane damagef (211 genes) 27 5 Triton-mediated membrane damageg (73 genes) 22 2 Relaxing the stringency to include genes upregulated by 3 of 4 drugs in Mtb in MØ – gives a profile similar to INH.
- 36. Interpretation and implications for drug development The drug hits the bug and reduces its fitness via a drug-specific route The reduction in fitness renders the bacterium more susceptible to its environment and host-derived stresses. This model contains an implied prediction: Compounds that block the ability of Mtb to deal with these host derived stresses should enhance the activity of anti-Mtb drugs.
- 37. Synergy: Exploitation of the host “stresses” that are intensified by drug pressure renders Mtb more susceptible to known drugs. Identifies the potential for exploiting host- derived pressures to enhance drug action in vivo
- 38. Human macrophages infected with Mtb (mCherry) and HIV (pseudotyped virus with GFP) Co-infections with HIV and M. tuberculosis HIV is the biggest risk factor for active tuberculosis, and this risk is impacted minimally by ART. Why?
- 39. Human macrophages infected with HIV (day 0) and Mtb CDC 1551 (day 3) Days post-infection with Mtb
- 40. Work with Henry Mwandumba and Kondwani Jambo Impact of HIV infection on alveolar macrophage function
- 41. FACS plots show control and HIV-infected human monocyte-derived following incubation with a FISH probe directed against HIV gag or gp120 mRNA sequence, in the Y axis. Fluorescent confocal micrograph of the FISH label of 8E5 cells is shown on the right Development of a mRNA FISH detection system for HIV Stellaris probes developed in collaboration with BioSearch Technologies Inc.
- 42. The Make-up of the Cellular Populations in the Lung The cells in the lung airways are approximately 75% macrophages and 25% lymphocytes. This is not altered by asymptomatic HIV infection.
- 43. HIV is preferentially located in the alveolar macrophages. In all individuals examined the % abundance of HIV-infected cells is higher in the macrophage population than it is in the lymphocyte population Jambo, K.C., Banda, D., Kankwatira, A., Sukumar, N., Allain, T., Heyderman, R.S., Russell, D.G., and Mwandumba, H.C. (2014) Small alveolar macrophages are infected preferentially by HIV and exhibit impaired phagocytic function. Mucosal Immunology. PMID: 24472847
- 44. HIV Infection in vivo causes a profound shift in CD phenotype. AMs from all 6 HIV+ve individuals examined show a down-regulation in expression of CD206 (Mannose receptor), HLA-DR, and CD71 (Transferrin receptor), while the expression of CD45 is unaltered.
- 45. Thanks to: Henry Mwandumba and Kondwani Jambo, MLW Labs, Blantyre, Malawi. The NIH: NIAID & NHLBI. Roadside snacks in Malawi