Recombinant MT0516 exhibits concentration- and time-dependent exopolyphosphatase activity.

<p>A. Polyacrylamide gel electrophoresis showing the fractions of recombinant His-tagged MT0516 protein (36.6 KDa), expressed in <i>E.coli</i> Arctic Express (DE3) BL 21. Lane 1: Molecular weight markers; Lanes 2-3: Supernatant and pellet, respectively, prior to Ni⁺⁺NTA column binding; Lanes 4-5: Elution fraction 1 and 2, respectively, eluted sequentially from Ni⁺⁺NTA column. B. Western blot using Penta-His antibody. Lane 1: Molecular weight markers. Lane 2: Elution fraction 2 after native purification demonstrating the expected size band (36.6 KDa). C. Exopolyphosphatase activity of recombinant MT0516 expressed as % hydrolysis of substrate (65-mer poly P) as a function of protein concentration (μg/ml). **p <0.001. D. Exopolyphosphatase activity of recombinant MT0516 expressed as % hydrolysis of substrate (65-mer poly P) as a function of time (hours). E =  elution fraction 2; AP =  alkaline phosphatase (positive control); NC =  negative control comprising supernatant obtained following IPTG induction of Arctic <i>E coli</i> transformed with empty vector. In each reaction, 1 μg/ml poly P was used as substrate. Data for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028076#pone-0028076-g005" target="_blank">Fig. 5C and 5D</a> are derived from individual experiments, each of which was performed three times with separate samples yielding similar results.</p

MT0516 is required for full virulence of Mtb in guinea pigs.

<p>A. Growth and survival of the mutant following low-dose aerosol infection in guinea pig lungs relative to wild-type and complement strains (*p≤0.01; **p≤0.001 for differences in bacillary counts between mutant- and wild-type-infected lungs). B. Gross pathology of Mtb-infected guinea pig lungs at Day 84 after aerosol infection. C. Microscopic examination of Mtb-infected lungs at Day 84 (H&E stain, 2× magnification. Insets: Ziehl-Neelsen staining, 50x magnification. CDC1551 =  wild-type strain; MT0516::Tn =  MT0516-deficent mutant; MT0516::Tn Comp =  MT0516::Tn complement strain.</p

Sequence of primers used in this study.

<p>Sequence of primers used in this study.</p

Protein modeling predicts that <i>M. tuberculosis</i> MT0516 is an exopolyphosphatase.

<p>The protein backbone ribbon structure was modeled by PHYRE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028076#pone.0028076-Kelley1" target="_blank">[23]</a>, showing the conserved hydrolase fold associated with exopolyphosphatases. Left-angled, top-down view of the interface canyon between domains I and II with the α helix 4 (within box) that houses the highly conserved active site E112 side chain opening into the canyon floor. The canyon walls are lined with β sheets.</p

Complementation of transposon mutant MT0516::Tn.

<p>A. Diagram of expected recombination between integrating plasmid and genomic DNA. B. PCR analysis of genomic DNA. A list of relevant primers is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028076#pone-0028076-t001" target="_blank">Table 1</a>. Lane 1: Molecular weight markers. Lanes 2-4: Amplification of the MT0516 gene from wild-type, MT0516::Tn, and MT0516::Tn Comp, respectively. The expected 1035-bp PCR product is present in Lanes 2 and 4 but absent in Lane 3. Lane 5: Molecular weight markers. Lanes 6-8: Amplification of the kanamycin resistance cassette from wild type, MT0516::Tn and MT0516::Tn Comp, respectively, yielding the expected 226-bp product only in Lanes 7 and 8. Lane 9: Molecular weight markers. Lanes 10-11: Amplification of the hygromycin resistance cassette from MT0516::Tn and MT0516::Tn Comp, respectively, yielding the expected 319-bp product only in Lane 11. C. Diagram of expected sizes of <i>EcoRI</i>-digested genomic fragments expected to hybridize to probe recognizing region of MT0516 coding sequence by Southern blot (D). Note that MT0516::Tn Comp is expected to have two different size fragments hybridizing to the MT0516 probe, including that found in MT0516::Tn (1.8 Kb) and a distinct fragment unique to the attB integration site (1.7 Kb). D. Southern blot detecting the presence of DNA fragments bound to the MT0516 hybridization probe. Lane 1: Molecular weight markers. Lane 2: Blank. Lanes 3-5: Wild type, MT0516::Tn, and MT:0516::Tn Comp, respectively.</p

Comparison of mean log₁₀ CFU/organ recovered from guinea pigs after aerosol infection with <i>Mtb</i> CDC1551.

<p>Comparison of mean log₁₀ CFU/organ recovered from guinea pigs after aerosol infection with <i>Mtb</i> CDC1551.</p

<em>Mycobacterium tuberculosis</em> Complex Enhances Susceptibility of CD4 T Cells to HIV through a TLR2-Mediated Pathway

<div><p>Among HIV-infected individuals, co-infection with <em>Mycobacterium tuberculosis</em> is associated with faster progression to AIDS. We investigated the hypothesis that <em>M. bovis</em> BCG and <em>M. tuberculosis</em> (Mtb complex) could enhance susceptibility of CD4+ cells to HIV infection. Peripheral blood mononuclear cells (PBMCs) collected from healthy donors were stimulated with <em>M. bovis</em> BCG, <em>M. tuberculosis</em> CDC1551 and <em>M. smegmatis</em> MC²155, and stimulated CD4+ cells were infected with R5-and X4-tropic single replication-competent pseudovirus. CD4+ cells stimulated with Mtb complex showed enhanced infection with R5- and X4-tropic HIV, compared to unstimulated cells or cells stimulated with <em>M. smegmatis</em> (p<0.01). Treatment with TLR2 siRNA reversed the increased susceptibility of CD4+ cells with R5- and X4-tropic virus induced by Mtb complex. These findings suggest that TB infection and/or BCG vaccination may be a risk factor for HIV acquisition.</p> </div

VEGF expression (seen in green) in guinea pig retinal pigmented epithelium (RPE) and photoreceptor outer segments as detected by immunohistochemistry (40×).

<p>Sections are from <i>Mtb</i>-infected eyes on Day 28 (A), Day 56 (B), and Day 84 (C) after aerosol infection and from uninfected control eyes (D). Photoreceptor outer segments (arrows) in panels B and C stain for VEGF; however, the photoreceptor layer in control eyes (D) and on Day 28 does not demonstrate VEGF expression. Retinal pigment epithelium (arrowhead) stains for VEGF at all time points.</p

<i>M. tuberculosis</i> complex increases susceptibility to HIV infectivity.

<p>The percentage of unstimulated CD4+ cells and CD4+ cells stimulated with PHA (50 µg/ml), <i>M. bovis</i> BCG (Copehnhagen), <i>M. tuberculosis</i> (CDC1551), <i>M. smegmatis</i> (MC²155) from individual subjects infected with X4-tropic pseudovirus (A) or R5-tropic pseudovirus (B). Results from individual donors are color-coded. Statistical analysis was performed using student T test (** p<0.005). Flow cytometry analysis of the representative CD4+ cells after infection with GFP-expressing pseudovirus (C).</p

The lungs of <i>Mtb</i>-infected guinea pigs show evidence of tissue hypoxia and VEGF expression.

<p>A. Hematoxylin-eosin stain of guinea pig lungs on Day 56 after aerosol infection revealing well-circumscribed granuloma primarily comprising lymphocytes and epithelioid histiocytes with central necrosis (2×), containing acid-fast bacilli (inset; 60×). B. VEGF staining within TB granulomas in the lungs of guinea pigs infected with <i>Mtb</i> CDC1551 on Day 56 post-infection (40×).</p