scholarly journals Respiratory Flexibility in Response to Inhibition of CytochromecOxidase in Mycobacterium tuberculosis

2014 ◽  
Vol 58 (11) ◽  
pp. 6962-6965 ◽  
Author(s):  
Kriti Arora ◽  
Bernardo Ochoa-Montaño ◽  
Patricia S. Tsang ◽  
Tom L. Blundell ◽  
Stephanie S. Dawes ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Energy Metabolism ◽  
New Drugs ◽  
Dependent Manner ◽  
Content Type ◽  
Strain Dependent

ABSTRACTWe report here a series of five chemically diverse scaffolds that havein vitroactivities on replicating and hypoxic nonreplicating bacilli by targeting the respiratorybc1complex inMycobacterium tuberculosisin a strain-dependent manner. Deletion of the cytochromebdoxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation inqcrB. These results highlight the promiscuity of thebc1complex and the risk of targeting energy metabolism with new drugs.

scholarly journals Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Matthew B. McNeil ◽  
Theresa O’Malley ◽  
Devon Dennison ◽  
Catherine D. Shelton ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
New Drugs ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Primary Mechanism ◽  
Resistant Mutants ◽  
Mycobacterial Cell Wall

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

scholarly journals Peptidoglycan Hydrolases RipA and Ami1 Are Critical for Replication and Persistence of Mycobacterium tuberculosis in the Host

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Claire Healy ◽  
Alexandre Gouzy ◽  
Sabine Ehrt
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Drug Target ◽  
New Drugs ◽  
Chemotherapeutic Regimen ◽  
Content Type ◽  
Cell Wall Polymer ◽  
Redundant Functions

ABSTRACT Synthesis and cleavage of the cell wall polymer peptidoglycan (PG) are carefully orchestrated processes and are essential for the growth and survival of bacteria. Yet, the function and importance of many enzymes that act on PG in Mycobacterium tuberculosis remain to be elucidated. We demonstrate that the activity of the N-acetylmuramyl-l-alanine amidase Ami1 is dispensable for cell division in M. tuberculosis in vitro yet contributes to the bacterium’s ability to persist during chronic infection in mice. Furthermore, the d,l-endopeptidase RipA, a predicted essential enzyme, is dispensable for the viability of M. tuberculosis but required for efficient cell division in vitro and in vivo. Depletion of RipA sensitizes M. tuberculosis to rifampin and to cell envelope-targeting antibiotics. Ami1 helps sustain residual cell division in cells lacking RipA, but the partial redundancy provided by Ami1 is not sufficient during infection, as depletion of RipA prevents M. tuberculosis from replicating in macrophages and leads to dramatic killing of the bacteria in mice. Notably, RipA is essential for persistence of M. tuberculosis in mice, suggesting that cell division is required during chronic mouse infection. Despite the multiplicity of enzymes acting on PG with redundant functions, we have identified two PG hydrolases that are important for M. tuberculosis to replicate and persist in the host. IMPORTANCE Tuberculosis (TB) is a major global heath burden, with 1.6 million people succumbing to the disease every year. The search for new drugs to improve the current chemotherapeutic regimen is crucial to reducing this global health burden. The cell wall polymer peptidoglycan (PG) has emerged as a very successful drug target in bacterial pathogens, as many currently used antibiotics target the synthesis of this macromolecule. However, the multitude of genes encoding PG-synthesizing and PG-modifying enzymes with apparent redundant functions has hindered the identification of novel drug targets in PG synthesis in Mycobacterium tuberculosis. Here, we demonstrate that two PG-cleaving enzymes are important for virulence of M. tuberculosis. In particular, the d,l-endopeptidase RipA represents a potentially attractive drug target, as its depletion results in the clearance of M. tuberculosis from the host and renders the bacteria hypersusceptible to rifampin, a frontline TB drug, and to several cell wall-targeting antibiotics.

scholarly journals Characterization and Transcriptome Analysis of Mycobacterium tuberculosis Persisters

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Iris Keren ◽  
Shoko Minami ◽  
Eric Rubin ◽  
Kim Lewis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Major Public Health Problem ◽  
Dependent Manner ◽  
Persister Cells ◽  
Large Reservoir ◽  
Content Type ◽  
Infected People ◽  
Latently Infected ◽  
Growing Population

ABSTRACTTuberculosis continues to be a major public health problem in many parts of the world. Significant obstacles in controlling the epidemic are the length of treatment and the large reservoir of latently infected people. Bacteria form dormant, drug-tolerant persister cells, which may be responsible for the difficulty in treating both acute and latent infections. We find that inMycobacterium  tuberculosis, low numbers of drug-tolerant persisters are present in lag and early exponential phases, increasing sharply at late exponential and stationary phases to make up ~1% of the population. This suggests that persister formation is governed by both stochastic and deterministic mechanisms. In order to isolate persisters, an exponentially growing population was treated withd-cycloserine, and cells surviving lysis were collected by centrifugation. A transcriptome of persisters was obtained by using hybridization to an Affymetrix array. The transcriptome shows downregulation of metabolic and biosynthetic pathways, consistent with a certain degree of dormancy. A set of genes was upregulated in persisters, and these are likely involved in persister formation and maintenance. A comparison of the persister transcriptome with transcriptomes obtained for severalin vitrodormancy models identified a small number of genes upregulated in all cases, which may represent a core dormancy response.IMPORTANCEIt is estimated that every third person on the planet is infected withMycobacterium tuberculosis. The two major problems in controllingM. tuberculosisare the length of the treatment and the large reservoir of latently infected people. Dormant persister cells may be responsible for both problems. We find thatM. tuberculosisproduces persistersin vitroin a growth phase-dependent manner. Persisters were isolated from an exponentially growing population, and their transcriptome shows a distinct pattern of dormancy. These results give the first insight intoM. tuberculosispersisters and point to possible mechanisms responsible for their formation.

scholarly journals Dual Mechanism of Action of 5-Nitro-1,10-Phenanthroline against Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Saqib Kidwai ◽  
Chan-Yong Park ◽  
Shradha Mawatwal ◽  
Prabhakar Tiwari ◽  
Myung Geun Jung ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mechanism Of Action ◽  
Chemotherapeutic Agents ◽  
Intracellular Bacteria ◽  
Dependent Manner ◽  
Content Type ◽  
Structure Activity ◽  
Dual Mechanism

ABSTRACT New chemotherapeutic agents with novel mechanisms of action are urgently required to combat the challenge imposed by the emergence of drug-resistant mycobacteria. In this study, a phenotypic whole-cell screen identified 5-nitro-1,10-phenanthroline (5NP) as a lead compound. 5NP-resistant isolates harbored mutations that were mapped to fbiB and were also resistant to the bicyclic nitroimidazole PA-824. Mechanistic studies confirmed that 5NP is activated in an F420-dependent manner, resulting in the formation of 1,10-phenanthroline and 1,10-phenanthrolin-5-amine as major metabolites in bacteria. Interestingly, 5NP also killed naturally resistant intracellular bacteria by inducing autophagy in macrophages. Structure-activity relationship studies revealed the essentiality of the nitro group for in vitro activity, and an analog, 3-methyl-6-nitro-1,10-phenanthroline, that had improved in vitro activity and in vivo efficacy in mice compared with that of 5NP was designed. These findings demonstrate that, in addition to a direct mechanism of action against Mycobacterium tuberculosis, 5NP also modulates the host machinery to kill intracellular pathogens.

scholarly journals The Combination of Sulfamethoxazole, Trimethoprim, and Isoniazid or Rifampin Is Bactericidal and Prevents the Emergence of Drug Resistance in Mycobacterium tuberculosis

2012 ◽  
Vol 56 (10) ◽  
pp. 5142-5148 ◽  
Author(s):  
Catherine Vilchèze ◽  
William R. Jacobs
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Multidrug Resistant ◽  
New Drugs ◽  
Drug Resistant ◽  
Content Type ◽  
The Past ◽  
Tb Treatment

ABSTRACTThe challenges of developing new drugs to treat tuberculosis (TB) are indicated by the relatively small number of candidates entering clinical trials in the past decade. To overcome these issues, we reexamined two FDA-approved antibacterial drugs, sulfamethoxazole (SMX) and trimethoprim (TMP), for use in TB treatment. SMX and TMP inhibit folic acid biosynthesis and are used in combination to treat infections of the respiratory, urinary, and gastrointestinal tracts. The MICs of SMX and TMP, alone and in combination, were determined for drug-susceptible, multidrug-resistant (MDR), and extensively drug-resistantMycobacterium tuberculosisstrains. While TMP alone was not effective againstM. tuberculosis, the combination of TMP and SMX was bacteriostatic againstM. tuberculosis. Surprisingly, the combination of SMX and TMP was also active against a subset of MDRM. tuberculosisstrains. Treatment ofM. tuberculosiswith TMP-SMX and a first-line anti-TB drug, either isoniazid or rifampin, was bactericidal, demonstrating that the combination of TMP and SMX with isoniazid or rifampin was not antagonistic. Moreover, the addition of SMX-TMP in combination with either isoniazid or rifampin also prevented the emergence of drug resistancein vitro. In conclusion, this study further illustrates the opportunity to reevaluate the activity of TMP-SMXin vivoto prevent the emergence of drug-resistantM. tuberculosis.

scholarly journals Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Jerome Prusa ◽  
Drake Jensen ◽  
Gustavo Santiago-Collazo ◽  
Steven S. Pope ◽  
Ashley L. Garner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mycobacterium Tuberculosis ◽  
Transcription Initiation ◽  
Dependent Manner ◽  
Interaction Domain ◽  
Structural Domains ◽  
Content Type ◽  
Group I

ABSTRACT The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RP o ) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP β′ and σ subunits, a core domain (CD) that contacts the RNAP β′ subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited in vivo studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis . We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RP o stabilization in vitro , while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA. IMPORTANCE Mycobacterium tuberculosis is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in M. tuberculosis transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general.

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

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
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.

scholarly journals Novel Pyrimidines as Antitubercular Agents

2018 ◽  
Vol 62 (3) ◽  
Author(s):  
Daigo Inoyama ◽  
Steven D. Paget ◽  
Riccardo Russo ◽  
Srinivasan Kandasamy ◽  
Pradeep Kumar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Small Molecules ◽  
New Drugs ◽  
Cross Resistance ◽  
Antitubercular Agents ◽  
Front Line ◽  
Content Type ◽  
Pharmacokinetic Profiles ◽  
Global Pandemic

ABSTRACTMycobacterium tuberculosisinfection is responsible for a global pandemic. New drugs are needed that do not show cross-resistance with the existing front-line therapeutics. A triazine antitubercular hit led to the design of a related pyrimidine family. The synthesis of a focused series of these analogs facilitated exploration of theirin vitroactivity,in vitrocytotoxicity, and physiochemical and absorption-distribution-metabolism-excretion properties. Select pyrimidines were then evaluated for their pharmacokinetic profiles in mice. The findings suggest a rationale for the further evolution of this promising series of antitubercular small molecules, which appear to share some similarities with the clinical compound PA-824 in terms of activation, while highlighting more general guidelines for the optimization of small-molecule antitubercular agents.

scholarly journals Strain-Dependent Inhibition of Clostridioides difficile by Commensal Clostridia Carrying the Bile Acid-Inducible (bai) Operon

2020 ◽  
Vol 202 (11) ◽  
Author(s):  
A. D. Reed ◽  
M. A. Nethery ◽  
A. Stewart ◽  
R. Barrangou ◽  
C. M. Theriot
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Dependent Manner ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Coculture System ◽  
The Relationship ◽  
Strain Dependent ◽  
Secondary Bile Acids

ABSTRACT Clostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. Gut microbiota-derived secondary bile acids and commensal Clostridia that carry the bile acid-inducible (bai) operon are associated with protection from C. difficile infection (CDI), although the mechanism is not known. In this study, we hypothesized that commensal Clostridia are important for providing colonization resistance against C. difficile due to their ability to produce secondary bile acids, as well as potentially competing against C. difficile for similar nutrients. To test this hypothesis, we examined the abilities of four commensal Clostridia carrying the bai operon (Clostridium scindens VPI 12708, C. scindens ATCC 35704, Clostridium hiranonis, and Clostridium hylemonae) to convert cholate (CA) to deoxycholate (DCA) in vitro, and we determined whether the amount of DCA produced was sufficient to inhibit the growth of a clinically relevant C. difficile strain. We also investigated the competitive relationships between these commensals and C. difficile using an in vitro coculture system. We found that inhibition of C. difficile growth by commensal Clostridia supplemented with CA was strain dependent, correlated with the production of ∼2 mM DCA, and increased the expression of bai operon genes. We also found that C. difficile was able to outcompete all four commensal Clostridia in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics. Future studies dissecting the regulation of the bai operon in vitro and in vivo and how this affects CDI will be important. IMPORTANCE Commensal Clostridia carrying the bai operon, such as C. scindens, have been associated with protection against CDI; however, the mechanism for this protection is unknown. Herein, we show four commensal Clostridia that carry the bai operon and affect C. difficile growth in a strain-dependent manner, with and without the addition of cholate. Inhibition of C. difficile by commensals correlated with the efficient conversion of cholate to deoxycholate, a secondary bile acid that inhibits C. difficile germination, growth, and toxin production. Competition studies also revealed that C. difficile was able to outcompete the commensals in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics.

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.

Sign in / Sign up

Export Citation Format

Share Document