scholarly journals Three Mycobacterium tuberculosis Rel Toxin-Antitoxin Modules Inhibit Mycobacterial Growth and Are Expressed in Infected Human Macrophages

2008 ◽  
Vol 191 (5) ◽  
pp. 1618-1630 ◽  
Author(s):  
Shaleen B. Korch ◽  
Heidi Contreras ◽  
Josephine E. Clark-Curtiss
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Protein Pair ◽  
Growth Arrest ◽  
Broth Culture ◽  
Human Macrophages ◽  
In Vivo Analysis ◽  
Mycobacterial Growth

ABSTRACT Mycobacterium tuberculosis protein pairs Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, here named RelBE, RelFG, and RelJK, respectively, were identified based on homology to the Escherichia coli RelBE toxin:antitoxin (TA) module. In this study, we have characterized each Rel protein pair and have established that they are functional TA modules. Overexpression of individual M. tuberculosis rel toxin genes relE, relG, and relK induced growth arrest in Mycobacterium smegmatis; a phenotype that was completely reversible by expression of their cognate antitoxin genes, relB, relF, and relJ, respectively. We also provide evidence that RelB and RelE interact directly, both in vitro and in vivo. Analysis of the genetic organization and regulation established that relBE, relFG, and relJK form bicistronic operons that are cotranscribed and autoregulated, in a manner unlike typical TA modules. RelB and RelF act as transcriptional activators, inducing expression of their respective promoters. However, RelBE, RelFG, and RelJK (together) repress expression to basal levels of activity, while RelJ represses promoter activity altogether. Finally, we have determined that all six rel genes are expressed in broth-grown M. tuberculosis, whereas relE, relF, and relK are expressed during infection of human macrophages. This is the first demonstration of M. tuberculosis expressing TA modules in broth culture and during infection of human macrophages.

scholarly journals Intracellular localisation of Mycobacterium tuberculosis affects efficacy of the antibiotic pyrazinamide

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pierre Santucci ◽  
Daniel J. Greenwood ◽  
Antony Fearns ◽  
Kai Chen ◽  
Haibo Jiang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Combination Chemotherapy ◽  
In Vitro Activity ◽  
In Vivo Efficacy ◽  
Human Macrophages ◽  
Ion Microscopy ◽  
Intracellular Localisation

AbstractTo be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis. However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. Here, we use correlative light, electron, and ion microscopy to investigate how various microenvironments within human macrophages affect the activity of pyrazinamide (PZA), a key antibiotic against TB. We show that PZA accumulates heterogeneously among individual bacteria in multiple host cell environments. Crucially, PZA accumulation and efficacy is maximal within acidified phagosomes. Bedaquiline, another antibiotic commonly used in combined TB therapy, enhances PZA accumulation via a host cell-mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy. Our results may explain the potent in vivo efficacy of PZA, compared to its modest in vitro activity, and its critical contribution to TB combination chemotherapy.

scholarly journals Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 290-299 ◽  
Author(s):  
Veeraraghavan Usha ◽  
Sudagar S. Gurcha ◽  
Andrew L. Lovering ◽  
Adrian J. Lloyd ◽  
Athina Papaemmanouil ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Genomic Sequence ◽  
Fold Increase ◽  
Inosine Monophosphate Dehydrogenase ◽  
Inosine Monophosphate ◽  
Genes Encoding ◽  
In Trans

In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue in M. tuberculosis and showed optimal activity at pH 8.5 and 37 °C. Mt-GuaB2 was inhibited significantly in vitro by a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2–0.5 μg ml−1. When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a diphenyl urea analogue showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologues (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compounds in vivo.

scholarly journals Somatostatin receptor 1,2 and 5 activation leads to C6 glioma growth arrest in vitro and in vivo; analysis of the intracellular pathways involved

2011 ◽  
Vol 84 (1) ◽  
Author(s):  
M. Gatti ◽  
A. Pattarozzi ◽  
R. Würth ◽  
F. Angeletti ◽  
F. Barbieri ◽  
...  
Keyword(s):  
Somatostatin Receptor ◽  
Combined Treatment ◽  
Growth Arrest ◽  
C6 Glioma ◽  
Cancer Growth ◽  
Cell Growth Arrest ◽  
Glioma Growth ◽  
In Vivo Analysis

We report that somatostain receptor (SSTR) 1,2 and 5 activation by selective agonista, cause C6 cell growth arrest through PTPn-dependent dephosphorylation of ERK1/2 in vitro and after xenografting in nude mice. Individual SSTR agonist desplayed different efficacy and potecy showing partial synergism by combined treatment. Since most tumor cells express multiple SSTRs, the activtion of all the subtypes may grant a better control of cancer growth.

scholarly journals Novel Conjugate of Moxifloxacin and Carboxymethylated Glucan with Enhanced Activity against Mycobacterium tuberculosis

2006 ◽  
Vol 50 (6) ◽  
pp. 1982-1988 ◽  
Author(s):  
Y. S. Schwartz ◽  
M. I. Dushkin ◽  
V. A. Vavilin ◽  
E. V. Melnikova ◽  
O. M. Khoschenko ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Targeted Delivery ◽  
Scavenger Receptors ◽  
Intracellular Pathogen ◽  
Macrophage Cell ◽  
Macrophage Receptors ◽  
Mycobacterial Growth ◽  
Dose Dependent

ABSTRACT Mycobacterium tuberculosis is an intracellular pathogen that persists within macrophages of the human host. One approach to improving the treatment of tuberculosis (TB) is the targeted delivery of antibiotics to macrophages using ligands to macrophage receptors. The moxifloxacin-conjugated dansylated carboxymethylglucan (M-DCMG) conjugate was prepared by chemically linking dansylcadaverine (D) and moxifloxacin (M) to carboxymethylglucan (CMG), a known ligand of macrophage scavenger receptors. The targeted delivery to macrophages and the antituberculosis activity of the conjugate M-DCMG were studied in vitro and in vivo. Using fluorescence microscopy, fluorimetry, and the J774 macrophage cell line, M-DCMG was shown to accumulate in macrophages through scavenger receptors in a dose-dependent (1 to 50 μg/ml) manner. After intravenous administration of M-DCMG into C57BL/6 mice, the fluorescent conjugate was concentrated in the macrophages of the lungs and spleen. Analyses of the pharmacokinetics of the conjugate demonstrated that M-DCMG was more rapidly accumulated and more persistent in tissues than free moxifloxacin. Importantly, therapeutic studies of mycobacterial growth in C57BL/6 mice showed that the M-DCMG conjugate was significantly more potent than free moxifloxacin.

scholarly journals Characterization of Mycobacterium smegmatis Expressing the Mycobacterium tuberculosis Fatty Acid Synthase I (fas1) Gene

2004 ◽  
Vol 186 (13) ◽  
pp. 4051-4055 ◽  
Author(s):  
Oren Zimhony ◽  
Catherine Vilchèze ◽  
William R. Jacobs
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Mycobacterium Smegmatis ◽  
Fatty Acid Synthase ◽  
Complex Interaction ◽  
Fatty Acid Elongation ◽  
Biochemical Studies

ABSTRACT Unlike most other bacteria, mycobacteria make fatty acids with the multidomain enzyme eukaryote-like fatty acid synthase I (FASI). Previous studies have demonstrated that the tuberculosis drug pyrazinamide and 5-chloro-pyrazinamide target FASI activity. Biochemical studies have revealed that in addition to C16:0, Mycobacterium tuberculosis FASI synthesizes C26:0 fatty acid, while the Mycobacterium smegmatis enzyme makes C24:0 fatty acid. In order to express M. tuberculosis FASI in a rapidly growing Mycobacterium and to characterize the M. tuberculosis FASI in vivo, we constructed an M. smegmatis Δfas1 strain which contained the M. tuberculosis fas1 homologue. The M. smegmatis Δfas1 (attB::M. tuberculosis fas1) strain grew more slowly than the parental M. smegmatis strain and was more susceptible to 5-chloro-pyrazinamide. Surprisingly, while the M. smegmatis Δfas1 (attB::M. tuberculosis fas1) strain produced C26:0, it predominantly produced C24:0. These results suggest that the fatty acid elongation that produces C24:0 or C26:0 in vivo is due to a complex interaction among FASI, FabH, and FASII and possibly other systems and is not solely due to FASI elongation, as previously suggested by in vitro studies.

scholarly journals Different Innate Ability of I/St and A/Sn Mice To Combat Virulent Mycobacterium tuberculosis: Phenotypes Expressed in Lung and Extrapulmonary Macrophages

2003 ◽  
Vol 71 (2) ◽  
pp. 697-707 ◽  
Author(s):  
Konstantin B. Majorov ◽  
Irina V. Lyadova ◽  
Tatiana K. Kondratieva ◽  
Eugeny B. Eruslanov ◽  
Elvira I. Rubakova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Inbred Strains ◽  
Peritoneal Macrophages ◽  
Lung Macrophages ◽  
Isolated Lung ◽  
Mycobacterial Antigens ◽  
Mycobacterial Growth

ABSTRACT Mice of the I/St and A/Sn inbred strains display a severe and moderate course, respectively, of disease caused by Mycobacterium tuberculosis. Earlier, we showed that the response to mycobacterial antigens in I/St mice compared to that in A/Sn mice is shifted toward Th2-like reactivity and a higher proliferative activity and turnover of T cells. However, the physiologic basis for different expressions of tuberculosis severity in these mice remains largely unknown. Here, we extend our previous observations with evidence that I/St interstitial lung macrophages are defective in the ability to inhibit mycobacterial growth and to survive following in vitro infection with M. tuberculosis H37Rv. A unique feature of this phenotype is its exclusive expression in freshly isolated lung macrophages. The defect is not displayed in ex vivo macrophages obtained from the peritoneal cavity nor in macrophages developed in vitro from progenitors extracted from various organs, including the lung itself. In addition, we show that, in sharp contrast to peritoneal macrophages, the mycobactericidal capacity of lung macrophages is not elevated in the presence of exogenous gamma interferon. Our data suggest that the in vivo differentiation in a particular anatomical microenvironment determines the pattern of macrophage-mycobacterium interaction. Thus, caution should be exercised when conclusions based upon the results obtained in a particular in vitro system are generalized to the functions of all phagocytes during M. tuberculosis infection.

scholarly journals Multi-Stress Induction of the Mycobacterium tuberculosis MbcTA Bactericidal Toxin-Antitoxin System

Toxins ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 329
Author(s):  
Kanchiyaphat Ariyachaokun ◽  
Anna D. Grabowska ◽  
Claude Gutierrez ◽  
Olivier Neyrolles
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Internal Standard ◽  
Physical Interaction ◽  
Genetic Dissection ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Promising Target

MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD+ and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (PmbcA) in a variety of stress conditions, including in vivo in mice and in macrophages.

scholarly journals Mesenchymal stem cells (MSCs) offer a drug tolerant and immune- privileged niche to Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Neharika Jain ◽  
Haroon Kalam ◽  
Lakshyaveer Singh ◽  
Vartika Sharma ◽  
Saurabh Kedia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Mycobacterium Tuberculosis ◽  
Prolonged Treatment ◽  
Phagosome Maturation ◽  
Tuberculosis Prevention ◽  
Prevention And Cure ◽  
Mycobacterial Growth

SummaryAnti-tuberculosis (TB) drugs while being highly potent in vitro require prolonged treatment to control Mycobacterium tuberculosis (Mtb) infections in vivo. We report here, mesenchymal stem cells (MSCs) shelter Mtb to help tolerate anti-TB drugs. MSCs uptake Mtb readily and allow them grow unabated despite having functional innate pathway of phagosome maturation. Unlike macrophage-resident ones, MSC-resident Mtb tolerates anti-TB drugs remarkably well, a phenomenon requiring proteins ABCC1, ABCG2 and vacuolar-type H+ATPases. Additionally, contrary to what is classically known, IFNγ and TNFα aid mycobacterial growth within MSCs. Mechanistically, evading drugs and inflammatory cytokines by MSC-resident Mtb is dependent on elevated PGE2 signaling, which we subsequently verified in vivo analyzing sorted CD45-CD73+SCA1+-MSCs from the lungs of infected mice. Moreover granulomas from human pulmonary and extra-pulmonary TB show presence of MSCs co-inhabiting with Mtb. Together the results show targeting the immune-privileged niche, provided by MSCs to Mtb, can revolutionize tuberculosis prevention and cure.

scholarly journals Mycobacterium tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, Is Negatively Regulated by Ser/Thr Phosphorylation

2013 ◽  
Vol 288 (23) ◽  
pp. 16546-16556 ◽  
Author(s):  
Jade Leiba ◽  
Karl Syson ◽  
Grégory Baronian ◽  
Isabelle Zanella-Cléon ◽  
Rainer Kalscheuer ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Streptomyces Coelicolor ◽  
Phosphorylation Site ◽  
Catalytic Efficiency ◽  
Forward Genetics ◽  
Phosphorylation Sites ◽  
Potential Therapeutic Target

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette–Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.

Tuberkulose - Screening

2011 ◽  
Vol 68 (7) ◽  
pp. 381-387
Author(s):  
Otto Schoch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Interferon Gamma ◽  
Wird Eine ◽  
Interferon Gamma Release Assays

Das primäre Ziel der Aktivitäten zur bevölkerungsbezogenen Tuberkulosekontrolle ist die Identifizierung von Patienten mit sputummikroskopisch positiver Lungentuberkulose. Wenn diese Patienten umgehend therapiert werden, haben sie nicht nur eine optimale Heilungschance, sondern übertragen auch den Krankheitserreger nicht weiter auf andere Personen. Das Screening, die systematische Suche nach Tuberkulose, erfolgt in der Regel radiologisch bei der Suche nach Erkrankten, während immunologische Teste bei der Suche nach einer Infektion mit Mycobacterium tuberculosis zur Anwendung kommen. Diese Infektion, die ein erhöhtes Risiko für die Entwicklung einer Tuberkulose-Erkrankung mit sich bringt, wird im Rahmen der Umgebungsuntersuchungen oder bei Hochrisikogruppen gesucht. Neben dem traditionellen in vivo Mantoux Hauttest stehen heute die neueren in vitro Blutteste, die sogenannten Interferon Gamma Release Assays (IGRA) zur Verfügung, die unter anderem den Vorteil einer höheren Spezifität mit sich bringen, weil die verwendeten Antigene der Mykobakterien-Wand beim Impfstamm Bacille Calmitte Guerin (BCG) und bei den meisten atypischen Mykobakterien nicht vorhanden sind. Zudem kann bei Immunsupprimierten dank einer mitgeführten Positivkontrolle eine Aussage über die Wahrscheinlichkeit eines falsch negativen Testresultates gemacht werden. Bei neu diagnostizierter Infektion mit Mycobacterium tuberculosis wird eine präventive Chemotherapie mit Isoniazid während 9 Monaten durchgeführt.

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