scholarly journals InhA inhibitors have activity against non-replicating Mycobacterium tuberculosis

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0239354
Author(s):  
Lindsay Flint ◽  
Aaron Korkegian ◽  
Tanya Parish
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Nutrient Starvation ◽  
Cell Wall Biosynthesis

We previously identified a diazaborine series with potential for development as a new tuberculosis drug. This series has activity in vitro and in vivo and targets cell wall biosynthesis via inhibition of InhA. The overall aim of this study was to determine whether InhA inhibitors have activity against non-replicating Mycobacterium tuberculosis. We tested the ability of two molecules of the diazaborine series to kill non-replicating M. tuberculosis in the nutrient starvation model; both molecules were bactericidal, reducing viability by >3 logs in 21 days. Activity showed similar kill rates to other InhA inhibitors (isoniazid and NITD-916). We conclude that inhibition of InhA is bactericidal against nutrient-starved non-replicating M. tuberculosis.

scholarly journals InhA inhibitors have activity against non-replicating Mycobacterium tuberculosis

2020 ◽  
Author(s):  
Lindsay Flint ◽  
Aaron Korkegian ◽  
Tanya Parish
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Nutrient Starvation ◽  
Cell Wall Biosynthesis

AbstractWe previously identified a diazaborine series with potential for development as a new tuberculosis drug. This series has activity in vitro and in vivo and targets cell wall biosynthesis via inhibition of InhA. We tested the ability of two molecules of the diazaborine series to kill non-replicating Mycobacterium tuberculosis in the nutrient starvation model; both molecules were bactericidal, reducing viability by >3 logs in 21 days. Activity was not inoculum-dependent and showed similar kill rates to other InhA inhibitors (isoniazid and NITD-916). We conclude that inhibition of InhA is bactericidal against nutrient-starved non-replicating M. tuberculosis.

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

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
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.

scholarly journals Inhibition of Mycobacterium tuberculosis Glutamine Synthetase as a Novel Antibiotic Strategy against Tuberculosis: Demonstration of Efficacy In Vivo

2003 ◽  
Vol 71 (1) ◽  
pp. 456-464 ◽  
Author(s):  
Günter Harth ◽  
Marcus A. Horwitz
Keyword(s):  
Weight Loss ◽  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Secretory Proteins ◽  
Cell Wall Biosynthesis ◽  
Conventional Antibiotics ◽  
Guinea Pig Model ◽  
Highly Virulent

ABSTRACT Tuberculosis remains one of humankind's greatest killers, and new therapeutic strategies are needed to combat the causative agent, Mycobacterium tuberculosis, which is rapidly developing resistance to conventional antibiotics. Using the highly demanding guinea pig model of pulmonary tuberculosis, we have investigated the feasibility of inhibiting M. tuberculosis glutamine synthetase (GS), an enzyme that plays a key role in both nitrogen metabolism and cell wall biosynthesis, as a novel antibiotic strategy. In guinea pigs challenged by aerosol with the highly virulent Erdman strain of M. tuberculosis, the GS inhibitor l-methionine-SR-sulfoximine (MSO) protected the animals against weight loss, a hallmark of tuberculosis, and against the growth of M. tuberculosis in the lungs and spleen; MSO reduced the CFU of M. tuberculosis at 10 weeks after challenge by ∼0.7 log unit compared with that in control animals. MSO acted synergistically with isoniazid in protecting animals against weight loss and bacterial growth, reducing the CFU in the lungs and spleen by ∼1.5 log units below the level seen with isoniazid alone. In the presence of ascorbate, which allows treatment with a higher dose, MSO was highly efficacious, reducing the CFU in the lungs and spleen by 2.5 log units compared with that in control animals. This study demonstrates that inhibition of M. tuberculosis GS is a feasible therapeutic strategy against this pathogen and supports the concept that M. tuberculosis enzymes involved in cell wall biosynthesis, including major secretory proteins, have potential as antibiotic targets.

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

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
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.

scholarly journals Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy

2021 ◽  
Author(s):  
Eun-Ik Koh ◽  
Nadine Ruecker ◽  
Megan K. Proulx ◽  
Vijay Soni ◽  
Kenan C. Murphy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Interactions ◽  
Carbon Metabolism ◽  
Drug Efficacy ◽  
Metabolic State ◽  
Chemical Genetic ◽  
Tuberculosis Therapy

AbstractCurrent chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug-drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilize conditional Mtb knockdown mutants of essential genes as an experimentally-tractable surrogate for drug treatment, and probe the relationship between Mtb carbon metabolism and chemical-genetic interactions (CGI). We examined the anti-tubercular drugs isoniazid, rifampicin and moxifloxacin, and found that CGI are differentially responsive to the metabolic state, defining both environment-independent and – dependent synergies. Specifically, growth on the in vivo-relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGI could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of novel chemical-genetic-environmental synergies that can be used to optimize drug-drug interactions as well as provide a framework for understanding in vitro correlates of in vivo efficacy.SignificanceEfforts to improve tuberculosis therapy include optimizing multi-drug regimens to take advantage of drug-drug synergies. However, the complex host environment has a profound effect on bacterial metabolic state and drug activity, making predictions of optimal drug combinations difficult. In this study, we leverage a newly developed library of conditional knockdown Mycobacterium tuberculosis mutants in which genetic depletion of essential genes mimics the effect of drug therapy. This tractable system allowed us to assess the effect of growth condition on predicted drug-drug interactions. We found that these interactions can be differentially sensitive to the metabolic state and select in vitro-defined synergies can be leveraged to accelerate bacterial killing during infection. These findings suggest new strategies for optimizing tuberculosis therapy.

scholarly journals An essential mycolate remodeling program for mycobacterial adaptation in host cells

2018 ◽  
Author(s):  
Eliza J.R. Peterson ◽  
Rebeca Bailo ◽  
Alissa C. Rothchild ◽  
Mario Arrieta-Ortiz ◽  
Amardeep Kaur ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
New Technology ◽  
Mycolic Acid ◽  
Host Cells ◽  
Macrophage Infection ◽  
Cell Wall Remodeling ◽  
Mycobacterial Cell Wall

AbstractThe success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA. Using Path-seq we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/A2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection.One Sentence SummaryNovel technology (Path-seq) discovers cell wall remodeling program during Mycobacterium tuberculosis infection of macrophages

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 Mycobacterium tuberculosis Resists Stress by Regulating PE19 Expression

2015 ◽  
Vol 84 (3) ◽  
pp. 735-746 ◽  
Author(s):  
Pavithra Ramakrishnan ◽  
Alisha M. Aagesen ◽  
John D. McKinney ◽  
Anna D. Tischler
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Transcriptional Profiling ◽  
Constitutive Expression ◽  
System Component ◽  
Uptake System ◽  
Aberrant Expression ◽  
Content Type

Mycobacterium tuberculosisrequires the phosphate-sensing signal transduction system Pst/SenX3-RegX3 to resist host immune responses. A ΔpstA1mutant lacking a Pst phosphate uptake system component is hypersensitive to diverse stress conditionsin vitroand is attenuatedin vivodue to constitutive expression of the phosphate starvation-responsive RegX3 regulon. Transcriptional profiling of the ΔpstA1mutant revealed aberrant expression of certainpeandppegenes. PE and PPE proteins, defined by conserved N-terminal domains containing Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs, account for a substantial fraction of theM. tuberculosisgenome coding capacity, but their functions are largely uncharacterized. Because some PE and PPE proteins localize to the cell wall, we hypothesized that overexpression of these proteins sensitizesM. tuberculosisto stress by altering cell wall integrity. To test this idea, we deletedpeandppegenes that were overexpressed by ΔpstA1bacteria. Deletion of a singlepegene,pe19, suppressed hypersensitivity of the ΔpstA1mutant to both detergent and reactive oxygen species. Ethidium bromide uptake assays revealed increased envelope permeability of the ΔpstA1mutant that was dependent on PE19. The replication defect of the ΔpstA1mutant in NOS2−/−mice was partially reversed by deletion ofpe19, suggesting that increased membrane permeability due to PE19 overexpression sensitizesM. tuberculosisto host immunity. Our data indicate that PE19, which comprises only a 99-amino-acid PE domain, has a unique role in the permeability of theM. tuberculosisenvelope that is regulated to resist stresses encountered in the host.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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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