The temporal response of the Mycobacterium tuberculosis gene regulatory network during growth arrest

The virulence of Mycobacterium tuberculosis depends on the ability of the bacilli to switch between replicative (growth) and non-replicative (dormancy) states in response to host immunity. However, the gene regulatory events associated with transition to dormancy are largely unknown. To address this question, we have assembled the largest M. tuberculosis transcriptional-regulatory network to date, and characterized the temporal response of this network during adaptation to stationary phase and hypoxia, using published microarray data. Distinct sets of transcriptional subnetworks (origons) were responsive at various stages of adaptation, showing a gradual progression of network response under both conditions. Most of the responsive origons were in common between the two conditions and may help define a general transcriptional signature of M. tuberculosis growth arrest. These results open the door for a systems-level understanding of transition to non-replicative persistence, a phenotypic state that prevents sterilization of infection by the host immune response and promotes the establishment of latent M. tuberculosis infection, a condition found in two billion people worldwide

Down-Regulation of EBV-LMP1 Radio-Sensitizes Nasal Pharyngeal Carcinoma Cells via NF-κB Regulated ATM Expression

BACKGROUND:The latent membrane protein 1 (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. In previous studies we experimentally demonstrated that down-regulation of LMP1 expression by DNAzymes could increase radiosensitivity both in cells and in a xenograft NPC model in mice. RESULTS:In this study we explored the molecular mechanisms underlying the radiosensitization caused by the down-regulation of LMP1 in nasopharyngeal carcinoma. It was confirmed that LMP1 could up-regulate ATM expression in NPCs. Bioinformatic analysis of the ATM ptomoter region revealed three tentative binding sites for NF-κB. By using a specific inhibitor of NF-κB signaling and the dominant negative mutant of IkappaB, it was shown that the ATM expression in CNE1-LMP1 cells could be efficiently suppressed. Inhibition of LMP1 expression by the DNAzyme led to attenuation of the NF-κB DNA binding activity. We further showed that the silence of ATM expression by ATM-targeted siRNA could enhance the radiosensitivity in LMP1 positive NPC cells. CONCLUSIONS:Together, our results indicate that ATM expression can be regulated by LMP1 via the NF-κB pathways through direct promoter binding, which resulted in the change of radiosensitivity in NPCs

Role of Alanine Dehydrogenase of <i>Mycobacterium tuberculosis</i> during Recovery from Hypoxic Nonreplicating Persistence

<div><p><i>Mycobacterium tuberculosis</i> can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of <i>M</i>. <i>tuberculosis</i> through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of <i>ald</i> was also induced <i>in vitro</i> by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels <i>in vivo</i> during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An <i>ald</i> knockout mutant of <i>M</i>. <i>tuberculosis</i> showed no reduction in anaerobic survival <i>in vitro</i>, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the <i>ald</i> mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation.</p></div

Transcriptional analysis of <i>ald in vivo</i>, and in response to stress conditions <i>in vitro</i>.

<p>(A) Expression of <i>ald</i> in the lungs of mice. Lungs were harvested from mice at the indicated time points post-infection. Total RNA was extracted and quantitation of bacterial transcripts was performed. Shown is the mean (± standard deviation) of normalized mRNA copy numbers relative to 16S rRNA from three mice at each time point. (B) Expression after exposure to the NO donor DETA/NO. (C) Expression of <i>ald</i> during extended incubation in the Wayne model. Circles indicate expression of <i>ald</i> relative to the 16S RNA, and squares indicate the optical density of the culture at 580 nm.</p

Enzyme Activities in <i>M</i>. <i>tuberculosis</i> H37Rv and Erdman.

<p>A: Glyoxylate reductive aminase activity. B: pyruvate reductive aminase activity. C:isocitrate lyase activity. D: malate synthase activity. Black bars are aerobic cultures, blue bars are NRP-1 and green bars are NRP-2. All units are μmoles/min/mg protein. The standard deviation is shown. Asterisks indicate statistical significance (p<0.05) of the comparison with the aerobic samples.</p

Expression of genes involved in the glyoxylate cycle.

<p>Total RNA from <i>M</i>. <i>tuberculosis</i> cultures in either aerobic; microaerobic NRP-1 or anaerobic NRP-2 was extracted and quantitation of transcripts performed. (A) H37Rv. (B) Erdman. Black bars–aerobic cultures. Blue bars–NRP-1, and green bars–NRP-2. RNA levels are expressed relative to the stable 16S rRNA. The standard deviation is shown. Asterisks indicate statistical significance (p<0.05) of the comparison with the aerobic samples.</p

Physiological parameters of <i>M</i>. <i>tuberculosis</i> during reaeration.

<p>(A) Metabolic activity after extended hypoxia. Reducing activity was determined with XTT. Data are shown in arbitrary units. (B) The ratio of NADH to NAD. (C) Oxygen consumption was measured by methylene blue decolorization. (D) ATP levels in arbitrary units normalized to cell number. Black circles–WT, green squares–RVW7, red triangles–RVW7 pAld-Gen21. Asterisks indicate statistical significance (p<0.05) of the comparison of RVW7 with WT.</p

Interaction of Ald with two metabolic pathways.

<p>Even-chain fatty acids are degraded to acetyl-CoA while odd-chain fatty acids also produce propionyl-CoA. Acetyl-CoA is oxidized by the (A) glyoxylate cycle and propionyl-CoA by the (B) methylcitrate cycle. Enzyme activities are indicated in italics. ACN–aconitase; ICL–isocitrate lyase; MLS–malate synthase; MD–malate dehydrogenase; CIT–citrate synthase; SDH–succinate dehydrogenase; FUM–fumarase; MCD–methylcitrate dehydratase; MCL methylisocitrate lyase; MCS–methylcitrate synthase; GxRA–glycine dehydrogenase; PvRA–alanine dehydrogenase. ICL and MCL activities are catalyzed by the same isocitrate lyase enzyme. GxRA and PvRA activities are produced by the same alanine dehydrogenase.</p