Effect of db-cAMP on <i>whiB1-7</i> expression.

<p><i>M.tb</i> CDC1551 was grown 24h in the presence of 500 µM db-cAMP. <i>whiB1</i> and to a lesser extent <i>−2</i> and <i>−4</i> were up-regulated.</p

Relative abundance of <i>whiB1-7</i> transcripts.

<p>mRNA levels of the seven <i>M.tb whiB</i> genes at mid-logarithmic growth in 7H9 presented as fold change in relation to <i>sigA</i> (set to 0). <i>WhiB1</i> was highly expressed and <i>whiB5</i> was under-expressed compared to the other <i>whiB</i> genes, which exhibited an expression level very close to that of <i>sigA</i>.</p

Effect of nitric oxide on <i>whiB1-7</i> expression.

<p><i>M.tb</i> CDC1551 was grown for 12 h in the presence of 500 µM DETA-NO. <i>whiB3, whiB6</i> and to a lesser extent, <i>whiB7</i> were upregulated.</p

<i>Rv2190c</i> expression peaks at log phase and is involved in the response to cell wall disruption.

<p><b>A.</b> Fold change of <i>Rv2190c</i> expression (relative to level at the start of the growth curve) over increasing cell density in 7H9 broth in wild-type <i>M. tuberculosis</i> CDC1551, normalized to <i>sigA</i> expression. pc: post-clumping. <b>B.</b> Fold-change of <i>Rv2190c</i> expression after exposure to oxidative (cumene), disulfide (diamide) and detergent (SDS) stress conditions, relative to expression levels prior to addition of stressors and normalized to <i>sigA</i>. <b>C.</b> Survival of WT, the <i>Rv2190c</i> mutant and complement <i>M. tuberculosis</i> strains following 24 hours of lysozyme exposure. <b>D.</b> PDIMs for <i>M. tuberculosis</i> WT and Rv2190c mutant strains analyzed by thin layer chromatography using hexane∶diethyl ether∶acetic acid solvent. <b>E.</b> PDIMs analyzed using petroleum ether∶diethyl ether solvent.</p

The <i>M. tuberculosis Rv2190c</i> mutant is attenuated <i>in vivo</i>.

<p><b>A. </b><i>M. tuberculosis</i> aerosol implantation in the lungs of BALB/c mice for the <i>Rv2190c</i> mutant, complement and WT (CDC1551) strains for the time-to-death experiment, determined day 1 post-infection. <b>B.</b> Survival curves for the time-to-death experiment. <b>C–D.</b> Lung (C) and spleen (D) CFU counts for BALB/c mice infected via aerosol with ∼3.4 log₁₀CFUs of WT, <i>Rv2190c</i> mutant and complement <i>M. tuberculosis</i> strains. <b>E.</b> Gross pathology of infected mouse lungs at 112 days post-infection.</p

The <i>M. tuberculosis Rv2190c</i> causes decreased lung histopathology in the mouse.

<p>Formalin-fixed lung sections from mice infected with WT, <i>Rv2190c</i> mutant and complement strains were stained with hematoxylin and eosin and visualized using light microscopy. Scale bar = 0.1 mm.</p

Immunogenic properties of the Rv2190c protein.

<p>N-terminal truncated Rv2190c was purified and analyzed for immunogenic properties by Dot-Blot ELISA against various sera. <b>A.</b> Immunogenic response in the human serum of an individual with latent TB (PPD-Pos.) (left panel) as opposed to a negligible response in a PPD negative human serum (right panel). <b>B.</b> Immunogenic response in serum from an <i>M. bovis</i> infected rabbit (left panel) and weak responses from sera raised against WhiB6 protein (middle panel) or pre-bleeds (right panel). i) 2ng of Rv2190c and ii) 2ng of BSA.</p

Intracellular cAMP levels increase within <i>M.tb.</i>-infected THP-1 human monocytic cells upon exposure to PDE-Is.

<p>The treated infected cells received 100 uM of PDE inhibitors (PDE-I 3, and 5 class) for 2 h followed by infection. UI: uninfected cells; the PDE5-I was 4-{[3′,4′-(Methylenedioxy)benzyl]amino}-6-methoxyquinazoline (MBM); and the PDE3-I was trequinsin. Results shown (mean and SD) represent two biological replicates, each with 2 technical replicates.</p

Interaction of PDE-Is with Rifampin.

<p>BALB/c mice were infected with 3.6 log₁₀CFU <i>M.tb.</i> and were treated daily by oral gavage (starting the day after infection) with 10 or 30 mg/kg cilostazol (C10 and C30, respectively), 10 mg/kg rifampin (R10), C10 plus R10, or C30 plus R10. Lung CFU counts were determined on days 14 and 28 post-infection.</p

Proportion of mice with no detectable CFUs during TB standard therapy with and without PDE-I.

<p>The proportions of mice with no detectable CFUs were determined by homogenizing and plating the entire lungs from 56 to 168 days of treatment. Treatment was terminated after 168 days, and groups of mice were kept for an additional 4 months (168+112). Shading indicates lung sterilization in all mice in the group. SD: standard drug therapy (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030749#pone-0030749-g004" target="_blank">Fig. 4</a> legend for details); S: 10 mg/kg sildenafil; C: 10 mg/kg cilostazol; CS: 10 mg/kg each of sildenafil and cilostazol.</p