scholarly journals Inhibiting Mycobacterium tuberculosis CoaBC by targeting a new allosteric site

2019 ◽  
Author(s):  
Vitor Mendes ◽  
Simon R. Green ◽  
Joanna C. Evans ◽  
Jeannine Hess ◽  
Michal Blaszczyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Biosynthetic Pathway ◽  
Model Organism ◽  
Allosteric Site ◽  
Cellular Processes ◽  
Coa Thioesters ◽  
Multiple Pathogens ◽  
Substantial Interest

AbstractCoenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent inhibitors of M. tuberculosis CoaB, which we show to bind to a novel cryptic allosteric site within CoaB.

scholarly journals Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vitor Mendes ◽  
Simon R. Green ◽  
Joanna C. Evans ◽  
Jeannine Hess ◽  
Michal Blaszczyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Biosynthetic Pathway ◽  
Model Organism ◽  
Allosteric Site ◽  
Cellular Processes ◽  
Coa Thioesters ◽  
Multiple Pathogens ◽  
Substantial Interest

AbstractCoenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

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 Transcriptional Inhibition of the F1F0-Type ATP Synthase Has Bactericidal Consequences on the Viability of Mycobacteria

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Matthew B. McNeil ◽  
Heath W. K. Ryburn ◽  
Liam K. Harold ◽  
Justin F. Tirados ◽  
Gregory M. Cook
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Atp Synthase ◽  
Bactericidal Activity ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Potent Inhibitor ◽  
Time Dependent ◽  
Content Type ◽  
Transcriptional Inhibition ◽  
Delayed Time

ABSTRACT Bedaquiline, an inhibitor of the mycobacterial ATP synthase, has revolutionized the treatment of Mycobacterium tuberculosis infection. Although a potent inhibitor, it is characterized by poorly understood delayed time-dependent bactericidal activity. Here, we demonstrate that in contrast to bedaquiline, the transcriptional inhibition of the ATP synthase in M. tuberculosis and Mycobacterium smegmatis has rapid bactericidal activity. These results validate the mycobacterial ATP synthase as a drug target with the potential for rapid bactericidal activity.

Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1647-1658 ◽  
Author(s):  
Narottam Acharya ◽  
Pradeep Kumar ◽  
Umesh Varshney
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Three Dimensional ◽  
Model Organism ◽  
Dna Glycosylase ◽  
Structural Elements ◽  
Dna Glycosylases ◽  
Hydrophobic Pocket ◽  
Uracil Dna Glycosylase ◽  
Simplex Virus

Uracil, a promutagenic base, appears in DNA either by deamination of cytosine or by incorporation of dUMP by DNA polymerases. This unconventional base in DNA is removed by uracil-DNA glycosylase (UDG). Interestingly, a bacteriophage-encoded short polypeptide, UDG inhibitor (Ugi), specifically inhibits UDGs by forming a tight complex. Three-dimensional structures of the complexes of Ugi with UDGs from Escherichia coli, human and herpes simplex virus have shown that two of the structural elements in Ugi, the hydrophobic pocket and the β1-edge, establish key interactions with UDGs. In this report the characterization of complexes of Ugi with UDGs from Mycobacterium tuberculosis, a pathogenic bacterium, and Mycobacterium smegmatis, a widely used model organism for the former, is described. Unlike the E. coli (Eco) UDG-Ugi complex, which is stable to treatment with 8 M urea, the mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea. Furthermore, the Ugi from the complexes of mycobacterial UDGs can be exchanged by the DNA substrate. Interestingly, while EcoUDG sequestered Ugi into the EcoUDG-Ugi complex when incubated with mycobacterial UDG-Ugi complexes, even a large excess of mycobacterial UDGs failed to sequester Ugi from the EcoUDG-Ugi complex. However, the M. tuberculosis (Mtu) UDG-Ugi complex was seen when MtuUDG was incubated with M. smegmatis (Msm) UDG-Ugi or EcoUDG(L191G)-Ugi complexes. The reversible nature of the complexes of Ugi with mycobacterial UDGs (which naturally lack some of the structural elements important for interaction with the β1-edge of Ugi) and with mutants of EcoUDG (which are deficient in interaction with the hydrophobic pocket of Ugi) highlights the significance of both classes of interaction in formation of UDG-Ugi complexes. Furthermore, it is shown that even though mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea, Ugi is still a potent inhibitor of UDG activity in mycobacteria.

scholarly journals 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase (IspC) from Mycobacterium tuberculosis: towards Understanding Mycobacterial Resistance to Fosmidomycin

2005 ◽  
Vol 187 (24) ◽  
pp. 8395-8402 ◽  
Author(s):  
Rakesh K. Dhiman ◽  
Merrill L. Schaeffer ◽  
Ann Marie Bailey ◽  
Charles A. Testa ◽  
Hataichanok Scherman ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Pathogenic Bacteria ◽  
Biosynthetic Pathway ◽  
Inhibitory Concentration ◽  
Forward Direction ◽  
Gram Negative Bacteria ◽  
Isopentenyl Diphosphate ◽  
Intrinsic Properties ◽  
Specificity Constant

ABSTRACT 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl diphosphate biosynthetic pathway in this organism. Therefore, the enzymatic properties of recombinant IspC from M. tuberculosis were characterized. Rv2870c from M. tuberculosis converts 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol 4-phosphate in the presence of NADPH. The enzymatic activity is dependent on the presence of Mg2+ ions and exhibits optimal activity between pH 7.5 and 7.9; the Km for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 μM, and the Km for NADPH was 29.7 μM. The specificity constant of Rv2780c in the forward direction is 1.5 × 106 M−1 min−1, and the reaction is inhibited by fosmidomycin, with a 50% inhibitory concentration of 310 nM. In addition, Rv2870c complements an inactivated chromosomal copy of IspC in Salmonella enterica, and the complemented strain is sensitive to fosmidomycin. Thus, M. tuberculosis resistance to fosmidomycin is not due to intrinsic properties of Rv2870c, and the enzyme appears to be a valid drug target in this pathogen.

scholarly journals Rv0100, a proposed acyl carrier protein in Mycobacterium tuberculosis: expression, purification and crystallization

2019 ◽  
Vol 75 (10) ◽  
pp. 646-651 ◽  
Author(s):  
Jasper Marc G. Bondoc ◽  
Hiten J. Gutka ◽  
Mashal M. Almutairi ◽  
Ryan Patwell ◽  
Maxwell W. Rutter ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Acid Biosynthesis ◽  
Acyl Carrier Proteins ◽  
Drug Discovery And Development ◽  
Novel Drug

Acyl carrier proteins (ACPs) are important components in fatty-acid biosynthesis in prokaryotes. Rv0100 is predicted to be an essential ACP in Mycobacterium tuberculosis, the pathogen that is the causative agent of tuberculosis, and therefore has the potential to be a novel antituberculosis drug target. Here, the successful cloning and purification of Rv0100 using Mycobacterium smegmatis as a host is reported. Crystals of the purified protein were obtained that diffracted to a resolution of 1.9 Å. Overall, this work lays the foundation for the future pursuit of drug discovery and development against this potentially novel drug target.

Genetic analysis of the β-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to β-lactam antibiotics

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 521-532 ◽  
Author(s):  
Anthony R. Flores ◽  
Linda M. Parsons ◽  
Martin S. Pavelka,
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Inhibitory Concentration ◽  
Cell Envelope ◽  
Model Organism ◽  
Resistance Mechanisms ◽  
Disc Diffusion ◽  
Peptidoglycan Biosynthesis ◽  
Strain H37rv

Mycobacteria produce β-lactamases and are intrinsically resistant to β-lactam antibiotics. In addition to the β-lactamases, cell envelope permeability and variations in certain peptidoglycan biosynthetic enzymes are believed to contribute to β-lactam resistance in these organisms. To allow the study of these additional mechanisms, mutants of the major β-lactamases, BlaC and BlaS, were generated in the pathogenic Mycobacterium tuberculosis strain H37Rv and the model organism Mycobacterium smegmatis strain PM274. The mutants M. tuberculosis PM638 (ΔblaC1) and M. smegmatis PM759 (ΔblaS1) showed an increase in susceptibility to β-lactam antibiotics, as determined by disc diffusion and minimal inhibitory concentration (MIC) assays. The susceptibility of the mutants, as assayed by disc diffusion tests, to penicillin-type β-lactam antibiotics was affected most, compared to the cephalosporin-type β-lactam antibiotics. The M. tuberculosis mutant had no detectable β-lactamase activity, while the M. smegmatis mutant had a residual type 1 β-lactamase activity. We identified a gene, blaE, encoding a putative cephalosporinase in M. smegmatis. A double β-lactamase mutant of M. smegmatis, PM976 (ΔblaS1ΔblaE : : res), had no detectable β-lactamase activity, but its susceptibility to β-lactam antibiotics was not significantly different from that of the ΔblaS1 parental strain, PM759. The mutants generated in this study will help determine the contribution of other β-lactam resistance mechanisms in addition to serving as tools to study the biology of peptidoglycan biosynthesis in these organisms.

scholarly journals Distinct Spatiotemporal Dynamics of Peptidoglycan Synthesis between Mycobacterium smegmatis and Mycobacterium tuberculosis

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Helene Botella ◽  
Guangli Yang ◽  
Ouathek Ouerfelli ◽  
Sabine Ehrt ◽  
Carl F. Nathan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mycobacterium Tuberculosis ◽  
Antimicrobial Resistance ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Bacterial Infections ◽  
Super Resolution ◽  
Spatiotemporal Dynamics ◽  
The World ◽  
Super Resolution Microscopy

ABSTRACT Peptidoglycan (PG), a polymer cross-linked by d-amino acid-containing peptides, is an essential component of the bacterial cell wall. We found that a fluorescent d-alanine analog (FDAA) incorporates chiefly at one of the two poles in Mycobacterium smegmatis but that polar dominance varies as a function of the cell cycle in Mycobacterium tuberculosis: immediately after cytokinesis, FDAAs are incorporated chiefly at one of the two poles, but just before cytokinesis, FDAAs are incorporated comparably at both. These observations suggest that mycobacterial PG-synthesizing enzymes are localized in functional compartments at the poles and septum and that the capacity for PG synthesis matures at the new pole in M. tuberculosis. Deeper knowledge of the biology of mycobacterial PG synthesis may help in discovering drugs that disable previously unappreciated steps in the process. IMPORTANCE People are dying all over the world because of the rise of antimicrobial resistance to medicines that could previously treat bacterial infections, including tuberculosis. Here, we used fluorescent d-alanine analogs (FDAAs) that incorporate into peptidoglycan (PG)—the synthesis of which is an attractive drug target—combined with high- and super-resolution microscopy to investigate the spatiotemporal dynamics of PG synthesis in M. smegmatis and M. tuberculosis. FDAA incorporation predominates at one of the two poles in M. smegmatis. In contrast, while FDAA incorporation into M. tuberculosis is also polar, there are striking variations in polar dominance as a function of the cell cycle. This suggests that enzymes involved in PG synthesis are localized in functional compartments in mycobacteria and that M. tuberculosis possesses a mechanism for maturation of the capacity for PG synthesis at the new pole. This may help in discovering drugs that cripple previously unappreciated steps in the process. IMPORTANCE People are dying all over the world because of the rise of antimicrobial resistance to medicines that could previously treat bacterial infections, including tuberculosis. Here, we used fluorescent d-alanine analogs (FDAAs) that incorporate into peptidoglycan (PG)—the synthesis of which is an attractive drug target—combined with high- and super-resolution microscopy to investigate the spatiotemporal dynamics of PG synthesis in M. smegmatis and M. tuberculosis. FDAA incorporation predominates at one of the two poles in M. smegmatis. In contrast, while FDAA incorporation into M. tuberculosis is also polar, there are striking variations in polar dominance as a function of the cell cycle. This suggests that enzymes involved in PG synthesis are localized in functional compartments in mycobacteria and that M. tuberculosis possesses a mechanism for maturation of the capacity for PG synthesis at the new pole. This may help in discovering drugs that cripple previously unappreciated steps in the process.

scholarly journals Phosphorylation on PstP regulates cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis

2019 ◽  
Author(s):  
Farah Shamma ◽  
Kadamba Papavinasasundaram ◽  
Samantha Y. Quintanilla ◽  
Aditya Bandekar ◽  
Christopher Sassetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Control Cell ◽  
Model Organism ◽  
Mycolic Acid ◽  
Regulatory Function ◽  
Antibiotic Tolerance ◽  
Bacterial Survival ◽  
Cell Wall Metabolism

AbstractMycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis.ImportanceRegulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

scholarly journals Phosphorylation on PstP regulates cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Farah Shamma ◽  
Kadamba Papavinasasundaram ◽  
Samantha Y. Quintanilla ◽  
Aditya Bandekar ◽  
Christopher Sassetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Control Cell ◽  
Model Organism ◽  
Mycolic Acid ◽  
Regulatory Function ◽  
Antibiotic Tolerance ◽  
Bacterial Survival ◽  
Cell Wall Metabolism

Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis. Importance Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

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