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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
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
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
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?
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.
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