Inhibition of CorA-Dependent Magnesium Homeostasis Is Cidal in Mycobacterium tuberculosis

ABSTRACT Mechanisms of magnesium homeostasis in Mycobacterium tuberculosis are poorly understood. Here, we describe the characterization of a pyrimidinetrione amide scaffold that disrupts magnesium homeostasis in the pathogen by direct binding to the CorA Mg2+/Co2+ transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg2+ ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions in vitro and were efficacious against the organism during macrophage infection. However, the compound lacked efficacy in infected mice, possibly due to limited exposure. Our results indicate that inhibition of Mg2+ homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.

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N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01310-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 6

Author(s):

Stanislav Huszár ◽

Vinayak Singh ◽

Alica Polčicová ◽

Peter Baráth ◽

María Belén Barrio ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Target ◽

Functional Characterization ◽

Drug Candidate ◽

Content Type ◽

Chemical Libraries ◽

Whole Cells ◽

Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

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The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00778-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4446-4452 ◽

Cited By ~ 37

Author(s):

Vadim Makarov ◽

João Neres ◽

Ruben C. Hartkoorn ◽

Olga B. Ryabova ◽

Elena Kazakova ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Nitro Group ◽

Covalent Bond ◽

Antimycobacterial Activity ◽

Content Type ◽

Sar Studies ◽

Covalent Bond Formation

ABSTRACT8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity againstMycobacterium tuberculosisin vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml againstM. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorablein vitroabsorption-distribution-metabolism-excretion/toxicity (ADME/T) andin vivopharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

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Transcriptional Profiling of Mycobacterium tuberculosis Exposed toIn VitroLysosomal Stress

Infection and Immunity ◽

10.1128/iai.00072-16 ◽

2016 ◽

Vol 84 (9) ◽

pp. 2505-2523 ◽

Cited By ~ 22

Author(s):

Wenwei Lin ◽

Paola Florez de Sessions ◽

Garrett Hor Keong Teoh ◽

Ahmad Naim Nazri Mohamed ◽

Yuan O. Zhu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Efflux Pump ◽

Metabolic Reprogramming ◽

Human Macrophage ◽

Host Immune System ◽

Activated Macrophages ◽

Intrinsic Resistance ◽

Macrophage Infection ◽

Content Type

Increasing experimental evidence supports the idea thatMycobacterium tuberculosishas evolved strategies to survive within lysosomes of activated macrophages. To further our knowledge ofM. tuberculosisresponse to the hostile lysosomal environment, we profiled the global transcriptional activity ofM. tuberculosiswhen exposed to the lysosomal soluble fraction (SF) prepared from activated macrophages. Transcriptome sequencing (RNA-seq) analysis was performed using various incubation conditions, ranging from noninhibitory to cidal based on the mycobacterial replication or killing profile. Under inhibitory conditions that led to the absence of apparent mycobacterial replication,M. tuberculosisexpressed a unique transcriptome with modulation of genes involved in general stress response, metabolic reprogramming, respiration, oxidative stress, dormancy response, and virulence. The transcription pattern also indicates characteristic cell wall remodeling with the possible outcomes of increased infectivity, intrinsic resistance to antibiotics, and subversion of the host immune system. Among the lysosome-specific responses, we identified theglgE-mediated 1,4 α-glucan synthesis pathway and a defined group of VapBC toxin/anti-toxin systems, both of which represent toxicity mechanisms that potentially can be exploited for killing intracellular mycobacteria. A meta-analysis including previously reported transcriptomic studies in macrophage infection andin vitrostress models was conducted to identify overlapping and nonoverlapping pathways. Finally, the Tap efflux pump-encoding geneRv1258cwas selected for validation. AnM. tuberculosis ΔRv1258cmutant was constructed and displayed increased susceptibility to killing by lysosomal SF and the antimicrobial peptide LL-37, as well as attenuated survival in primary murine macrophages and human macrophage cell line THP-1.

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In Vitro Isolation and Characterization of Oxazolidinone-Resistant Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01296-17 ◽

2017 ◽

Vol 61 (10) ◽

Cited By ~ 15

Author(s):

Matthew B. McNeil ◽

Devon D. Dennison ◽

Catherine D. Shelton ◽

Tanya Parish

Keyword(s):

Mycobacterium Tuberculosis ◽

23S Rrna ◽

Clinical Settings ◽

Content Type ◽

Isolation And Characterization ◽

Mutant Strains ◽

Ribosomal Protein L3 ◽

Resistant Strains

ABSTRACT Oxazolidinones are promising candidates for the treatment of Mycobacterium tuberculosis infections. We isolated linezolid-resistant strains from H37Rv (Euro-American) and HN878 (East-Asian) strains; resistance frequencies were similar in the two strains. Mutations were identified in ribosomal protein L3 (RplC) and the 23S rRNA (rrl). All mutant strains were cross resistant to sutezolid; a subset was cross resistant to chloramphenicol. Mutations in rrl led to growth impairment and decreased fitness that may limit spread in clinical settings.

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Characterization of Large Deletion Mutants of Mycobacterium tuberculosis Selected for Isoniazid Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00792-20 ◽

2020 ◽

Vol 64 (9) ◽

Author(s):

Catherine Vilchèze ◽

Rajagopalan Saranathan ◽

Brian Weinrick ◽

William R. Jacobs

Keyword(s):

Oxidative Stress ◽

Mycobacterium Tuberculosis ◽

Large Deletion ◽

Growth Defect ◽

Intracellular Growth ◽

Isoniazid Resistance ◽

Content Type ◽

Genomic Deletions

ABSTRACT Large genomic deletions (LGDs) (6 to 63 kbp) were observed in isoniazid-resistant Mycobacterium tuberculosis mutants derived from four M. tuberculosis strains. These LGDs had no growth defect in vitro but could be defective in intracellular growth and showed various sensitivities toward oxidative stress despite lacking katG. The LGD regions comprise 74 genes, mostly of unknown function, that may be important for M. tuberculosis intracellular growth and protection against oxidative stress.

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Characterization of Mutations Conferring Resistance to Rifampin inMycobacterium tuberculosisClinical Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01093-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 4

Author(s):

Tomasz Jagielski ◽

Zofia Bakuła ◽

Anna Brzostek ◽

Alina Minias ◽

Radosław Stachowiak ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Content Type ◽

Control Programs ◽

Gene Coding ◽

H37rv Strain ◽

Different Types ◽

Resistant Strains

ABSTRACTResistance ofMycobacterium tuberculosisto rifampin (RMP), mediated by mutations in therpoBgene coding for the beta-subunit of RNA polymerase, poses a serious threat to the efficacy of clinical management and, thus, control programs for tuberculosis (TB). The contribution of many individualrpoBmutations to the development and level of RMP resistance remains elusive. In this study, the incidence of mutations throughout therpoBgene among 115Mycobacterium tuberculosisclinical isolates, both resistant and susceptible to RMP, was determined. Of the newly discoveredrpoBmutations, the role of three substitutions in the causation of RMP resistance was empirically tested. The results fromin vitromutagenesis experiments were combined with the assessment of the prevalence ofrpoBmutations, and their reciprocal co-occurrences, across globalM. tuberculosispopulations. Twenty-two different types of mutations in therpoBgene were identified and distributed among 58 (89.2%) RMP-resistant strains. The MICs of RMP were within the range of 40 to 800 mg/liter, with MIC50and MIC90values of 400 and 800 mg/liter, respectively. None of the mutations (Gln429His, Met434Ile, and Arg827Cys) inspected for their role in the development of RMP resistance produced an RMP-resistant phenotype in isogenicM. tuberculosisH37Rv strain-derived mutants. These mutations are supposed to compensate for fitness impairment incurred by other mutations directly associated with drug resistance.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

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.

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Characterization of T Cells Specific for CFP-10 and ESAT-6 in Mycobacterium tuberculosis-Infected Mauritian Cynomolgus Macaques

Infection and Immunity ◽

10.1128/iai.01009-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 5

Author(s):

Amy Ellis ◽

Alexis Balgeman ◽

Mark Rodgers ◽

Cassaundra Updike ◽

Jaime Tomko ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

Mhc Class Ii ◽

Nonhuman Primates ◽

Content Type ◽

Host Immune Responses ◽

Cell Responses ◽

Cynomolgus Macaques ◽

Mhc Haplotype

ABSTRACT Nonhuman primates can be used to study host immune responses to Mycobacterium tuberculosis. Mauritian cynomolgus macaques (MCMs) are a unique group of animals that have limited major histocompatibility complex (MHC) genetic diversity, such that MHC-identical animals can be infected with M. tuberculosis. Two MCMs homozygous for the relatively common M1 MHC haplotype were bronchoscopically infected with 41 CFU of the M. tuberculosis Erdman strain. Four other MCMs, which had at least one copy of the M1 MHC haplotype, were infected with a lower dose of 3 CFU M. tuberculosis. All animals mounted similar T-cell responses to CFP-10 and ESAT-6. Two epitopes in CFP-10 were characterized, and the MHC class II alleles restricting them were determined. A third epitope in CFP-10 was identified but exhibited promiscuous restriction. The CFP-10 and ESAT-6 antigenic regions targeted by T cells in MCMs were comparable to those seen in cases of human M. tuberculosis infection. Our data lay the foundation for generating tetrameric molecules to study epitope-specific CD4 T cells in M. tuberculosis-infected MCMs, which may guide future testing of tuberculosis vaccines in nonhuman primates.

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Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00165-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 18

Author(s):

Zhaojing Zong ◽

Wei Jing ◽

Jin Shi ◽

Shu'an Wen ◽

Tingting Zhang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Novel Mutation ◽

Multidrug Resistant ◽

23S Rrna ◽

Drug Resistant ◽

Content Type ◽

Extensively Drug Resistant ◽

Significant Difference ◽

Mdr Tb

ABSTRACT Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis. In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

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Preclinical Evidence of Nanomedicine Formulation to Target Mycobacterium tuberculosis at Its Bone Marrow Niche

Pathogens ◽

10.3390/pathogens9050372 ◽

2020 ◽

Vol 9 (5) ◽

pp. 372 ◽

Cited By ~ 1

Author(s):

Jaishree Garhyan ◽

Surender Mohan ◽

Vinoth Rajendran ◽

Rakesh Bhatnagar

Keyword(s):

Bone Marrow ◽

Mycobacterium Tuberculosis ◽

In Vitro Release ◽

Bone Marrow Niche ◽

Mice Model ◽

Latently Infected ◽

Marrow Mesenchymal Stem Cells

One-third of the world’s population is estimated to be latently infected with Mycobacterium tuberculosis (Mtb). Recently, we found that dormant Mtb hides in bone marrow mesenchymal stem cells (BM-MSCs) post-chemotherapy in mice model and in clinical subjects. It is known that residual Mtb post-chemotherapy may be responsible for increased relapse rates. However, strategies for Mtb clearance post-chemotherapy are lacking. In this study, we engineered and formulated novel bone-homing PEGylated liposome nanoparticles (BTL-NPs) which actively targeted the bone microenvironment leading to Mtb clearance. Targeting of BM-resident Mtb was carried out through bone-homing liposomes tagged with alendronate (Ald). BTL characterization using TEM and DLS showed that the size of bone-homing isoniazid (INH) and rifampicin (RIF) BTLs were 100 ± 16.3 nm and 84 ± 18.4 nm, respectively, with the encapsulation efficiency of 69.5% ± 4.2% and 70.6% ± 4.7%. Further characterization of BTLs, displayed by sustained in vitro release patterns, increased in vivo tissue uptake and enhanced internalization of BTLs in RAW cells and CD271+BM-MSCs. The efficacy of isoniazid (INH)- and rifampicin (RIF)-loaded BTLs were shown using a mice model where the relapse rate of the tuberculosis was decreased significantly in targeted versus non-targeted groups. Our findings suggest that BTLs may play an important role in developing a clinical strategy for the clearance of dormant Mtb post-chemotherapy in BM cells.

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