scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Mutations in fbiD (Rv2983) as a novel determinant of resistance to pretomanid and delamanid in Mycobacterium tuberculosis

2020 ◽  
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Solid Media ◽  
Resistant Mutants ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole pro-drugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: fbiC (56%), fbiA (15%), ddn (12%), fgd (4%) and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983, a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance, but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals In Vitro Study of Stepwise Acquisition of rv0678 and atpE Mutations Conferring Bedaquiline Resistance

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Nabila Ismail ◽  
Nazir A. Ismail ◽  
Shaheed V. Omar ◽  
Remco P. H. Peters
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Solid Medium ◽  
In Vitro Study ◽  
Whole Genome ◽  
Content Type ◽  
Phenotypic Resistance ◽  
Atcc Strain ◽  
High Level ◽  
Level Resistance

ABSTRACT Bedaquiline resistance within Mycobacterium tuberculosis may arise through efflux-based (rv0678) or target-based (atpE) pathway mutations. M. tuberculosis mutant populations from each of five sequential steps in a passaging approach, using a pyrazinamide-resistant ATCC strain, were subjected to MIC determinations and whole-genome sequencing. Exposure to increasing bedaquiline concentrations resulted in increasing phenotypic resistance (up to >2 μg/ml) through MIC determination on solid medium (Middlebrook 7H10). rv0678 mutations were dynamic, while atpE mutations were fixed, once occurring. We present the following hypothesis for in vitro emergence of bedaquiline resistance: rv0678 mutations may be the first transient step in low-level resistance acquisition, followed by high-level resistance due to fixed atpE mutations.

scholarly journals Plasmodium falciparum Resistance to a Lead Benzoxaborole Due to Blocked Compound Activation and Altered Ubiquitination or Sumoylation

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kirthana M. V. Sindhe ◽  
Wesley Wu ◽  
Jenny Legac ◽  
Yong-Kang Zhang ◽  
Eric E. Easom ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Stress Responses ◽  
Mechanisms Of Action ◽  
Antimalarial Drugs ◽  
New Drugs ◽  
Loss Of Function ◽  
Content Type ◽  
High Level ◽  
Level Resistance

ABSTRACT New antimalarial drugs are needed. The benzoxaborole AN13762 showed excellent activity against cultured Plasmodium falciparum, against fresh Ugandan P. falciparum isolates, and in murine malaria models. To gain mechanistic insights, we selected in vitro for P. falciparum isolates resistant to AN13762. In all of 11 independent selections with 100 to 200 nM AN13762, the 50% inhibitory concentration (IC50) increased from 18–118 nM to 180–890 nM, and whole-genome sequencing of resistant parasites demonstrated mutations in prodrug activation and resistance esterase (PfPARE). The introduction of PfPARE mutations led to a similar level of resistance, and recombinant PfPARE hydrolyzed AN13762 to the benzoxaborole AN10248, which has activity similar to that of AN13762 but for which selection of resistance was not readily achieved. Parasites further selected with micromolar concentrations of AN13762 developed higher-level resistance (IC50, 1.9 to 5.0 μM), and sequencing revealed additional mutations in any of 5 genes, 4 of which were associated with ubiquitination/sumoylation enzyme cascades; the introduction of one of these mutations, in SUMO-activating enzyme subunit 2, led to a similar level of resistance. The other gene mutated in highly resistant parasites encodes the P. falciparum cleavage and specificity factor homolog PfCPSF3, previously identified as the antimalarial target of another benzoxaborole. Parasites selected for resistance to AN13762 were cross-resistant with a close analog, AN13956, but not with standard antimalarials, AN10248, or other benzoxaboroles known to have different P. falciparum targets. Thus, AN13762 appears to have a novel mechanism of antimalarial action and multiple mechanisms of resistance, including loss of function of PfPARE preventing activation to AN10248, followed by alterations in ubiquitination/sumoylation pathways or PfCPSF3. IMPORTANCE Benzoxaboroles are under study as potential new drugs to treat malaria. One benzoxaborole, AN13762, has potent activity and promising features, but its mechanisms of action and resistance are unknown. To gain insights into these mechanisms, we cultured malaria parasites with nonlethal concentrations of AN13762 and generated parasites with varied levels of resistance. Parasites with low-level resistance had mutations in PfPARE, which processes AN13762 into an active metabolite; PfPARE mutations prevented this processing. Parasites with high-level resistance had mutations in any of a number of enzymes, mostly those involved in stress responses. Parasites selected for AN13762 resistance were not resistant to other antimalarials, suggesting novel mechanisms of action and resistance for AN13762, a valuable feature for a new class of antimalarial drugs.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

scholarly journals Rv3131, a gene encoding nitroreductase, is essential for metronidazole activation in Mycobacterium tuberculosis under hypoxic condition

2020 ◽  
Author(s):  
Wenzhu Dong ◽  
Jin Shi ◽  
Ping Chu ◽  
Rongmei Liu ◽  
Shu’an Wen ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Anaerobic Bacteria ◽  
Aerobic Condition ◽  
Hypoxic Condition ◽  
Significant Inhibition ◽  
Wild Type ◽  
Minimal Inhibitory Concentrations ◽  
High Level ◽  
Nitroreductase Activity

Abstract ObjectivesThe impressive potency of metronidazole (MTZ) against anaerobic bacteria indicates the potential for killing anaerobic Mtb. However, how MTZ is activated in Mtb still remains unknown. We aimed to characterize the endogenous nitroreductase responsible for MTZ activation in anaerobic Mtb.MethodsThe minimal inhibitory concentrations (MICs) of Mtb isolates against MTZ were determined by microplate Alamar Blue assay. Intracellular anti-TB activities of MTZ and pyrazinamide (PZA) were tested in THP-1 cells infected by Mycobacterium tuberculosis (Mtb) H37Rv with a multiplicity of infection (MOI) of 10. The nitroreductase activity of purified wild-type Rv3131 and mutants were measured under anaerobic conditions generated by glucose oxidase/catalase system. Two-tailed unpaired Student’s t test was used to compare the difference between various groups.Results180 Mtb isolates (81.8%, 180/220) had MIC values higher than 16 μg/mL, and 40 had MIC values of 16 μg/mL, demonstrating high-level resistance to MTZ under aerobic condition. The number of viable bacteria in macrophages treated with MTZ was dramatically decreased by 71.3% after 5-day MTZ treatment, indicating significant inhibition of MTZ against anaerobic Mtb. In vitro biochemical analysis demonstrated that Rv3131 exhibited the NADPH oxidase activity under anaerobic condition. The substitutions of Cys75Ser and Cys279Ser could maintain 41.7% and 71.1% of enzyme activity compared to wild-type protein, respectively.ConclusionsOur data demonstrate that MTZ has more potent efficacy against intracellular Mtb than PZA. Rv3131 is identified as a nitroreductase enzyme in the activation of MTZ, and Cys75 of Rv3131 is the major active residue for nitroreductase activity.

scholarly journals Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Jordan B. Jastrab ◽  
Marie I. Samanovic ◽  
Richard Copin ◽  
Bo Shopsin ◽  
K. Heran Darwin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Heat Shock ◽  
Protein Quality ◽  
Normal Growth ◽  
Culture Conditions ◽  
Growth Defect ◽  
Loss Of Function ◽  
Content Type ◽  
Accessory Factor

ABSTRACT Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled by Mycobacterium tuberculosis proteasome accessory factor E (PafE). Recently, we found that a Mycobacterium tuberculosis pafE mutant has slowed growth in vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR (heat shock protein repressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterized pafE suppressor mutants that grew similarly to pafE + bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of a pafE mutant were caused primarily by the inability of the proteasome to degrade HspR. IMPORTANCE Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence of M. tuberculosis. In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress.

scholarly journals Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

2016 ◽  
Vol 60 (9) ◽  
pp. 5232-5237 ◽  
Author(s):  
Xia Yu ◽  
Guirong Wang ◽  
Suting Chen ◽  
Guomei Wei ◽  
Yuanyuan Shang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Bacterial Species ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Wild Type ◽  
Resistance Mutations ◽  
Content Type ◽  
Lowenstein Jensen

ABSTRACTAntofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying itsin vitroactivity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinicalMycobacterium tuberculosisstrains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions ofgyrA(Rv0006) andgyrB(Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.

scholarly journals Contribution of rpoB Mutations to Development of Rifamycin Cross-Resistance in Mycobacterium tuberculosis

1998 ◽  
Vol 42 (7) ◽  
pp. 1853-1857 ◽  
Author(s):  
D. L. Williams ◽  
L. Spring ◽  
L. Collins ◽  
L. P. Miller ◽  
L. B. Heifets ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cross Resistance ◽  
In Vitro Mutagenesis ◽  
Missense Mutations ◽  
Resistance Mutations ◽  
Development Of Resistance ◽  
High Level ◽  
The Impact ◽  
Level Resistance

ABSTRACT The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of rpoB, the gene encoding the β-subunit of the DNA-dependent RNA polymerase of Mycobacterium tuberculosis, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutantrpoB alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific rpoBmutant alleles on the development of rifamycin resistance, mutations were incorporated into the rpoB gene of M. tuberculosis H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant M. tuberculosis clones containing plasmids with specific mutations in either codon 531 or 526 of rpoB exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specificrpoB mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of rpoB genotyping for predicting rifamycin-resistant phenotypes.

scholarly journals Rapidly Correcting Frameshift Mutations in the Mycobacterium tuberculosis orn Gene Produce Reversible Ethambutol Resistance and Small-Colony-Variant Morphology

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Hassan Safi ◽  
Subramanya Lingaraju ◽  
Shuyi Ma ◽  
Seema Husain ◽  
Mainul Hoque ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Frameshift Mutation ◽  
Relative Increase ◽  
Wild Type ◽  
Content Type ◽  
Reading Frame ◽  
Small Colony Variant ◽  
Large Colony ◽  
Small Colony ◽  
High Level

ABSTRACT We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosis orn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis. However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 μg/ml) produces an SCV with an EMB MIC of 32 μg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 μg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.

scholarly journals Point Mutations within the Fatty Acid Synthase Type II Dehydratase Components HadA or HadC Contribute to Isoxyl Resistance in Mycobacterium tuberculosis

2012 ◽  
Vol 57 (1) ◽  
pp. 629-632 ◽  
Author(s):  
Laila Gannoun-Zaki ◽  
Laeticia Alibaud ◽  
Laurent Kremer
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Point Mutations ◽  
Mycolic Acid ◽  
Type Ii ◽  
Content Type ◽  
High Level ◽  
Fas Ii ◽  
Level Resistance

ABSTRACTThe mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO inMycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.

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