scholarly journals Identifying Vulnerable Pathways in Mycobacterium tuberculosis by Using a Knockdown Approach

2011 ◽  
Vol 77 (14) ◽  
pp. 5040-5043 ◽  
Author(s):  
Paul Carroll ◽  
Marie-Claire Faray-Kele ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Content Type ◽  
Recombinant Strains

ABSTRACTWe constructed recombinant strains ofMycobacterium tuberculosisin which expression of specific genes was downregulated to identify vulnerable drug targets. Growth phenotypes in macrophages and culture were used to rank targets: thedprE1,clpP1, andfadD32operons were the best targets andglnA1,glnE,pknL,regX3, andsenX3were poor targets.

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

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
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.

scholarly journals Usnic Acid Treatment Changes the Composition of Mycobacterium tuberculosis Cell Envelope and Alters Bacterial Redox Status

mSystems ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Elwira Sieniawska ◽  
Rafal Sawicki ◽  
Wieslaw Truszkiewicz ◽  
Andrey S. Marchev ◽  
Milen I. Georgiev
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Acid Treatment ◽  
Cell Envelope ◽  
Redox Homeostasis ◽  
Usnic Acid ◽  
Lipid Synthesis ◽  
Redox Balance ◽  
Resistance Mechanisms ◽  
Content Type

ABSTRACT Mycobacterium tuberculosis developed efficient adaptation mechanisms in response to different environmental conditions. This resulted in the ability to survive in human macrophages and in resistance to numerous antibiotics. To get insight into bacterial responses to potent antimycobacterial natural compounds, we tested how usnic acid, a lichen-derived secondary metabolite, would influence mycobacteria at transcriptomic and metabolomic levels. The analysis of expression of sigma factors revealed a profound impact of usnic acid on one of the primary genetic regulatory systems of M. tuberculosis. Combined liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses allowed us to observe the perturbations in metabolic pathways, as well as in lipid composition, which took place within 24 h of exposure. Early bacterial response was related to redox homeostasis, lipid synthesis, and nucleic acid repair. Usnic acid treatment provoked disturbances of redox state in mycobacterial cells and increased production of structural elements of the cell wall and cell membrane. In addition, to increase the number of molecules related to restoration of redox balance, the rearrangements of the cell envelope were the first defense mechanisms observed under usnic acid treatment. IMPORTANCE The evaluation of mechanisms of mycobacterial response to natural products has been barely studied. However, it might be helpful to reveal bacterial adaptation strategies, which are eventually crucial for the discovery of new drug targets and, hence, understanding the resistance mechanisms. This study showed that the first-line mycobacterial defense against usnic acid, a potent antimicrobial agent, is the remodeling of the cell envelope and restoring redox homeostasis. Transcriptomic data correlated with metabolomics analysis. The observed metabolic changes appeared similar to those exerted by antibiotics.

scholarly journals Deletion of Rv2571c Confers Resistance to Arylamide Compounds in Mycobacterium tuberculosis

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Catherine D. Shelton ◽  
Matthew B. McNeil ◽  
Julie V. Early ◽  
Thomas R. Ioerger ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Stop Codon ◽  
Fusaric Acid ◽  
Premature Stop Codon ◽  
Acid Resistance ◽  
New Drugs ◽  
Wild Type ◽  
Content Type ◽  
Global Health Problem

ABSTRACT Tuberculosis, caused by Mycobacterium tuberculosis, is an urgent global health problem requiring new drugs, new drug targets, and an increased understanding of antibiotic resistance. We have determined the mode of resistance to be a series of arylamide compounds in M. tuberculosis. We isolated M. tuberculosis resistant mutants to two arylamide compounds which are inhibitory to growth under host-relevant conditions (butyrate as a sole carbon source). Thirteen mutants were characterized, and all had mutations in Rv2571c; mutations included a premature stop codon and frameshifts as well as nonsynonymous polymorphisms. We isolated a further 10 strains with mutations in Rv2571c with resistance. Complementation with a wild-type copy of Rv2571c restored arylamide sensitivity. Overexpression of Rv2571c was toxic in both wild-type and mutant backgrounds. We constructed M. tuberculosis strains with an unmarked deletion of the entire Rv2571c gene by homologous recombination and confirmed that these were resistant to the arylamide series. Rv2571c is a member of the aromatic amino acid transport family and has a fusaric acid resistance domain which is associated with compound transport. Since loss or inactivation of Rv2571c leads to resistance, we propose that Rv2571c is involved in the import of arylamide compounds.

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 Systems Biology-Based Identification of Mycobacterium tuberculosis Persistence Genes in Mouse Lungs

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Noton K. Dutta ◽  
Nirmalya Bandyopadhyay ◽  
Balaji Veeramani ◽  
Gyanu Lamichhane ◽  
Petros C. Karakousis ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Systems Biology ◽  
Biological Networks ◽  
Drug Targets ◽  
Experimental Testing ◽  
Data Types ◽  
Term Survival ◽  
Content Type ◽  
Mouse Lungs

ABSTRACTIdentifyingMycobacterium tuberculosispersistence genes is important for developing novel drugs to shorten the duration of tuberculosis (TB) treatment. We developed computational algorithms that predictM. tuberculosisgenes required for long-term survival in mouse lungs. As the input, we used high-throughputM. tuberculosismutant library screen data, mycobacterial global transcriptional profiles in mice and macrophages, and functional interaction networks. We selected 57 unique, genetically defined mutants (18 previously tested and 39 untested) to assess the predictive power of this approach in the murine model of TB infection. We observed a 6-fold enrichment in the predicted set ofM. tuberculosisgenes required for persistence in mouse lungs relative to randomly selected mutant pools. Our results also allowed us to reclassify several genes as required forM. tuberculosispersistencein vivo. Finally, the new results implicated additional high-priority candidate genes for testing. Experimental validation of computational predictions demonstrates the power of this systems biology approach for elucidatingM. tuberculosispersistence genes.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis (TB), has a genetic repertoire that permits it to persist in the face of host immune responses. Identification of such persistence genes could reveal novel drug targets and elucidate mechanisms by which the organism eludes the immune system and resists drugs. Genetic screens have identified a total of 31 persistence genes, but to date only 15% of the ~4,000M. tuberculosisgenes have been tested experimentally. In this paper, as an alternative to brute force experimental screens, we describe computational methods that predict new persistence genes by combining known examples with growing databases of biological networks. Experimental testing demonstrated that these predictions are highly accurate, validating the computational approach and providing new information aboutM. tuberculosispersistence in host tissues. Using the new experimental results as additional input highlights additional genes for testing. Our approach can be extended to other data types and target organisms to characterize host-pathogen interactions relevant to this and other infectious diseases.

scholarly journals Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Paras Jain ◽  
Tsungda Hsu ◽  
Masayoshi Arai ◽  
Karolin Biermann ◽  
David S. Thaler ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Single Step ◽  
Open Reading Frame ◽  
Model Systems ◽  
Effective Vaccine ◽  
Temperature Sensitive ◽  
Content Type ◽  
Reading Frame ◽  
New Methods

ABSTRACTSpecialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulentMycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library ofM. tuberculosisnull strains, where each strain is deleted for a single defined open reading frame inM. tuberculosis.IMPORTANCEThis work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulentM. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.

scholarly journals Comparative Analyses of Nonpathogenic, Opportunistic, and Totally Pathogenic Mycobacteria Reveal Genomic and Biochemical Variabilities and Highlight the Survival Attributes of Mycobacterium tuberculosis

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Syed Asad Rahman ◽  
Yadvir Singh ◽  
Sakshi Kohli ◽  
Javeed Ahmad ◽  
Nasreen Z. Ehtesham ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Complete Sequence ◽  
Size Analysis ◽  
Comparative Genomic ◽  
Mycobacterial Species ◽  
Content Type ◽  
Alternative Pathways ◽  
Mycobacterium Indicus Pranii ◽  
Pathway Analyses

ABSTRACT Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size—their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. IMPORTANCE The complete sequence analysis of Mycobacterium indicus pranii, a novel species of Mycobacterium shown earlier to have strong immunomodulatory properties and currently in use for the treatment of leprosy, places it evolutionarily at the point of transition to pathogenicity. With the purpose of establishing the importance of M. indicus pranii in providing insight into the virulence mechanism of tuberculous and nontuberculous mycobacteria, we carried out comparative genomic and proteomic analyses of 44 mycobacterial species representing nonpathogenic (NP), opportunistic (OP), and totally pathogenic (TP) mycobacteria. Our results clearly placed M. indicus pranii as an ancestor of the M. avium complex. Analyses of comparative metabolic pathways between M. indicus pranii (NP), M. tuberculosis (TP), and M. intracellulare (OP) pointed to the presence of novel alternative pathways in M. tuberculosis with implications for pathogenesis and survival in the human host and identification of new drug targets.

scholarly journals Identification of 4-Amino-Thieno[2,3-d]Pyrimidines as QcrB Inhibitors in Mycobacterium tuberculosis

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Gregory A. Harrison ◽  
Anne E. Mayer Bridwell ◽  
Megh Singh ◽  
Keshav Jayaraman ◽  
Leslie A. Weiss ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Electron Transport ◽  
Electron Transport Chain ◽  
Drug Target ◽  
Drug Targets ◽  
Bacterial Infections ◽  
Nonsynonymous Mutation ◽  
Content Type ◽  
Transport Chain

ABSTRACT Antibiotic resistance is a global crisis that threatens our ability to treat bacterial infections, such as tuberculosis, caused by Mycobacterium tuberculosis. Of the 10 million cases of tuberculosis in 2017, approximately 19% of new cases and 43% of previously treated cases were caused by strains of M. tuberculosis resistant to at least one frontline antibiotic. There is a clear need for new therapies that target these genetically resistant strains. Here, we report the discovery of a new series of antimycobacterial compounds, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit the growth of M. tuberculosis. To elucidate the mechanism by which these compounds inhibit M. tuberculosis, we selected for mutants resistant to a representative 4-amino-thieno[2,3-d]pyrimidine and sequenced these strains to identify the mutations that confer resistance. We isolated a total of 12 resistant mutants, each of which harbored a nonsynonymous mutation in the gene qcrB, which encodes a subunit of the electron transport chain (ETC) enzyme cytochrome bc1 oxidoreductase, leading us to hypothesize that 4-amino-thieno[2,3-d]pyrimidines target this enzyme complex. We found that addition of 4-amino-thieno[2,3-d]pyrimidines to M. tuberculosis cultures resulted in a decrease in ATP levels, supporting our model that these compounds inhibit the M. tuberculosis ETC. Furthermore, 4-amino-thieno[2,3-d]pyrimidines had enhanced activity against a mutant of M. tuberculosis deficient in cytochrome bd oxidase, which is a hallmark of cytochrome bc1 inhibitors. Therefore, 4-amino-thieno[2,3-d]pyrimidines represent a novel series of QcrB inhibitors that build on the growing number of chemical scaffolds that are able to inhibit the mycobacterial cytochrome bc1 complex. IMPORTANCE The global tuberculosis (TB) epidemic has been exacerbated by the rise in drug-resistant TB cases worldwide. To tackle this crisis, it is necessary to identify new vulnerable drug targets in Mycobacterium tuberculosis, the causative agent of TB, and develop compounds that can inhibit the bacterium through novel mechanisms of action. The QcrB subunit of the electron transport chain enzyme cytochrome bc1 has recently been validated to be a potential drug target. In the current work, we report the discovery of a new class of QcrB inhibitors, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit M. tuberculosis growth in vitro. These compounds are chemically distinct from previously reported QcrB inhibitors, and therefore, 4-amino-thieno[2,3-d]pyrimidines represent a new scaffold that can be exploited to inhibit this drug target.

scholarly journals Genome analyses of 174 strains of Mycobacterium tuberculosis provide insight into the evolution of drug resistance and reveal potential drug targets

2021 ◽  
Vol 7 (3) ◽  
Author(s):  
Helianthous Verma ◽  
Shekhar Nagar ◽  
Shivani Vohra ◽  
Shubhanshu Pandey ◽  
Devi Lal ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Drug Targets ◽  
Type Species ◽  
Close Association ◽  
Drug Resistant ◽  
Potential Drug ◽  
Sequence Alignments ◽  
Content Type ◽  
Link Type

Mycobacterium tuberculosis is a known human pathogen that causes the airborne infectious disease tuberculosis (TB). Every year TB infects millions of people worldwide. The emergence of multi-drug resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) M. tuberculosis strains against the first- and second-line anti-TB drugs has created an urgent need for the development and implementation of new drug strategies. In this study, the complete genomes of 174 strains of M. tuberculosis are analysed to understand the evolution of molecular drug target (MDT) genes. Phylogenomic placements of M. tuberculosis strains depicted close association and temporal clustering. Selection pressure analysis by deducing the ratio of non-synonymous to synonymous substitution rates (dN/dS) in 51 MDT genes of the 174 M . tuberculosis strains led to categorizing these genes into diversifying (D, dN/dS>0.70), moderately diversifying (MD, dN/dS=0.35–0.70) and stabilized (S, dN/dS<0.35) genes. The genes rpsL, gidB, pncA and ahpC were identified as diversifying, and Rv0488, kasA, ndh, ethR, ethA, embR and ddn were identified as stabilized genes. Furthermore, sequence similarity networks were drawn that supported these divisions. In the multiple sequence alignments of diversifying and stabilized proteins, previously reported resistance mutations were checked to predict sensitive and resistant strains of M. tuberculosis . Finally, to delineate the potential of stabilized or least diversified genes/proteins as anti-TB drug targets, protein–protein interactions of MDT proteins with human proteins were analysed. We predict that kasA (dN/dS=0.29), a stabilized gene that encodes the most host-interacting protein, KasA, should serve as a potential drug target for the treatment of TB.

Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

2012 ◽  
Vol 12 (7) ◽  
pp. 766-796 ◽  
Author(s):  
Benjamin P. Duckworth ◽  
Kathryn M. Nelson ◽  
Courtney C. Aldrich
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets
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