Ectopic expression of DosR induces the DosR regulon.

<p>Scatterplot displaying transcript levels of all <i>M. tuberculosis</i> genes after 24 hours of treatment with either 10 ng/mL Atc (induced) or an equivalent volume of sterile DMSO (uninduced). Three biological replicates were RMA-normalized and the median pixel intensity data are plotted on a log₂ scale. Genes of the DosR regulon are represented as dark gray circles. Significantly induced genes (moderated t-test with Benjamini-Hochberg FDR correction, p<0.05) not part of the DosR regulon are presented as black diamonds, and the <i>dosR</i> transcript is indicated with a star.</p

Integrated Modeling of Gene Regulatory and Metabolic Networks in <i>Mycobacterium tuberculosis</i>

<div><p><i>Mycobacterium tuberculosis</i> (MTB) is the causative bacterium of tuberculosis, a disease responsible for over a million deaths worldwide annually with a growing number of strains resistant to antibiotics. The development of better therapeutics would greatly benefit from improved understanding of the mechanisms associated with MTB responses to different genetic and environmental perturbations. Therefore, we expanded a genome-scale regulatory-metabolic model for MTB using the Probabilistic Regulation of Metabolism (PROM) framework. Our model, <i>MTB</i>PROM2.0, represents a substantial knowledge base update and extension of simulation capability. We incorporated a recent ChIP-seq based binding network of 2555 interactions linking to 104 transcription factors (TFs) (representing a 3.5-fold expansion of TF coverage). We integrated this expanded regulatory network with a refined genome-scale metabolic model that can correctly predict growth viability over 69 source metabolite conditions and predict metabolic gene essentiality more accurately than the original model. We used <i>MTB</i>PROM2.0 to simulate the metabolic consequences of knocking out and overexpressing each of the 104 TFs in the model. <i>MTB</i>PROM2.0 improves performance of knockout growth defect predictions compared to the original PROM MTB model, and it can successfully predict growth defects associated with TF overexpression. Moreover, condition-specific models of <i>MTB</i>PROM2.0 successfully predicted synergistic growth consequences of overexpressing the TF <i>whiB4</i> in the presence of two standard anti-TB drugs. <i>MTB</i>PROM2.0 can screen <i>in silico</i> condition-specific transcription factor perturbations to generate putative targets of interest that can help prioritize future experiments for therapeutic development efforts.</p></div

Nonpathogenic SIV and Pathogenic HIV Infections Associate with Disparate Innate Cytokine Signatures in Response to <i>Mycobacterium bovis</i> BCG

<div><p>Infections with mycobacteria, including <i>Mycobacterium tuberculosis</i> (Mtb) and <i>Mycobacterium bovis (M</i>. <i>bovis)</i> BCG, are a leading cause of morbidity and mortality for HIV-infected persons. In contrast to HIV, nonpathogenic SIV infections of sooty mangabeys are characterized by a lack of clinical disease including an absence of opportunistic infections. The goal of this study was to identify innate immune responses to <i>M</i>. <i>bovis</i> BCG maintained during nonpathogenic lentiviral infections through a comparison of functional responses during pathogenic HIV or nonpathogenic SIV infections. Monocytes were evaluated for their ability to express key anti-mycobacterial cytokines TNF-α and IL-12 following a six-hour <i>ex vivo</i> BCG exposure. While HIV-infection was associated with a decreased percentage of IL-12-producing monocytes, nonpathogenic SIV-infection was associated with an increased percentage of monocytes producing both cytokines. Gene expression analysis of PBMC following <i>ex vivo</i> BCG exposure identified differential expression of NK cell-related genes and several cytokines, including IFN-γ and IL-23, between HIV-infected and control subjects. In contrast, SIV-infected and uninfected-control mangabeys exhibited no significant differences in gene expression after BCG exposure. Finally, differential gene expression patterns were identified between species, with mangabeys exhibiting lower IL-6 and higher IL-17 in response to BCG when compared to humans. Overall, this comparison of immune responses to <i>M</i>. <i>bovis</i> BCG identified unique immune signatures (involving cytokines IL-12, TNF-α, IL-23, IL-17, and IL-6) that are altered during HIV, but maintained or increased during nonpathogenic SIV infections. These unique cytokine and transcriptome signatures provide insight into the differential immune responses to Mycobacteria during pathogenic HIV-infection that may be associated with an increased incidence of mycobacterial co-infections.</p></div

Whole blood monocyte proinflammatory cytokine production in HIVneg and HIV+ donors or SIVneg and SIV+ mangabeys following exposure to <i>M</i>. <i>bovis</i> BCG.

<p>(A) Representative flow cytometry plots and gating strategy of cytokine-producing monocytes, which were first defined as being live and CD3 negative (not shown), CD14+, and producing TNF-α, IL-12, or both following 6h exposure to BCG. (B-G) Percentage of TNF-α+ (B, E), IL-12+ (C, F), or double-positive (TNF-α+ and IL-12+) monocytes (D, G) in HIVneg (unfilled circles) and ART-naive HIV+ (filled circles) humans or SIVneg (unfilled squares) and SIV+ (filled squares) mangabeys following ex vivo BCG exposure. Lines represent the medians for each group (*p<0.05, Mann-Whitney).</p

TF overexpression growth defect prediction performance.

<p>Performance of <i>MTB</i>PROM2.0 at predicting TF overexpression growth defects compared to two alternative methods: (1) iMAT and (2) whether the overexpressing TF repressed any essential metabolic genes. Performance is quantified by the MCC.</p

Model of differential cellular responses during the innate immune response to BCG during pathogenic HIV or non-pathogenic SIV infection.

<p>Cytokines that are differentially expressed between HIVneg and HIV+ humans (A) or SIVneg and SIV+ mangabeys (B) are presented without boxes and are in bold type font. Arrow(s) represent the up or down modulation of these immune modulators in response to BCG, and fold change (FC) is presented as numeric values. Cytokines expressed differently between human subjects and mangabeys are boxed (B).</p

Genes associated with HIV-status-specific effects following BCG exposure ex vivo.

<p>Genes associated with HIV-status-specific effects following BCG exposure ex vivo.</p

Whole PBMC cytokine gene regulation in HIVneg or HIV+ donors following BCG exposure. Plots demonstrate regulation of key proinflammatory.

<p>(A), Th1 (B), Th17 (C) and immunoregulatory genes (D) following 4h BCG exposure. Log2 copy number of RNA in an unstimulated control (U) or matched BCG-stimulated (B) samples in either humans (circles) or mangabeys (squares). The lower bound of the gray shaded portion of the graph represents the mean of the negative controls, and the upper bound represents two standard deviations above the mean, which ultimately determined the cutoff for expression. The most highly up- and down-regulated genes in humans or mangabeys are displayed via a heat map (E). FC: fold change; BD: below detection.</p

<i>Mycobacterium tuberculosis</i> Ser/Thr Protein Kinase B Mediates an Oxygen-Dependent Replication Switch

<div><p>The majority of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) infections are clinically latent, characterized by drug tolerance and little or no bacterial replication. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Here, we tested the role of serine/threonine phosphorylation in the <i>Mtb</i> response to altered oxygen status, using an <i>in vitro</i> model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of <i>Mtb</i> in reaeration. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. <i>Mtb</i> replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability and gross morphological defects in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the <i>Mtb</i> life cycle—active disease, latency, and reactivation.</p></div

Selected genes that demonstrate HIV status-specific effects of BCG exposure.

<p>Fold change in the expression of 199 immunologic genes in response to 4h BCG exposure of PBMC from HIVneg (circles) or ART-naïve HIV+ (squares) was assessed via Nanostring gene expression analysis. Twelve genes remained significant following correction for multiple comparisons at an FDR threshold <10% (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158149#pone.0158149.t001" target="_blank">Table 1</a>). Following the identification of these genes, further validation of the differences in response between HIVneg and HIV+ subjects was identified via a paired t-test: PRF1 (HIVneg p = 0.0045, HIV+ p = n.s), and NKp46 (HIVneg p = 0.0008, HIV+ p = n.s) perform cellular effector functions, and CCR5 (HIVneg p = 0.0029, HIV+ p = n.s) is an HIV coreceptor.</p