Mycobacterium tuberculosis strain 18b, a useful non-virulent streptomycin dependent mutant to study latent tuberculosis as well as for in vivo and in vitro testing of anti-tuberculosis drugs

Tuberculosis ◽  
2016 ◽  
Vol 99 ◽  
pp. 54-55 ◽  
Author(s):  
Antonio Campos-Neto
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Latent Tuberculosis ◽  
In Vitro Testing ◽  
Tuberculosis Strain ◽  
Mycobacterium Tuberculosis Strain

scholarly journals Ekspresi Protein Resuscitation promoting factor-D Mycobacterium tuberculosis pada sel CHO-K1 secara in-vitro

2020 ◽  
Vol 9 (1) ◽  
pp. 1-7
Author(s):  
Wahyu Agung Pratama ◽  
Andriansjah Rukmana ◽  
Fithriyah Sjatha
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mammalian Cells ◽  
Expression System ◽  
Latent Tuberculosis ◽  
Beijing Strain ◽  
Active Infection ◽  
Tuberculosis Strain ◽  
Humoral Immune ◽  
Mycobacterium Tuberculosis Strain

Abstrak Protein Resuscitation promoting factor–D (Rpf-D) merupakan protein transmembran dari Mycobacterium tuberculosis dan dieskpresikan pada fase reaktivasi infeksi laten menjadi aktif. Protein Rpf-D tersusun atas 154 asam amino berukuran 16 kDa yang dikode oleh 456 basa dari famili gen-Rpf. Pada studi sebelumnya, kami telah berhasil mengklona gen rpfD dari Mycobacterium tuberculosis strain Beijing kedalam plasmid pcDNA3.1, dikenal sebagai pcDNA3.1-rpfD. Dalam studi ini, kami mentransfeksi sel mamalia CHO-K1 dengan plasmid rekombinan pcDNA3.1-rpfD dan dilanjutkan dengan pewarnaan imunologis menggunakan serum mencit yang telah diimunisasi dengan pcDNA3.1-rpfD sebagai vaksin DNA. Hasil pewarnaan menunjukkan bahwa protein RpfD mampu diekspresikan pada sel mamalia. Selain itu, protein rekombinan yang diekspresikan ini terbukti bersifat imunogenik dan mampu menginduksi respon imun humoral pada hewan uji mencit Balb/C berupa IgG1, IgG2a, IgG2b dan IgG3. Hasil penelitian ini menunjukan potensi pemanfaatan protein rekombinan RpfD untuk penelitian lanjutan pengembangan vaksin ataupun uji diagnosis tuberkulosis. Kata kunci: Resuscitation promoting factor-D, Mycobacterium tuberculosis, sel CHO-K1, transfeksi, immunostaining Abstract Resuscitation promoting factor-D (Rpf-D) protein is known as a component of Mycobacterium tuberculosis cell wall which highly expressed in reactivation state of latent tuberculosis to turn in to an active infection. Rpf-D protein is a small protein consist of 154 amino acids encoded by 465 nucleotides in Rpf-gene family and having 16 kDa in size. In our previous study, we had successfully cloned Rpf-D gene of Mycobacterium tuberculosis Beijing strain into pcDNA3.1 plasmid expression system, which named as pcDNA3.1-rpfD later on. In this study, we transfect pcDNA3.1-rpfD into CHO-K1 mammalian cell line followed by immunostaining using mice sera immunized with pcDNA3.1-rpfD as DNA vaccine. Our result shows that our recombinant pcDNA3.1-rpfD construct can express recombinant RpfD protein in mammalian cells. On the other hand, this expressed recombinant protein has been proven to be immunogenic and able to induce a humoral immune response in Balb/C mice especially IgG1, IgG2a, IgG2b, and IgG3. Our study has shown the potency of recombinant RpfD protein which can be further developed as vaccine or diagnostic approach for tuberculosis. Keywords: Resuscitation promoting factor-D, Mycobacterium tuberculosis, CHO-K1 cells, transfection, immunostaining

scholarly journals In VivoIS6110Profile Changes in a Mycobacterium tuberculosis Strain as Determined by Tracking over 14 Years

2015 ◽  
Vol 53 (7) ◽  
pp. 2359-2361 ◽  
Author(s):  
María Isabel Millán-Lou ◽  
Isabel Otal ◽  
María Luisa Monforte ◽  
María Asunción Vitoria ◽  
María José Revillo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Homologous Recombination ◽  
Origin Of Replication ◽  
Content Type ◽  
Tuberculosis Strain ◽  
Mycobacterium Tuberculosis Strain

Transposition and homologous recombination of IS6110appear inMycobacterium tuberculosisalongin vivosequential infections. These events were checked in different clones of a successful strain,M. tuberculosisZaragoza, with the focus on a variant in which integration of a copy of IS6110in the origin of replication (oriC) region occurred.

IN VITRO MODELS OF MYCOBACTERIUM TUBERCULOSIS DORMANCY, AND IN VIVO MODELS OF LATENT TUBERCULOSIS INFECTION

2019 ◽  
Vol 64 (5) ◽  
pp. 299-307
Author(s):  
M. V. Fursov ◽  
I. A. Dyatlov ◽  
V. D. Potapov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Latent Tuberculosis Infection ◽  
Latent Tuberculosis ◽  
In Vitro Models ◽  
Tuberculosis Infection ◽  
Published Data ◽  
In Vivo Models ◽  
Dormant State

Modeling of tuberculosis infection is carried out in order to clarify various aspects of the tuberculosis pathogenesis, as well as the testing of new anti-tuberculosis drugs. The characteristic of in vitro models (n = 16) for Mycobacterium tuberculosis dormant state and in vivo models (n = 14) for the latent tuberculosis infection involving several animal species published to date are presented in this review. A brief description of the models and the results obtained by the authors are presented. The analysis of the published data reflects the list of methodological procedures that allow researchers to study the mechanism of the transition of M. tuberculosis cells to a dormant state and reverse to metabolically active state, as well as the process of conversion of active tuberculosis infection to a latent tuberculosis and reactivation.

scholarly journals Inhibition of Respiration by Nitric Oxide Induces a Mycobacterium tuberculosis Dormancy Program

2003 ◽  
Vol 198 (5) ◽  
pp. 705-713 ◽  
Author(s):  
Martin I. Voskuil ◽  
Dirk Schnappinger ◽  
Kevin C. Visconti ◽  
Maria I. Harrell ◽  
Gregory M. Dolganov ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mycobacterium Tuberculosis ◽  
Latent Tuberculosis ◽  
No Synthase ◽  
No Production ◽  
Respiratory Pathway ◽  
Latently Infected ◽  
Synthase Inhibitor

An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis.

scholarly journals Streptomycin-Starved Mycobacterium tuberculosis 18b, a Drug Discovery Tool for Latent Tuberculosis

2012 ◽  
Vol 56 (11) ◽  
pp. 5782-5789 ◽  
Author(s):  
Ming Zhang ◽  
Claudia Sala ◽  
Ruben C. Hartkoorn ◽  
Neeraj Dhar ◽  
Alfonso Mendoza-Losana ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Testing ◽  
Latent Tuberculosis ◽  
Microplate Assay ◽  
Experimental Tuberculosis ◽  
Systematic Analysis ◽  
Content Type

ABSTRACTMycobacterium tuberculosis18b, a streptomycin (STR)-dependent mutant that enters a viable but nonreplicating state in the absence of STR, has been developed as a simple model for drug testing against dormant bacilli. Here, we further evaluated the STR-starved 18b (SS18b) model bothin vitroandin vivoby comparing the behavior of 22 approved and experimental tuberculosis drugs. Using the resazurin reduction microplate assay (REMA), rifampin (RIF), rifapentine (RPT), TMC207, clofazimine (CFM), and linezolid (LIN) were found to be active against SS18bin vitro, and their bactericidal activity was confirmed by determining the number of CFU. A latent 18b infection was established in mice, and some of the above-mentioned drugs were used for treatment, either alone or in combination with RIF. RIF, RPT, TMC207, CFM, and pyrazinamide (PZA) were all activein vivo, while cell wall inhibitors were not. A comparative kinetic study of rifamycin efficacy was then undertaken, and the results indicated that RPT clears latent 18b infection in mice faster than RIF. Intrigued by the opposing responses of live and dormant 18b cells to cell wall inhibitors, we conducted a systematic analysis of 14 such inhibitors using REMA. This uncovered an SS18b signature (CWPRED) that accurately predicted the activities of cell wall inhibitors and performed well in a blind study. CWPRED will be useful for establishing the mode of action of compounds with unknown targets, while the SS18b system should facilitate the discovery of drugs for treating latent tuberculosis.

scholarly journals One-pot Synthesis of Hollongdione from Dipterocarpol

2014 ◽  
Vol 9 (10) ◽  
pp. 1934578X1400901 ◽  
Author(s):  
Irina E. Smirnova ◽  
Oxana B. Kazakova ◽  
Do Thi Thu Huong ◽  
El'za M. Minnibaeva ◽  
Alexandr N. Lobov ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
One Pot ◽  
Steroid Molecule ◽  
Tuberculosis Strain ◽  
X Ray ◽  
One Pot Synthesis ◽  
First Time ◽  
Strain H37rv ◽  
Mycobacterium Tuberculosis Strain

A one-pot synthesis of a hybrid triterpenoid-steroid molecule, hollongdione (22,23,24,25,26,27-hexanordammar-3,20-dion), was achieved in a yield of 89%, based on the selective dehydration of dipterocarpol following ozonolysis. The structure of hollongdione was confirmed by X-ray analysis for the first time. Dammar-20(22),24(25)-dien inhibited the growth of Mycobacterium tuberculosis (strain H37Rv) in vitro with a MIC of 50 μg/mL.

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.

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

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.

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