Perturbation of Cytochrome c Maturation Reveals Adaptability of the Respiratory Chain in Mycobacterium tuberculosis

ABSTRACTMycobacterium tuberculosisdepends on aerobic respiration for growth and utilizes anaa3-type cytochromecoxidase for terminal electron transfer. Cytochromecmaturation in bacteria requires covalent attachment of heme to apocytochromec, which occurs outside the cytoplasmic membrane. We demonstrate that inM. tuberculosisthe thioredoxin-like protein Rv3673c, which we named CcsX, is required for heme insertion in cytochromec. Inactivation of CcsX resulted in loss ofc-type heme absorbance, impaired growth and virulence ofM. tuberculosis, and induced cytochromebdoxidase. This suggests that the bioenergetically less efficientbdoxidase can compensate for deficient cytochromecoxidase activity, highlighting the flexibility of theM. tuberculosisrespiratory chain. A spontaneous mutation in the active site of vitamin K epoxide reductase (VKOR) suppressed phenotypes of the CcsX mutant and abrogated the activity of the disulfide bond-dependent alkaline phosphatase, which shows that VKOR is the major disulfide bond catalyzing protein in the periplasm ofM. tuberculosis.IMPORTANCEMycobacterium tuberculosisrequires oxygen for growth; however, the biogenesis of respiratory chain components in mycobacteria has not been explored. Here, we identified a periplasmic thioredoxin, CcsX, necessary for heme insertion into cytochromec. We investigated the consequences of disrupting cytochromecmaturation (CCM) for growth and survival ofM. tuberculosis in vitroand for its pathogenesis. Appearance of a second-site suppressor mutation in the periplasmic disulfide bond catalyzing protein VKOR indicates the strong selective pressure for a functional cytochromecoxidase. The observation thatM. tuberculosisis able to partially compensate for defective CCM by upregulation of the cytochromebdoxidase exposes a functional role of this alternative terminal oxidase under normal aerobic conditions and during pathogenesis. This suggests that targeting both oxidases simultaneously might be required to effectively disrupt respiration inM. tuberculosis.

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Rv2223c, an acid inducible carboxyl-esterase of Mycobacterium tuberculosis enhanced the growth and survival of Mycobacterium smegmatis

Future Microbiology ◽

10.2217/fmb-2019-0162 ◽

2019 ◽

Vol 14 (16) ◽

pp. 1397-1415

Author(s):

Pratibha Maan ◽

Jagdeep Kaur

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Intracellular Survival ◽

Growth And Survival ◽

Enhanced Expression ◽

Stress Environment ◽

In Vitro Stress ◽

Carboxyl Esterase

Aim: To elucidate the role of Rv2223c in Mycobacterium tuberculosis. Methods: Purified recombinant Rv2223c protein was characterized. Expression of rv2223c in the presence of different stress environment and subcellular localization were performed in M. tuberculosis H37Ra and Mycobacterium smegmatis ( MS_2223c). Effect of its overexpression on growth rate, infection and intracellular survival in THP-1/PBMC cells were studied. Results: rRv2223c demonstrated esterase activity with preference for pNP-octanoate and hydrolyzed trioctanoate to di- and mono-octanoate. Expression of rv2223c was upregulated in acidic and nutritive stress conditions. rRv2223c was identified in extracellular and cell wall fractions. MS_2223c exhibited enhanced growth, survival during in vitro stress, infection and intracellular survival. Conclusions: Rv2223c is a secretary, carboxyl-esterase, with enhanced expression under acidic and nutritive stress condition and might help in intracellular survival of bacteria.

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Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.01135-09 ◽

2010 ◽

Vol 192 (8) ◽

pp. 2220-2227 ◽

Cited By ~ 41

Author(s):

Bavesh D. Kana ◽

Garth L. Abrahams ◽

Nackmoon Sung ◽

Digby F. Warner ◽

Bhavna G. Gordhan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Alkylating Agents ◽

Spontaneous Mutation ◽

Dna Polymerases ◽

Binding Motif ◽

Dependent Manner ◽

Mouse Macrophages ◽

Y Family

ABSTRACT The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N 2-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

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CD157 Confers Host Resistance to Mycobacterium tuberculosis via TLR2-CD157-PKCzeta-Induced Reactive Oxygen Species Production

mBio ◽

10.1128/mbio.01949-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 3

Author(s):

Qianting Yang ◽

Mingfeng Liao ◽

Wenfei Wang ◽

Mingxia Zhang ◽

Qi Chen ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Mycobacterium Tuberculosis ◽

Host Resistance ◽

Ros Production ◽

Oxygen Species ◽

Effective Strategy ◽

Content Type ◽

Protein Levels

ABSTRACT Recruitment of monocytes to the infection site is critical for host resistance against Mycobacterium tuberculosis. CD157 has a crucial role in neutrophil and monocyte transendothelial migration and adhesion, but its role in tuberculosis (TB) is unclear. Here, we show that both mRNA and protein levels of Cd157 are significantly increased during M. tuberculosis infection. Deficiency of Cd157 impaired host response to M. tuberculosis infection by increasing bacterial burden and inflammation in the lung in the murine TB model. In vitro experiments show that the bactericidal ability was compromised in Cd157 knockout (KO) macrophages, which was due to impaired M. tuberculosis-induced reactive oxygen species (ROS) production. We further reveal that CD157 interacts with TLR2 and PKCzeta and facilitates M. tuberculosis-induced ROS production in Cd157 KO macrophages, which resulted in enhanced M. tuberculosis killing. For the clinic aspect, we observe that the expression of CD157 decreases after effective anti-TB chemotherapy. CD157 is specifically increased in pleural fluid in tuberculous pleurisy patients compared to pneumonia and lung cancer patients. Interestingly, the levels of soluble CD157 (sCD157) correlate with human peripheral monocyte-derived macrophage bactericidal activity. Exogenous application of sCD157 could compensate for macrophage bactericidal ability and restore ROS production. In conclusion, we have identified a novel protective immune function of CD157 during M. tuberculosis infection via TLR2-dependent ROS production. Application of sCD157 might be an effective strategy for host-directed therapy against TB in those with insufficient CD157 production. IMPORTANCE Tuberculosis, a chronic bacterial disease caused by Mycobacterium tuberculosis, remains a major global health problem. CD157, a dual-function receptor and β-NAD+-metabolizing ectoenzyme, promotes cell polarization, regulates chemotaxis induced through the high-affinity fMLP receptor, and controls transendothelial migration. The role of CD157 in TB pathogenesis remains unknown. In this study, we find that both mRNA and protein levels of CD157 are significantly increased in TB. Deficiency of CD157 impaired host defense against M. tuberculosis infection both in vivo and in vitro, which is mediated by an interaction among CD157, TLR2, and PKCzeta. This interaction facilitates M. tuberculosis-induced macrophagic ROS production, which enhances macrophage bactericidal activity. Interestingly, the sCD157 level in plasma is reversibly associated with MDM M. tuberculosis killing activity. By uncovering the role of CD157 in pathogenesis of TB for the first time, our work demonstrated that application of soluble CD157 might be an effective strategy for host-directed therapy against TB.

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Quorum Sensing Extracellular Death Peptides Enhance the Endoribonucleolytic Activities of Mycobacterium tuberculosis MazF Toxins

mBio ◽

10.1128/mbio.00685-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 3

Author(s):

Akanksha Nigam ◽

Sathish Kumar ◽

Hanna Engelberg-Kulka

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Cell Death ◽

Mycobacterium Tuberculosis ◽

Quorum Sensing ◽

Inhibitory Effect ◽

Content Type ◽

Bacterial Cell Death

ABSTRACT mazEF is a toxin-antitoxin module located on chromosomes of most bacteria. MazF toxins are endoribonucleases antagonized by MazE antitoxins. Previously, we characterized several quorum sensing peptides called " e xtracellular d eath f actors" (EDFs). When secreted from bacterial cultures, EDFs induce interspecies cell death. EDFs also enhance the endoribonucleolytic activity of Escherichia coli MazF. Mycobacterium tuberculosis carries several mazEF modules. Among them, the endoribonucleolytic activities of MazF proteins mt-1, mt-3, and mt-6 were identified. MazF-mt6 and MazF-mt-3 cleave M. tuberculosis rRNAs. Here we report the in vitro effects of EDFs on the endoribonucleolytic activities of M. tuberculosis MazFs. Escherichia coli EDF ( Ec EDF) and the three Pseudomonas aeruginosa EDFs ( Pa EDFs) individually enhance the endoribonucleolytic activities of MazF-mt6 and MazF-mt3 and overcome the inhibitory effect of MazE-mt3 or MazE-mt6 on the endoribonucleolytic activities of the respective toxins. We propose that these EDFs can serve as a basis for a novel class of antibiotics against M. tuberculosis . IMPORTANCE Mycobacterium tuberculosis is one of the leading causes of death from infectious disease. M. tuberculosis is highly drug resistant, and drug delivery to the infected site is very difficult. In previous studies, we showed that e xtracellular d eath f actors (EDFs) can work as quorum sensing molecules which participate in interspecies bacterial cell death. In this study, we demonstrated the role of different EDFs in the endoribonucleolytic activities of M. tuberculosis MazFs. Escherichia coli EDF ( Ec EDF) and the three Pseudomonas aeruginosa EDFs ( Pa EDFs) individually enhance the endoribonucleolytic activities of MazF-mt6 and MazF-mt3. The current report provides a basis for the use of the EDF peptides Ec EDF and Pa EDF as novel antibiotics against M. tuberculosis .

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Arylvinylpiperazine Amides, a New Class of Potent Inhibitors Targeting QcrB ofMycobacterium tuberculosis

mBio ◽

10.1128/mbio.01276-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 30

Author(s):

Caroline S. Foo ◽

Andréanne Lupien ◽

Maryline Kienle ◽

Anthony Vocat ◽

Andrej Benjak ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Parent Compound ◽

New Drugs ◽

Cross Resistance ◽

Terminal Oxidase ◽

Content Type ◽

Allelic Exchange ◽

Isolation And Characterization

ABSTRACTNew drugs are needed to control the current tuberculosis (TB) pandemic caused by infection withMycobacterium tuberculosis. We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agentsin vitro. All five compounds showed good activity againstM. tuberculosisin vitroand in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization ofM. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in theqcrBgene encoding a subunit of cytochromebc1oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochromebc1-aa3is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles ofM. tuberculosistreated with AX-35 were similar to those generated by other cytochromebc1oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochromebdin respiratory adaptation. In the absence of cytochromebdoxidase, AX-35 was bactericidal againstM. tuberculosis. Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.IMPORTANCENew drugs againstMycobacterium tuberculosisare urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potentin vitroandin vivoactivities againstM. tuberculosis. AX compounds target the QcrB subunit of the cytochromebc1terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochromebdoxidase upon QcrB inhibition. In the absence of cytochromebdoxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.

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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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Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01341-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 6873-6881 ◽

Cited By ~ 13

Author(s):

Kathryn Winglee ◽

Shichun Lun ◽

Marco Pieroni ◽

Alan Kozikowski ◽

William Bishai

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Efflux Pump ◽

Transcriptional Regulator ◽

Cross Resistance ◽

Loss Of Function ◽

Strong Activity ◽

Content Type ◽

Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

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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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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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