Structural and biochemical characterization on the cognate and heterologous interactions of the MazEF-mt9 TA system

ABSTRACTThe toxin-antitoxin (TA) modules widely exist in bacteria, and their activities are associated with the persister phenotype of the pathogen Mycobacterium tuberculosis (M. tb). M. tb causes Tuberculosis, a contagious and severe airborne disease. There are ten MazEF TA systems in M. tb, which play important roles in stress adaptation. How the antitoxins antagonize toxins in M. tb or how the ten TA systems crosstalk to each other are of interests, but the detailed molecular mechanisms are largely unclear. MazEF-mt9 is a unique member among the MazEF families due to its tRNase activity, which is usually carried out by the VapC family toxins. Here we present the cocrystal structure of the MazEF-mt9 complex at 2.7 Å. By characterizing the association mode between the TA pairs through various characterization techniques, we found that MazF-mt9 not only bound its cognate antitoxin, but also the non-cognate antitoxin MazE-mt1, a phenomenon that could be also observed in vivo. Based on our structural and biochemical work, we proposed that the cognate and heterologous interactions among different TA systems work together to relieve MazF-mt9’s toxicity to M. tb cells, which may facilitate their adaptation to the stressful conditions encountered during host infection.IMPORTANCETuberculosis (TB) is one of the most severe contagious diseases. Caused by Mycobacterium tuberculosis (M. tb), it poses a serious threat to human health. Additionally, TB is difficult to cure because of the multipledrug-resistant (MDR) and extensively drug-resistant (XDR) M. tb strains. Toxin-antitoxin (TA) systems have been discovered to widely exist in prokaryotic organisms with diverse roles, normally composed of a pair of molecules that antagonize each other. M. tb has ten MazEF systems, and some of them have been proved to be directly associated with the genesis of persisters and drug-resistance of M. tb. We here report the MazEF-mt9 complex structure, and thoroughly characterized the interactions between MazF-mt9 with MazEs within or outside the MazEF-mt9 family. Our study not only revealed the crosstalks between TA families and its significance to M. tb survival but also offers insights into potential anti-TB drug design.

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A vitamin B 12 transporter in Mycobacterium tuberculosis

Open Biology ◽

10.1098/rsob.120175 ◽

2013 ◽

Vol 3 (2) ◽

pp. 120175 ◽

Cited By ~ 49

Author(s):

Krishnamoorthy Gopinath ◽

Česlovas Venclovas ◽

Thomas R. Ioerger ◽

James C. Sacchettini ◽

John D. McKinney ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Type System ◽

Mycobacterial Infection ◽

Vitamin B ◽

Abc Protein ◽

Genome Wide ◽

Host Infection ◽

Obligate Pathogen

Vitamin B 12 -dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis , an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B 12 in vitro , it is uncertain whether the organism is able to scavenge B 12 during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B 12 transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B 12 . A small proportion of these mapped to Rv1314c , identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B 12 assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B 12 acquisition for an ATP-binding cassette (ABC)-type protein previously investigated as the mycobacterial BacA homologue. Moreover, targeted deletion of Rv1819c eliminated the ability of M. tuberculosis to transport B 12 and related corrinoids in vitro . Our results establish an alternative to the canonical BtuCD-type system for B 12 uptake in M. tuberculosis , and elucidate a role in B 12 metabolism for an ABC protein implicated in chronic mycobacterial infection.

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Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to β-Lactams

mBio ◽

10.1128/mbio.01767-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 17

Author(s):

Shichun Lun ◽

David Miranda ◽

Andre Kubler ◽

Haidan Guo ◽

Mariama C. Maiga ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Synthetic Lethality ◽

Multidrug Resistant ◽

Cell Wall Biosynthesis ◽

Drug Resistant ◽

Content Type ◽

Innate Resistance ◽

Extensively Drug Resistant

ABSTRACT Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe’s innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. IMPORTANCE The global emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains has threatened public health worldwide, yet the pipeline of new tuberculosis drugs under development remains limited. One strategy to cope with the urgent need for new antituberculosis agents is to repurpose existing, approved antibiotics. The carbapenem class of β-lactam antibiotics has been proposed as one such class of drugs. Our study identifies molecular determinants of innate resistance to β-lactam drugs in M. tuberculosis, and we demonstrate that functional loss of one of these genes enables successful treatment of M. tuberculosis with β-lactams in the mouse model.

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Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706139114 ◽

2017 ◽

Vol 114 (28) ◽

pp. 7426-7431 ◽

Cited By ~ 69

Author(s):

Nitin P. Kalia ◽

Erik J. Hasenoehrl ◽

Nurlilah B. Ab Rahman ◽

Vanessa H. Koh ◽

Michelle L. T. Ang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Development ◽

Synthetic Lethality ◽

Electron Flow ◽

Clinical Stage ◽

Atp Synthesis ◽

Drug Candidate ◽

Respiratory Oxidases ◽

Host Infection

The recent discovery of small molecules targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1:aa3 consists of a menaquinone:cytochrome c reductase (bc1) and a cytochrome aa3-type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1:aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1:aa3, as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.

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The promises and limitations of N-acetylcysteine as a potentiator of first-line and second-line tuberculosis drugs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01703-20 ◽

2021 ◽

Author(s):

Catherine Vilchèze ◽

William R. Jacobs

Keyword(s):

Mycobacterium Tuberculosis ◽

Liver Injury ◽

Multidrug Resistant ◽

Human Macrophage ◽

Second Line ◽

Macrophage Cell ◽

First Line ◽

Host Infection

N-acetylcysteine (NAC) is most commonly used for the treatment of acetaminophen overdose and acetaminophen-induced liver injury. In patients infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), NAC is given to treat hepatotoxicity induced by TB drugs. We had previously shown that cysteine, a derivative of NAC, potentiated the activity of isoniazid, a first-line TB drug, by preventing the emergence of INH resistance and persistence in M. tuberculosis in vitro. Herein, we demonstrate that in vitro, NAC has the same boosting activity with various combinations of first- and second-line TB drugs against drug-susceptible and multidrug-resistant M. tuberculosis strains. Similar to cysteine, NAC increased M. tuberculosis respiration. However, in M. tuberculosis-infected mice, the addition of NAC did not augment the activity of first- or second-line TB drugs. A comparison of the activity of NAC combined with TB drugs in murine and human macrophage cell lines revealed that studies in mice might not be recapitulated during host infection in vivo.

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Evaluation of Carbapenems for Treatment of Multi- and Extensively Drug-Resistant Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01489-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01489-18 ◽

Cited By ~ 4

Author(s):

Sander P. van Rijn ◽

Marlanka A. Zuur ◽

Richard Anthony ◽

Bob Wilffert ◽

Richard van Altena ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

International Travel ◽

Future Research ◽

Drug Resistant ◽

Content Type ◽

Resistant Tuberculosis ◽

Extensively Drug Resistant ◽

Susceptibility Tests

ABSTRACT Multi- and extensively drug-resistant tuberculosis (M/XDR-TB) has become an increasing threat not only in countries where the TB burden is high but also in affluent regions, due to increased international travel and globalization. Carbapenems are earmarked as potentially active drugs for the treatment of Mycobacterium tuberculosis. To better understand the potential of carbapenems for the treatment of M/XDR-TB, the aim of this review was to evaluate the literature on currently available in vitro, in vivo, and clinical data on carbapenems in the treatment of M. tuberculosis and to detect knowledge gaps, in order to target future research. In February 2018, a systematic literature search of PubMed and Web of Science was performed. Overall, the results of the studies identified in this review, which used a variety of carbapenem susceptibility tests on clinical and laboratory strains of M. tuberculosis, are consistent. In vitro, the activity of carbapenems against M. tuberculosis is increased when used in combination with clavulanate, a BLaC inhibitor. However, clavulanate is not commercially available alone, and therefore, it is impossible in practice to prescribe carbapenems in combination with clavulanate at this time. Few in vivo studies have been performed, including one prospective, two observational, and seven retrospective clinical studies to assess the effectiveness, safety, and tolerability of three different carbapenems (imipenem, meropenem, and ertapenem). We found no clear evidence at the present time to select one particular carbapenem among the different candidate compounds to design an effective M/XDR-TB regimen. Therefore, more clinical evidence and dose optimization substantiated by hollow-fiber infection studies are needed to support repurposing carbapenems for the treatment of M/XDR-TB.

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The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

mBio ◽

10.1128/mbio.00333-17 ◽

2017 ◽

Vol 8 (2) ◽

Cited By ~ 9

Author(s):

E. F. Perkowski ◽

K. E. Zulauf ◽

D. Weerakoon ◽

J. D. Hayden ◽

T. R. Ioerger ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Host Environment ◽

Bacterial Proteins ◽

Novel Method ◽

Host Infection ◽

Membrane Cell ◽

Significant Effort

ABSTRACT Exported proteins of bacterial pathogens function both in essential physiological processes and in virulence. Past efforts to identify exported proteins were limited by the use of bacteria growing under laboratory (in vitro) conditions. Thus, exported proteins that are exported only or preferentially in the context of infection may be overlooked. To solve this problem, we developed a genome-wide method, named EXIT (exported in vivo technology), to identify proteins that are exported by bacteria during infection and applied it to Mycobacterium tuberculosis during murine infection. Our studies validate the power of EXIT to identify proteins exported during infection on an unprecedented scale (593 proteins) and to reveal in vivo induced exported proteins (i.e., proteins exported significantly more during in vivo infection than in vitro). Our EXIT data also provide an unmatched resource for mapping the topology of M. tuberculosis membrane proteins. As a new approach for identifying exported proteins, EXIT has potential applicability to other pathogens and experimental conditions. IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis. IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis.

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The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146217 ◽

1993 ◽

Vol 51 ◽

pp. 74-75

Author(s):

Jason R. Swedlow ◽

Neil Osheroff ◽

Tim Karr ◽

John W. Sedat ◽

David A. Agard

Keyword(s):

Topoisomerase Ii ◽

Biochemical Characterization ◽

Developmental Cycle ◽

Dna Topoisomerase Ii ◽

Chromosomal Distribution ◽

Dna Topoisomerase ◽

Ideal System ◽

Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

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SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

Biochemical Journal ◽

10.1042/bcj20190674 ◽

2020 ◽

Vol 477 (1) ◽

pp. 173-189 ◽

Cited By ~ 1

Author(s):

Marco Pedretti ◽

Carolina Conter ◽

Paola Dominici ◽

Alessandra Astegno

Keyword(s):

Excision Repair ◽

Complex Structure ◽

Binding Motif ◽

C Terminus ◽

Repair Protein ◽

Nucleotide Excision ◽

Ef Hand ◽

Molecular Determinants ◽

Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

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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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