scholarly journals The Veterinary Anti-Parasitic Selamectin Is a Novel Inhibitor of the Mycobacterium tuberculosis DprE1 Enzyme

2022 ◽  
Vol 23 (2) ◽  
pp. 771
Author(s):  
José Manuel Ezquerra-Aznárez ◽  
Giulia Degiacomi ◽  
Henrich Gašparovič ◽  
Giovanni Stelitano ◽  
Josè Camilla Sammartino ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Point Mutations ◽  
Anthelmintic Activity ◽  
Docking Studies ◽  
Metabolic Labeling ◽  
Biochemical Assays ◽  
Mutant Strains ◽  
In Vitro Interactions

Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-β-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.

scholarly journals The veterinary anti-parasitic selamectin is a novel inhibitor of the mycobacterial DprE1 enzyme

2021 ◽  
Author(s):  
José Manuel Ezquerra-Aznárez ◽  
Giulia Degiacomi ◽  
Henrich Gašparovič ◽  
Giovanni Stelitano ◽  
José Camilla Sammartino ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Point Mutations ◽  
Anthelmintic Activity ◽  
Docking Studies ◽  
Metabolic Labeling ◽  
Mycobacterial Species ◽  
Macrocyclic Lactones ◽  
Biochemical Assays ◽  
Mutant Strains

AbstractAvermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-β-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.

scholarly journals Design and synthesis of new 3-((7-chloroquinolin-4-yl)amino)thiazolidin-4-one analogs as Mycobacterium tuberculosis DNA gyrase inhibitors

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Preeti S. Salve ◽  
Prajakta Parchure ◽  
Lisel Araujo ◽  
Rohini S. Kavalapure ◽  
Sunil S. Jalalpure ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Dna Gyrase ◽  
Docking Studies ◽  
Human Populations ◽  
Antitubercular Agents ◽  
Major Health ◽  
Design And Synthesis ◽  
Gyrase Inhibitors

Abstract Background Tuberculosis is evidently a major health threat among human populations worldwide. The current study presents the synthesis of new 3-((7-chloroquinolin-4-yl)amino)thiazolidin-4-one analogs (4a–o) as potential Mycobacterium tuberculosis DNA gyrase inhibitors. DNA gyrase regulates DNA topology in MTB and has been a target of choice for antibacterial therapy. With this in mind, the synthesized derivatives (4a–o) were subjected to in vitro antitubercular evaluation by the MABA method and were tested for MTB DNA gyrase inhibition by supercoiling assay. Results All the synthesized compounds displayed inhibition of MTB within the MIC range of 1.56–12.5 μM. Further, out of the selected compounds that underwent DNA gyrase inhibition, compound 4o proved to be a potent lead molecule by displaying 82% of enzyme inhibition at 1 μM. All the synthesized derivatives also underwent molecular docking studies to comprehend their hypothetical binding interactions with Mycobacterium smegmatis GyrB. Conclusion All the results suggested that most of the synthesized derivatives inhibited Mycobacterium tuberculosis, and some 3-((7-chloroquinolin-4-yl)amino)thiazolidin-4-one analogs could act as leads for the development of antitubercular agents.

scholarly journals Expression of Mycobacterium smegmatisPyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

2000 ◽  
Vol 182 (19) ◽  
pp. 5479-5485 ◽  
Author(s):  
Helena I. M. Boshoff ◽  
Valerie Mizrahi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Intracellular Localization ◽  
Wild Type ◽  
Gene Encoding ◽  
Allelic Exchange ◽  
Mutant Strains ◽  
Established Role ◽  
Amidase Gene

ABSTRACT A pyrazinamidase (PZase)-deficient pncA mutant ofMycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role ofpncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the M. tuberculosis pncA gene into the pncAmutant complemented its PZase/nicotinamidase defect. In bothpzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. ThepzaA-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type andpncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 μg/ml in all cases) and rendered Escherichia colihypersensitive for growth at low pH.

scholarly journals Hypoxanthine-Guanine Phosphoribosyltransferase Is Dispensable for Mycobacterium smegmatis Viability

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.

scholarly journals The Redox Cofactor F420Protects Mycobacteria from Diverse Antimicrobial Compounds and Mediates a Reductive Detoxification System

2016 ◽  
Vol 82 (23) ◽  
pp. 6810-6818 ◽  
Author(s):  
Thanavit Jirapanjawat ◽  
Blair Ney ◽  
Matthew C. Taylor ◽  
Andrew C. Warden ◽  
Shahana Afroze ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Mycobacterium Smegmatis ◽  
Antimicrobial Compounds ◽  
Loss Of Function ◽  
Detoxifying Enzymes ◽  
Content Type ◽  
Wide Range ◽  
Detoxification System ◽  
Mutant Strains

ABSTRACTA defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F420. This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F420enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacteriumMycobacterium smegmatis. Mutant strains unable to synthesize or reduce F420were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F420mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover,M. smegmatisstrains unable to make F420H2lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F420loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F420enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.IMPORTANCEThis study reveals that a unique microbial cofactor, F420, is critical for antimicrobial resistance in the environmental actinobacteriumMycobacterium smegmatis. We show that a superfamily of redox enzymes, the F420H2-dependent reductases, can reduce diverse antimicrobialsin vitroandin vivo.M. smegmatisstrains unable to make or reduce F420become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F420to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F420H2-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.

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

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.

scholarly journals Hypoxic Response of Mycobacterium tuberculosis Studied by Metabolic Labeling and Proteome Analysis of Cellular and Extracellular Proteins

2002 ◽  
Vol 184 (13) ◽  
pp. 3485-3491 ◽  
Author(s):  
Ida Rosenkrands ◽  
Richard A. Slayden ◽  
Janne Crawford ◽  
Claus Aagaard ◽  
Clifton E. Barry ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proteome Analysis ◽  
Peptide Mass Fingerprinting ◽  
Extracellular Proteins ◽  
Metabolic Labeling ◽  
Low Oxygen ◽  
Hypoxic Response ◽  
Peptide Mass ◽  
Oxygen Conditions

ABSTRACT The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli.

The anthelmintic activity of 1,10-phenanthroline, a metalloenzyme inhibitor

2019 ◽  
Author(s):  
S.M.A. Abidi ◽  
Kavita Singh ◽  
A. Rehman ◽  
R. Ullah ◽  
L. Rehman ◽  
...  
Keyword(s):  
Enzyme Inhibitor ◽  
Anthelmintic Activity ◽  
In Vivo Studies ◽  
Parasitic Helminths ◽  
Tegumental Surface ◽  
Biochemical Assays ◽  
Direct Exposure ◽  
Surface Changes

Paramphistomosis is a chronic, debilitating parasitic disease of livestock prevalent in the tropical and sub-tropical countries. Globally there is a heavy reliance on anthelmintics but concerns over drug resistance encourage the search for new leads. Metalloproteinases play a significant role in the biology and life cycle of parasitic helminths. The efficacy of metalloproteinase inhibitor, 1,10-Phenanthroline (1,10-phe) which is commonly used as a specific enzyme inhibitor in biochemical assays, was tested in vitro on Gigantocotyle explanatum tegument as a marker of anthelmintic action. The scanning electron microscopy revealed that the tegumental surface exhibited considerable changes in the worms treated with the metalloenzyme inhibitor, 1,10-phe. The untreated control worms appeared normal showing smooth tegumental surface with abundant dome shaped papillae in the anterior to mid region, while their density was less around the acetabulum which serves as a hold-fast organ helping the worms to remain attached in biliary passage. The 1,10-phe produced significant tegumental damage when the liver amphistomes were in vitro exposed to this compound at 12.5 µM concentration. The surface changes appeared in the form of edematous ridges with prominent furrows and erosion of the dome shaped papillae with rosette shaped deep lesions as a result of which deep parenchymatous tissues were exposed. The collapse of sensory bulbs as well as sloughing of tegument, particularly in the anterior-mid region was observed. The nature of damage could be comparable to various anthelmintics used in previous studies. To the best of our knowledge this is the first report of direct exposure of amphistome worms to zinc metallo-enzyme inhibitor, however, further in vivo studies are required to ascertain the anthelmintic efficacy of 1,10-phe.

scholarly journals In Vitro Isolation and Characterization of Oxazolidinone-Resistant Mycobacterium tuberculosis

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Matthew B. McNeil ◽  
Devon D. Dennison ◽  
Catherine D. Shelton ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
23S Rrna ◽  
Clinical Settings ◽  
Content Type ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Ribosomal Protein L3 ◽  
Resistant Strains

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

scholarly journals Two New Point Mutations at A2062 Associated with Resistance to 16-Membered Macrolide Antibiotics in Mutant Strains ofMycoplasma hominis

2001 ◽  
Vol 45 (10) ◽  
pp. 2958-2960 ◽  
Author(s):  
Pio Maria Furneri ◽  
Giancarlo Rappazzo ◽  
Maria Pia Musumarra ◽  
Patrizia Di Pietro ◽  
Lucrezia S. Catania ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Mycoplasma Hominis ◽  
23S Rrna ◽  
Macrolide Antibiotics ◽  
Rrna Gene ◽  
In Vitro Exposure ◽  
Mutant Strains ◽  
23S Rrna Gene

ABSTRACT We describe two mutants of Mycoplasma hominis PG-21 which show resistance to 16-membered macrolides but susceptibility to lincosamides, obtained by in vitro exposure to increasing doses of josamycin. The 23S rRNA gene showed that each had a mutation (A2062G and A2062T) corresponding to nucleotide 2062 in Escherichia coli, which was associated with the acquired phenotype.

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