1. Mycobacterium tuberculosis Senses Host-Derived Carbon Monoxide during Macrophage Infection 
Michael U. Shiloh, Paolo Manzanillo, Jeffery S. Cox 
Cell Host & Microbe 
Volume 3, Issue 5, Pages (May 2008)
DOI: /j.chom
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Heme Oxygenase Is Induced in Macrophages and Mice by MTB Infection
(A) Naive bone marrow-derived macrophages were infected with MTB, and global macrophage gene expression was determined by microarray. Each time point (Cy5) was compared to a pooled reference (Cy3) and normalized to time zero. Shown is the average response of mouse HO-1 from four independent experiments. Scale bar indicates the mean of the log ratio (635/532). One-way ANOVA resulted in a p value of <0.0001, and comparison of time 0 with 4, 8, 16, and 24 hr yielded p values < 0.05 for all comparisons.
(B) Naive macrophages were either mock treated (−) or infected with MTB (+), and HO-1 accumulation was determined by western blotting with polyclonal anti-HO-1 antibodies and anti-GAPDH antibody as loading control.
(C) BALB/c mice were infected with MTB by IV injection, and organs were harvested after 10 days. Paraffin sections were incubated with rabbit anti-HO-1 antibodies followed by biotinylated goat anti-rabbit antibody and DAB staining, which stains positive cells brown. Cells were counterstained with hematoxylin. Arrows indicate Kupffer cells (3) and alveolar macrophages (4). Scale bars, 200 μm (1 and 2) or 50 μm (3 and 4).
Cell Host & Microbe 2008 3, DOI: ( /j.chom )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2 Carbon Monoxide Induces the MTB Dormancy Regulon In Vitro
(A) MTB growing in vitro was exposed to 20 (16 nM), 200 (160 nM), and 2000 (1.6 μm) ppm of CO gas in the headspace for 48 hr; RNA was harvested; and gene expression was determined by microarray analysis. CO-treated MTB samples were labeled with Cy5 (red) and from untreated MTB samples with Cy3 (green). Green and red spots indicate a gene whose transcription was repressed or induced at least 2-fold relative to untreated samples in at least two arrays. Scale bar indicates the mean of the log ratio (635/532). Two representative experiments of more than four similar experiments are shown.
(B) Quantitative PCR (qPCR) analysis of two MTB transcripts, fdxA (blue) and hspX (red), from MTB grown in the presence of CO. Each sample was normalized to 16S RNA as an internal control. Fold induction (±SD) was calculated by dividing all samples by the untreated sample (CO 0 ppm = 1-fold). Data are representative of three similar experiments. One-way ANOVA resulted in a p value of < ∗p < 0.05, ◊p < 0.01, and †p < compared to time zero.
Cell Host & Microbe 2008 3, DOI: ( /j.chom )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
The MTB DosS/T/R Signal Transduction System Transmits the CO Signal
(A) MTB wild-type, ΔdosR, and ΔdosR compdosR were treated with DETA-NO (100 μM) or CO (20,000 ppm, 16 μM) for 60 min. Shown are the genes of the dormancy regulon only. Scale bar indicates the mean of the log ratio (635/532). Data are representative of two similar experiments.
(B) MTB wild-type, ΔdosS, ΔdosS comp dosS, ΔdosT, ΔdosT comp dosT, ΔdosS/ΔdosT, and ΔdosS/ΔdosT comp dosT were treated with DETA-NO (100 μM) or CO (20,000 ppm, 16 μM) for 60 min. Shown are the genes of the dormancy regulon only. Scale bar indicates the mean of the log ratio (635/532). Data are representative of two similar experiments.
(C and D) Response of wild-type, ΔdosS, and ΔdosT mutants to either DETA-NO (C) or CO (D) for 3 hr at the indicated doses as determined by qPCR for fdxA. Each sample was normalized to 16S RNA as an internal control. Fold induction (±SD) was calculated by dividing each experimental sample by the untreated sample for its respective genotype. One-way ANOVA resulted in a p value of < for both (C) and(D). ∗p < versus MTB wild-type at each CO concentration. Data are representative of two similar experiments.
Cell Host & Microbe 2008 3, DOI: ( /j.chom )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
Absence of Macrophage Heme Oxygenase Reduces MTB Dormancy Gene Induction
(A) Bone marrow-derived macrophages from wild-type, HO1−/−, or NOS2−/− mice were infected with MTB. Twenty-four hours after infection, macrophages were lysed, bacterial RNA was isolated, and amplified antisense RNA was generated. Samples were labeled with Cy5, and a pooled reference of all samples was labeled with Cy3 and hybridized against MTB microarrays. All samples were normalized by the MTB arrays from WT macrophages. Scale bar indicates the mean of the log ratio (635/532).
(B) aRNA prepared as described above was subjected to qPCR for three MTB genes of the dormancy regulon: Rv2630, fdxA, and hspX. Genes were normalized to 16S RNA as an internal control. Included for comparison is the gene expression of MTB growing in vitro. One of two similar experiments is shown. One way ANOVA gave p values of < for all three genes. The asterisk indicates p < compared to WT macrophages.
(C) Bone marrow macrophages were infected as described above, and HO-1 was detected by western blotting after 24 hr of infection.
Cell Host & Microbe 2008 3, DOI: ( /j.chom )
Copyright © 2008 Elsevier Inc. Terms and Conditions