A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems

Toxin-antitoxin systems are widespread stress-responsive elements, many of whose functions remain largely unknown. Here, we characterize the four DUF1814-family nucleotidyltransferase-like toxins (MenT1–4) encoded by the human pathogen Mycobacterium tuberculosis. Toxin MenT3 inhibited growth of M. tuberculosis when not antagonized by its cognate antitoxin, MenA3. We solved the structures of toxins MenT3 and MenT4 to 1.6 and 1.2 Å resolution, respectively, and identified the biochemical activity and target of MenT3. MenT3 blocked in vitro protein expression and prevented tRNA charging in vivo. MenT3 added pyrimidines (C or U) to the 3′-CCA acceptor stems of uncharged tRNAs and exhibited strong substrate specificity in vitro, preferentially targeting tRNASer from among the 45 M. tuberculosis tRNAs. Our study identifies a previously unknown mechanism that expands the range of enzymatic activities used by bacterial toxins, uncovering a new way to block protein synthesis and potentially treat tuberculosis and other infections.

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The Copper-Responsive RicR Regulon Contributes to Mycobacterium tuberculosis Virulence

mBio ◽

10.1128/mbio.00876-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 34

Author(s):

Xiaoshan Shi ◽

Richard A. Festa ◽

Thomas R. Ioerger ◽

Susan Butler-Wu ◽

James C. Sacchettini ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Microbial Growth ◽

Human Pathogen ◽

Content Type ◽

Cu Toxicity ◽

Cu Homeostasis ◽

Cu Resistance ◽

Life On Earth

ABSTRACTAs with most life on Earth, the transition metal copper (Cu) is essential for the viability of the human pathogenMycobacterium tuberculosis. However, infected hosts can also use Cu to control microbial growth. Several Cu-responsive pathways are present inM. tuberculosis, including the regulated in copper repressor (RicR) regulon, which is unique to pathogenic mycobacteria. In this work, we describe the contribution of each RicR-regulated gene to Cu resistancein vitroand to virulence in animals. We found that the deletion or disruption of individual RicR-regulated genes had no impact on virulence in mice, although several mutants had Cu hypersensitivity. In contrast, a mutant unable to activate the RicR regulon was not only highly susceptible to Cu but also attenuated in mice. Thus, these data suggest that several genes of the RicR regulon are required simultaneously to combat Cu toxicityin vivoor that this regulon is also important for resistance against Cu-independent mechanisms of host defense.IMPORTANCEMycobacterium tuberculosisis the causative agent of tuberculosis, killing millions of people every year. Therefore, understanding the biology ofM. tuberculosisis crucial for the development of new therapies to treat this devastating disease. Our studies reveal that although host-supplied Cu can suppress bacterial growth,M. tuberculosishas a unique pathway, the RicR regulon, to defend against Cu toxicity. These findings suggest that Cu homeostasis pathways in both the host and the pathogen could be exploited for the treatment of tuberculosis.

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Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex

Nature Communications ◽

10.1038/s41467-021-27787-3 ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Mikhail Kavalchuk ◽

Ahmad Jomaa ◽

Andreas U. Müller ◽

Eilika Weber-Ban

Keyword(s):

Electron Microscopy ◽

Mycobacterium Tuberculosis ◽

Early Stage ◽

Structural Basis ◽

Human Pathogen ◽

Core Particle ◽

Cryo Electron Microscopy ◽

Complementary Interactions

AbstractProteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.

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Anionic Complex with Efficient Expression and Good Safety Profile for mRNA Delivery

Pharmaceutics ◽

10.3390/pharmaceutics13010126 ◽

2021 ◽

Vol 13 (1) ◽

pp. 126

Author(s):

Eri Hamada ◽

Tomoaki Kurosaki ◽

Junya Hashizume ◽

Hitomi Harasawa ◽

Hiroo Nakagawa ◽

...

Keyword(s):

Protein Expression ◽

Messenger Rna ◽

Charge Ratio ◽

Attractive Alternative ◽

Target Cells ◽

Anionic Complex ◽

In Vitro Protein Expression ◽

Vitro Protein

We previously found that a complex comprising plasmid DNA (pDNA), polyethylenimine (PEI), and γ-polyglutamic acid (γ-PGA) had high transgene efficiency without cytotoxicity in vitro and in vivo. However, messenger RNA (mRNA) remains an attractive alternative to pDNA. In this study, we developed a safe and effective delivery system for mRNA to prevent its degradation and efficiently deliver it into target cells. Various cationic and anionic complexes were produced containing PEI, γ-PGA, and an mRNA encoding firefly luciferase. Their physicochemical properties and cytotoxicities were analyzed and the in vitro and in vivo protein expression were determined. The cationic mRNA/PEI complex showed high in vitro protein expression with strong cytotoxicity. The anionic complex was constructed as mRNA/PEI8/γ-PGA12 complex with a theoretical charge ratio of 1:8:12 based on the phosphate groups of the mRNA, nitrogen groups of PEI, and carboxylate groups of γ-PGA. It was stable and showed high in vitro protein expression without cytotoxicity. After intravenous administration of mRNA/PEI8/γ-PGA12 complex to mice, high protein expression was observed in the spleen and liver and slight expression was observed in the lung over 24 h. Thus, the newly constructed mRNA/PEI8/γ-PGA12 complex provides a safe and effective strategy for the delivery of mRNA.

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Tuberkulose - Screening

Therapeutische Umschau ◽

10.1024/0040-5930/a000181 ◽

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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Applicability of Instability Index for In vitro Protein Stability Prediction

Protein and Peptide Letters ◽

10.2174/0929866526666190228144219 ◽

2019 ◽

Vol 26 (5) ◽

pp. 339-347 ◽

Cited By ~ 4

Author(s):

Dilani G. Gamage ◽

Ajith Gunaratne ◽

Gopal R. Periyannan ◽

Timothy G. Russell

Keyword(s):

Protein Stability ◽

Caulobacter Crescentus ◽

Effect Of Temperature ◽

Instability Index ◽

Dipeptide Composition ◽

Experimental Conditions ◽

The Stability ◽

Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

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Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

Current Molecular Pharmacology ◽

10.2174/1874467211666181025141114 ◽

2019 ◽

Vol 12 (1) ◽

pp. 27-49 ◽

Cited By ~ 6

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.

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Intracellular localisation of Mycobacterium tuberculosis affects efficacy of the antibiotic pyrazinamide

Nature Communications ◽

10.1038/s41467-021-24127-3 ◽

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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Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis

Nature Communications ◽

10.1038/s41467-021-21748-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Poushali Chakraborty ◽

Sapna Bajeli ◽

Deepak Kaushal ◽

Bishan Dass Radotra ◽

Ashwani Kumar

Keyword(s):

Mycobacterium Tuberculosis ◽

Immune System ◽

Biofilm Formation ◽

Antimycobacterial Activity ◽

Drug Tolerance ◽

Host Immune System ◽

Tissue Sections ◽

Slow Growing

AbstractTuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

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In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins

PLoS ONE ◽

10.1371/journal.pone.0005185 ◽

2009 ◽

Vol 4 (4) ◽

pp. e5185 ◽

Cited By ~ 61

Author(s):

A. Sesilja Aranko ◽

Sara Züger ◽

Edith Buchinger ◽

Hideo Iwaï

Keyword(s):

Protein Ligation ◽

Naturally Occurring ◽

Vitro Protein

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Use of an in Vitro Protein Synthesizing System to Test the Mode of Action of Chloracetamides

Weed Science ◽

10.1017/s0043174500055417 ◽

1980 ◽

Vol 28 (3) ◽

pp. 334-340 ◽

Cited By ~ 26

Author(s):

Luanne M. Deal ◽

J. T. Reeves ◽

B. A. Larkins ◽

F. D. Hess

Keyword(s):

Protein Synthesis ◽

Sand Culture ◽

Leucine Incorporation ◽

Insoluble Protein ◽

In Vitro System ◽

A Cell ◽

Protein Incorporation ◽

Vitro Protein

The effects of chloracetamides on protein synthesis were studied both in vivo and in vitro. Four chloracetamide herbicides, alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], CDAA (N–N-diallyl-2-chloroacetamide), and propachlor (2-chloro-N-isopropylacetanilide) were tested for inhibition of [3H]-leucine incorporation into protein. Incorporation of3H-leucine into trichloroacetic acid (TCA)-insoluble protein was inhibited in oat (Avena sativaL. ‘Victory’) seedlings grown in sand culture and treated 12 h at 1 × 10−4M with these chloracetamides. The herbicides were also tested in a cell-free protein synthesizing system containing polyribosomes purified from oat root cytoplasm. These herbicides had no effect on the rates of polypeptide elongation nor on the synthesis of specific polypeptides when herbicides (1 × 10−4M) were added directly to the system. Polypeptide formation was inhibited 89% when 1 × 10−4M cycloheximide was added during translation. Cytoplasmic polyribosomes were isolated from oat roots treated 12 h with 1 × 10−4M herbicide. Translation rates and products were not altered when these polyribosomes were added to the in vitro system. Protein synthesis is inhibited when tested in an in vivo system; however, the inhibition does not occur during the translation of mRNA into protein.

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