Characterizing the Effects of Glutathione as an Immunoadjuvant in the Treatment of Tuberculosis

ABSTRACT Mycobacterium tuberculosis is the etiological agent that is responsible for causing tuberculosis (TB), which continues to affect millions of people worldwide, and the rate of resistance of M. tuberculosis to antibiotics is ever increasing. We tested the synergistic effects of N-acetyl cysteine (NAC; the precursor molecule for the synthesis of glutathione [GSH]) and individual first-line antibiotics typically given for the treatment of TB, such as isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), to improve the ability of macrophages to control intracellular M. tuberculosis infection. GSH, a pleiotropic antioxidant molecule, has previously been shown to display both antimycobacterial and immune-enhancing effects. Our results indicate that there was not only an increase in beneficial immunomodulatory effects but also a greater reduction in the intracellular viability of M. tuberculosis when macrophages were treated with the combination of antibiotics (INH, RIF, EMB, or PZA) and NAC.

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Characterizing the Effects of Glutathione as an Immunoadjuvant in the Treatment of Tuberculosis

10.1101/335323 ◽

2018 ◽

Author(s):

Ruoqiong Cao ◽

Garrett Teskey ◽

Hicret Islamoglu ◽

Rachel Abrahem ◽

Karo Gyurjian ◽

...

Keyword(s):

Synergistic Effects ◽

Etiological Agent ◽

Immunomodulatory Effects ◽

First Line ◽

Precursor Molecule ◽

Resistance To Antibiotics ◽

Acetyl Cysteine ◽

Antioxidant Molecule

AbstractMycobacteriumtuberculosis(M.tb) is the etiological agent that is responsible for causing tuberculosis (TB), which continues to affect millions of people worldwide, with an ever-increasing resistance to antibiotics. We tested the synergistic effects of N-acetyl cysteine (NAC, the precursor molecule for the synthesis of glutathione) and individual first-line antibiotics typically given for the treatment of TB such as Isoniazid (INH), Rifampicin (RIF), Ethambutol (EMB) and Pyrazinamide (PZA) to improve the ability of macrophages to control intracellularM.tbinfection. Glutathione (GSH), a pleiotropic antioxidant molecule has been previously shown to display both antimycobacterial and immune-enhancing effects. Our results indicate that there was not only an increase in beneficial immunomodulatory effects, but a greater reduction in the intracellular viability ofM. tbwhen macrophages were treated with the combination of antibiotics (INH/RIF/EMB or PZA) and NAC.

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Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02130-16 ◽

2016 ◽

Vol 61 (2) ◽

Cited By ~ 8

Author(s):

Brandon C. Rosen ◽

Nicholas A. Dillon ◽

Nicholas D. Peterson ◽

Yusuke Minato ◽

Anthony D. Baughn

Keyword(s):

Mycobacterium Tuberculosis ◽

Coenzyme A ◽

Fatty Acyl ◽

Wild Type ◽

Intrinsic Resistance ◽

First Line ◽

Minimum Concentration ◽

Content Type ◽

Acyl Coenzyme A ◽

Long Chain

ABSTRACT Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis.

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Phenotypically Adapted Mycobacterium tuberculosis Populations from Sputum Are Tolerant to First-Line Drugs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01380-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2476-2483 ◽

Cited By ~ 26

Author(s):

Obolbek Turapov ◽

Benjamin D. O'Connor ◽

Asel A. Sarybaeva ◽

Caroline Williams ◽

Hemu Patel ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Liquid Medium ◽

Culture Supernatant ◽

Ex Vivo ◽

First Line ◽

Content Type ◽

Host Environment ◽

Solid Media ◽

Bactericidal Effects

ABSTRACTTuberculous sputum contains multipleMycobacterium tuberculosispopulations with different requirements for isolationin vitro. These include cells that form colonies on solid media (plateableM. tuberculosis), cells requiring standard liquid medium for growth (nonplateableM. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependentM. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of theseM. tuberculosispopulations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10reductions), and SN-dependentM. tuberculosiswas more tolerant to streptomycin and isoniazid than the plateable and nonplateableM. tuberculosisstrains. Susceptibility of plateableM. tuberculosisto bactericidal drugs was significantly increased after passagein vitro; thus, tolerance observed in the sputum samples from the population groups was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simpleex vivosystem for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection.

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Pantothenate and Pantetheine Antagonize the Antitubercular Activity of Pyrazinamide

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04028-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7258-7263 ◽

Cited By ~ 43

Author(s):

Nicholas A. Dillon ◽

Nicholas D. Peterson ◽

Brandon C. Rosen ◽

Anthony D. Baughn

Keyword(s):

Mycobacterium Tuberculosis ◽

Nicotinic Acid ◽

Active Form ◽

Laboratory Strain ◽

Antitubercular Activity ◽

First Line ◽

Auxotrophic Strain ◽

Content Type ◽

Related Compounds ◽

Pyrazinoic Acid

ABSTRACTPyrazinamide (PZA) is a first-line tuberculosis drug that inhibits the growth ofMycobacterium tuberculosisvia an as yet undefined mechanism. AnM. tuberculosislaboratory strain that was auxotrophic for pantothenate was found to be insensitive to PZA and to the active form, pyrazinoic acid (POA). To determine whether this phenotype was strain or condition specific, the effect of pantothenate supplementation on PZA activity was assessed using prototrophic strains ofM. tuberculosis. It was found that pantothenate and other β-alanine-containing metabolites abolished PZA and POA susceptibility, suggesting that POA might selectively target pantothenate synthesis. However, when the pantothenate-auxotrophic strain was cultivated using a subantagonistic concentration of pantetheine in lieu of pantothenate, susceptibility to PZA and POA was restored. In addition, we found that β-alanine could not antagonize PZA and POA activity against the pantothenate-auxotrophic strain, indicating that the antagonism is specific to pantothenate. Moreover, pantothenate-mediated antagonism was observed for structurally related compounds, includingn-propyl pyrazinoate, 5-chloropyrazinamide, and nicotinamide, but not for nicotinic acid or isoniazid. Taken together, these data demonstrate that while pantothenate can interfere with the action of PZA, pantothenate synthesis is not directly targeted by PZA. Our findings suggest that targeting of pantothenate synthesis has the potential to enhance PZA efficacy and possibly to restore PZA susceptibility in isolates withpanD-linked resistance.

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Detection of Mycobacterium tuberculosis pncA Mutations by the Nipro Genoscholar PZA-TB II Assay Compared to Conventional Sequencing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01871-17 ◽

2017 ◽

Vol 62 (1) ◽

Cited By ~ 3

Author(s):

Melisa J. Willby ◽

Maria Wijkander ◽

Joshua Havumaki ◽

Kartee Johnson ◽

Jim Werngren ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Confidence Interval ◽

Coding Region ◽

First Line ◽

Content Type ◽

Treatment Regimens ◽

Molecular Tests ◽

Initiation Of Therapy ◽

Probe Assay ◽

Detection Of Mutations

ABSTRACT Pyrazinamide (PZA) is a standard component of first-line treatment regimens for Mycobacterium tuberculosis and is included in treatment regimens for drug-resistant M. tuberculosis whenever possible. Therefore, it is imperative that susceptibility to PZA be assessed reliably prior to the initiation of therapy. Currently available growth-based PZA susceptibility tests are time-consuming, and results can be inconsistent. Molecular tests have been developed for most first-line antituberculosis drugs; however, a commercial molecular test is not yet available for rapid detection of PZA resistance. Recently, a line probe assay, the Nipro Genoscholar PZA-TB II assay, was developed for the detection of mutations within the pncA gene, including the promoter region, that are likely to lead to PZA resistance. The sensitivity and specificity of this assay were evaluated by two independent laboratories, using a combined total of 249 strains with mutations in pncA or its promoter and 21 strains with wild-type pncA. Overall, the assay showed good sensitivity (93.2% [95% confidence interval, 89.3 to 95.8%]) and moderate specificity (91.2% [95% confidence interval, 77.0 to 97.0%]) for the identification of M. tuberculosis strains predicted to be resistant to PZA on the basis of the presence of mutations (excluding known PZA-susceptible mutations) in the pncA coding region or promoter. The assay shows promise for the molecular prediction of PZA resistance.

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Galectin-8 Senses Phagosomal Damage and Recruits Selective Autophagy Adapter TAX1BP1 To Control Mycobacterium tuberculosis Infection in Macrophages

mBio ◽

10.1128/mbio.01871-20 ◽

2021 ◽

Author(s):

Samantha L. Bell ◽

Kayla L. Lopez ◽

Jeffery S. Cox ◽

Kristin L. Patrick ◽

Robert O. Watson

Keyword(s):

Mycobacterium Tuberculosis ◽

Infectious Diseases ◽

Tuberculosis Infection ◽

Harsh Environment ◽

Intracellular Pathogens ◽

First Line ◽

Mycobacterium Tuberculosis Infection ◽

Content Type ◽

Selective Autophagy ◽

Global Population

Mycobacterium tuberculosis (Mtb) infects one-quarter of the global population and causes one of the deadliest infectious diseases worldwide. Macrophages are the first line of defense against Mtb infection and are typically incredibly efficient at destroying intracellular pathogens, but Mtb has evolved to survive and replicate in this harsh environment.

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Vitamin C Potentiates the Killing of Mycobacterium tuberculosis by the First-Line Tuberculosis Drugs Isoniazid and Rifampin in Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02165-17 ◽

2018 ◽

Vol 62 (3) ◽

Cited By ~ 9

Author(s):

Catherine Vilchèze ◽

John Kim ◽

William R. Jacobs

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Vitamin C ◽

Mouse Serum ◽

First Line ◽

Content Type ◽

Tb Treatment ◽

High Concentrations

ABSTRACT The treatment of drug-susceptible tuberculosis (TB) is long and cumbersome. Mismanagement of TB treatment can lead to the emergence of drug resistance in patients, so shortening the treatment duration could significantly improve TB chemotherapy and prevent the development of drug resistance. We previously discovered that high concentrations of vitamin C sterilize cultures of drug-susceptible and drug-resistant Mycobacterium tuberculosis . Here, we tested subinhibitory concentration of vitamin C in combination with TB drugs against M. tuberculosis in vitro and in a mouse model of M. tuberculosis infection. In vivo , we showed that the vitamin C level in mouse serum can be increased by intraperitoneal injection of vitamin C to reach vitamin C levels close to the concentrations required for activity in vitro . Although vitamin C had no activity by itself in M. tuberculosis -infected mice, the combination of vitamin C with the first-line TB drugs isoniazid and rifampin reduced the bacterial burden in the lungs of M. tuberculosis -infected mice faster than isoniazid and rifampin combined in two independent experiments. These experiments suggest that the addition of vitamin C to first-line TB drugs could shorten TB treatment. Vitamin C, an inexpensive and nontoxic compound, could easily be added to the TB pharmacopeia to substantially improve chemotherapy outcome, which would have a significant impact on the worldwide TB community.

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Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01550-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2032-2041 ◽

Cited By ~ 236

Author(s):

Patricia J. Campbell ◽

Glenn P. Morlock ◽

R. David Sikes ◽

Tracy L. Dalton ◽

Beverly Metchock ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Dna Sequencing ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Second Line ◽

First Line ◽

Content Type ◽

Line Drug

ABSTRACTThe emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced:rpoB(for resistance to RIF),katGandinhA(INH),pncA(PZA),embB(EMB),gyrA(CIP and OFX), andrrs,eis, andtlyA(KAN, AMK, and CAP). A total of 314 clinicalMycobacterium tuberculosiscomplex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% forrpoB, 85.4% and 100% forkatG, 16.5% and 100% forinhA, 90.6% and 100% forkatGandinhAtogether, 84.6% and 85.8% forpncA, and 78.6% and 93.1% forembB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in theM. tuberculosiscomplex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

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Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions andpncAGene Sequencing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05894-11 ◽

2011 ◽

Vol 56 (3) ◽

pp. 1253-1257 ◽

Cited By ~ 35

Author(s):

J. Werngren ◽

E. Sturegård ◽

P. Juréen ◽

K. Ängeby ◽

S. Hoffner ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Critical Concentration ◽

Gene Mutations ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Clinical Isolates ◽

Wild Type ◽

First Line ◽

Content Type ◽

Resistant Strains

ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistantMycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates ofMycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinicalM. tuberculosisisolates and compared the results topncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and nopncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances betweenpncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated topncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

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