Triosephosphate Isomerase Is Dispensable In Vitro yet Essential for Mycobacterium tuberculosis To Establish Infection

ABSTRACTTriosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This reaction is required for glycolysis and gluconeogenesis, andtpihas been predicted to be essential for growth ofMycobacterium tuberculosis. However, when studying a conditionally regulatedtpiknockdown mutant, we noticed that depletion of TPI reduced growth ofM. tuberculosisin media containing a single carbon source but not in media that contained both a glycolytic and a gluconeogenic carbon source. We used such two-carbon-source media to isolate atpideletion (Δtpi) mutant. The Δtpimutant did not survive with single carbon substrates but grew like wild-type (WT)M. tuberculosisin the presence of both a glycolytic and a gluconeogenic carbon source.13C metabolite tracing revealed the accumulation of TPI substrates in Δtpiand the absence of alternative triosephosphate isomerases and metabolic bypass reactions, which confirmed the requirement of TPI for glycolysis and gluconeogenesis inM. tuberculosis. The Δtpistrain was furthermore severely attenuated in the mouse model of tuberculosis, suggesting thatM. tuberculosiscannot simultaneously access sufficient quantities of glycolytic and gluconeogenic carbon substrates to establish infection in mice.IMPORTANCEThe importance of central carbon metabolism for the pathogenesis ofM. tuberculosishas recently been recognized, but the consequences of depleting specific metabolic enzymes remain to be identified for many enzymes. We investigated triosephosphate isomerase (TPI) because it is central to both glycolysis and gluconeogenesis and had been predicted to be essential for growth ofM. tuberculosis. This work identified metabolic conditions that make TPI dispensable forM. tuberculosisgrowth in culture and proved thatM. tuberculosisrelies on a single TPI enzyme and has no metabolic bypass for the TPI-dependent interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in glycolysis and gluconeogenesis. Finally, we demonstrate that TPI is essential for growth of the pathogen in mouse lungs.

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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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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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Mycobacterium tuberculosis Alters the Metalloprotease Activity of the COP9 Signalosome

mBio ◽

10.1128/mbio.01278-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 7

Author(s):

Lia Danelishvili ◽

Lmar Babrak ◽

Sasha J. Rose ◽

Jamie Everman ◽

Luiz E. Bermudez

Keyword(s):

Mycobacterium Tuberculosis ◽

Cop9 Signalosome ◽

Virulence Determinants ◽

Bacterial Survival ◽

Phagocytic Cells ◽

Content Type ◽

Metalloprotease Activity ◽

Macrophage Protein

ABSTRACT Inhibition of apoptotic death of macrophages by Mycobacterium tuberculosis represents an important mechanism of virulence that results in pathogen survival both in vitro and in vivo. To identify M. tuberculosis virulence determinants involved in the modulation of apoptosis, we previously screened a transposon bank of mutants in human macrophages, and an M. tuberculosis clone with a nonfunctional Rv3354 gene was identified as incompetent to suppress apoptosis. Here, we show that the Rv3354 gene encodes a protein kinase that is secreted within mononuclear phagocytic cells and is required for M. tuberculosis virulence. The Rv3354 effector targets the metalloprotease (JAMM) domain within subunit 5 of the COP9 signalosome (CSN5), resulting in suppression of apoptosis and in the destabilization of CSN function and regulatory cullin-RING ubiquitin E3 enzymatic activity. Our observation suggests that alteration of the metalloprotease activity of CSN by Rv3354 possibly prevents the ubiquitin-dependent proteolysis of M. tuberculosis-secreted proteins. IMPORTANCE Macrophage protein degradation is regulated by a protein complex called a signalosome. One of the signalosomes associated with activation of ubiquitin and protein labeling for degradation was found to interact with a secreted protein from M. tuberculosis, which binds to the complex and inactivates it. The interference with the ability to inactivate bacterial proteins secreted in the phagocyte cytosol may have crucial importance for bacterial survival within the phagocyte.

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Ambroxol Induces Autophagy and Potentiates Rifampin Antimycobacterial Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01019-18 ◽

2018 ◽

Vol 62 (9) ◽

Cited By ~ 10

Author(s):

Seong Won Choi ◽

Yuexi Gu ◽

Ryan Scott Peters ◽

Padmini Salgame ◽

Jerrold J. Ellner ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Antimycobacterial Activity ◽

Lead Compound ◽

Content Type ◽

Tuberculosis Model

ABSTRACT Host-directed therapy in tuberculosis is a potential adjunct to antibiotic chemotherapy directed at Mycobacterium tuberculosis. Ambroxol, a lead compound, emerged from a screen for autophagy-inducing drugs. At clinically relevant doses, ambroxol induced autophagy in vitro and in vivo and promoted mycobacterial killing in macrophages. Ambroxol also potentiated rifampin activity in a murine tuberculosis model.

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De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01343-16 ◽

2016 ◽

Vol 61 (2) ◽

Cited By ~ 29

Author(s):

Jees Sebastian ◽

Sharmada Swaminath ◽

Rashmi Ravindran Nair ◽

Kishor Jakkala ◽

Atul Pradhan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Hydroxyl Radical ◽

Prolonged Exposure ◽

De Novo ◽

Random Mutagenesis ◽

Content Type ◽

Genome Wide ◽

Resistant Mutants ◽

Phase Population

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

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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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Sulfamethoxazole Susceptibility of Mycobacterium tuberculosis Isolates from HIV-Infected Ugandan Adults with Tuberculosis Taking Trimethoprim-Sulfamethoxazole Prophylaxis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01101-15 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5844-5846 ◽

Cited By ~ 3

Author(s):

Sam Ogwang ◽

Caryn E. Good ◽

Brenda Okware ◽

Mary Nsereko ◽

Michael R. Jacobs ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

Sub Saharan Africa ◽

Content Type ◽

Pulmonary Tb ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Sub Saharan

ABSTRACTAdditional drugs are needed for the treatment of multidrug-resistant tuberculosis (TB). Sulfamethoxazole has been shown to havein vitroactivity againstMycobacterium tuberculosis; however, there is concern about resistance given the widespread use of trimethoprim-sulfamethoxazole prophylaxis among HIV-infected patients in sub-Saharan Africa. Thirty-eight of 40Mycobacterium tuberculosisisolates (95%) from pretreatment sputum samples from Ugandan adults with pulmonary TB, including HIV-infected patients taking trimethoprim-sulfamethoxazole prophylaxis, were susceptible with MICs of ≤38.4 μg/ml.

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Vitamin A Metabolism by Dendritic Cells Triggers an Antimicrobial Response againstMycobacterium tuberculosis

mSphere ◽

10.1128/msphere.00327-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 3

Author(s):

Elliot W. Kim ◽

Avelino De Leon ◽

Zhichun Jiang ◽

Roxana A. Radu ◽

Adrian R. Martineau ◽

...

Keyword(s):

Dendritic Cells ◽

Mycobacterium Tuberculosis ◽

Retinoic Acid ◽

Immune System ◽

Vitamin A ◽

Family Member ◽

Serum Vitamin ◽

Normal Lung ◽

Content Type

ABSTRACTEpidemiological evidence correlates low serum vitamin A (retinol) levels with increased susceptibility to active tuberculosis (TB); however, retinol is biologically inactive and must be converted into its bioactive form, all-transretinoic acid (ATRA). Given that ATRA triggers a Niemann-Pick type C2 (NPC2)-dependent antimicrobial response againstMycobacterium tuberculosis, we investigated the mechanism by which the immune system converts retinol into ATRA at the site of infection. We demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived dendritic cells (DCs), but not macrophages, express enzymes in the vitamin A metabolic pathway, including aldehyde dehydrogenase 1 family, member a2 (ALDH1A2) and short-chain dehydrogenase/reductase family, member 9 (DHRS9), enzymes capable of the two-step conversion of retinol into ATRA, which is subsequently released from the cell. Additionally, mRNA and protein expression levels of ALDH1A2 and DC marker CD1B were lower in tuberculosis lung tissues than in normal lung. The conditioned medium from DCs cultured with retinol stimulated antimicrobial activity fromM. tuberculosis-infected macrophages, as well as the expression of NPC2 in monocytes, which was blocked by specific inhibitors, including retinoic acid receptor inhibitor (RARi) orN,N-diethylaminobenzaldehyde (DEAB), an ALDH1A2 inhibitor. These results indicate that metabolism of vitamin A by DCs transactivates macrophage antimicrobial responses.IMPORTANCETuberculosis (TB) is the leading cause of death by a single infectious agent worldwide. One factor that contributes to the success of the microbe is the deficiency in immunomodulatory nutrients, such as vitamin A (retinol), which are prevalent in areas where TB is endemic. Clinical trials show that restoration of systemic retinol levels in active TB patients is ineffective in mitigating the disease; however, laboratory studies demonstrate that activation of the vitamin A pathway inMycobacterium tuberculosis-infected macrophages triggers an antimicrobial response. Therefore, the goal of this study was to determine the link between host retinol levels and retinoic acid-mediated antimicrobial responses againstM. tuberculosis. By combining establishedin vitromodels within situstudies of lung tissue from TB patients, this study demonstrates that the innate immune system utilizes transcellular metabolism leading to activation between dendritic cells and macrophages as a means to combat the pathogen.

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Hypoxanthine-Guanine Phosphoribosyltransferase Is Dispensable for Mycobacterium smegmatis Viability

Journal of Bacteriology ◽

10.1128/jb.00710-19 ◽

2019 ◽

Vol 202 (5) ◽

Author(s):

Zdeněk Knejzlík ◽

Klára Herkommerová ◽

Dana Hocková ◽

Iva Pichová

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Model Organism ◽

Specific Inhibitor ◽

Purine Salvage ◽

Content Type ◽

Acyclic Nucleoside Phosphonates ◽

Hypoxanthine Guanine Phosphoribosyltransferase

ABSTRACT Purine metabolism plays a ubiquitous role in the physiology of Mycobacterium tuberculosis and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is essential for M. tuberculosis growth in vitro; however, its precise role in M. tuberculosis physiology is unclear. Membrane-permeable prodrugs of specifically designed HGPRT inhibitors arrest the growth of M. tuberculosis and represent potential new antituberculosis compounds. Here, we investigated the purine salvage pathway in the model organism Mycobacterium smegmatis. Using genomic deletion analysis, we confirmed that HGPRT is the only guanine and hypoxanthine salvage enzyme in M. smegmatis but is not required for in vitro growth of this mycobacterium or survival under long-term stationary-phase conditions. We also found that prodrugs of M. tuberculosis HGPRT inhibitors displayed an unexpected antimicrobial activity against M. smegmatis that is independent of HGPRT. Our data point to a different mode of mechanism of action for these inhibitors than was originally proposed. IMPORTANCE Purine bases, released by the hydrolytic and phosphorolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. The hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the formation of guanosine-5′-monophosphate from guanine and inosine-5′-monophosphate from hypoxanthine, represents a potential target for specific inhibitor development. Deletion of the HGPRT gene (Δhgprt) in the model organism Mycobacterium smegmatis confirmed that this enzyme is not essential for M. smegmatis growth. Prodrugs of acyclic nucleoside phosphonates (ANPs), originally designed against HGPRT from Mycobacterium tuberculosis, displayed anti-M. smegmatis activities comparable to those obtained for M. tuberculosis but also inhibited the Δhgprt M. smegmatis strain. These results confirmed that ANPs act in M. smegmatis by a mechanism independent of HGPRT.

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