scholarly journals Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Genetics of dTDP-Rhamnose Synthetic Enzymes and Development of a Microtiter Plate-Based Screen for Inhibitors of Conversion of dTDP-Glucose to dTDP-Rhamnose

2001 ◽  
Vol 45 (5) ◽  
pp. 1407-1416 ◽  
Author(s):  
Yufang Ma ◽  
Richard J. Stern ◽  
Michael S. Scherman ◽  
Varalakshmi D. Vissa ◽  
Wenxin Yan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Enzyme Assay ◽  
Enzyme Inhibitors ◽  
Microtiter Plate ◽  
Structural Motif ◽  
Chemical Library ◽  
Microtiter Plate Assay ◽  
Synthetic Enzymes ◽  
Genes Encoding

ABSTRACT An l-rhamnosyl residue plays an essential structural role in the cell wall of Mycobacterium tuberculosis. Therefore, the four enzymes (RmlA to RmlD) that form dTDP-rhamnose from dTTP and glucose-1-phosphate are important targets for the development of new tuberculosis therapeutics. M. tuberculosis genes encoding RmlA, RmlC, and RmlD have been identified and expressed inEscherichia coli. It is shown here that genes for only one isotype each of RmlA to RmlD are present in the M. tuberculosis genome. The gene for RmlB is Rv3464. Rv3264c was shown to encode ManB, not a second isotype of RmlA. Using recombinant RmlB, -C, and -D enzymes, a microtiter plate assay was developed to screen for inhibitors of the formation of dTDP-rhamnose. The three enzymes were incubated with dTDP-glucose and NADPH to form dTDP-rhamnose and NADP+ with a concomitant decrease in optical density at 340 nm (OD340). Inhibitor candidates were monitored for their ability to lower the rate of OD340change. To test the robustness and practicality of the assay, a chemical library of 8,000 compounds was screened. Eleven inhibitors active at 10 μM were identified; four of these showed activities against whole M. tuberculosis cells, with MICs from 128 to 16 μg/ml. A rhodanine structural motif was present in three of the enzyme inhibitors, and two of these showed activity against wholeM. tuberculosis cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth ofM. tuberculosis in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 μg/ml.

scholarly journals Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Development of a Microtiter Plate-Based Screen for UDP-Galactopyranose Mutase and Identification of an Inhibitor from a Uridine-Based Library

2003 ◽  
Vol 47 (1) ◽  
pp. 378-382 ◽  
Author(s):  
Michael S. Scherman ◽  
Katharine A. Winans ◽  
Richard J. Stern ◽  
Victoria Jones ◽  
Carolyn R. Bertozzi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targeting ◽  
Enzyme Inhibitor ◽  
Microtiter Plate ◽  
Biosynthetic Enzyme ◽  
Cell Wall Synthesis ◽  
Chemical Library ◽  
Microtiter Plate Assay ◽  
Plate Assay

ABSTRACT A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.

scholarly journals Quantitative measurement of antibiotic resistance in Mycobacterium tuberculosis reveals genetic determinants of resistance and susceptibility in a target gene approach

2021 ◽  
Author(s):  
◽  
Joshua J Carter
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Quantitative Measurement ◽  
Target Gene ◽  
Microtiter Plate ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Genetic Determinants ◽  
Microtiter Plate Assay ◽  
Health Organization

AbstractThe World Health Organization goal of universal drug susceptibility testing for patients with tuberculosis is most likely to be achieved through molecular diagnostics; however, to date these have focused largely on first-line drugs, and always on predicting binary susceptibilities. Here, we used whole genome sequencing and a quantitative microtiter plate assay to relate genomic mutations to minimum inhibitory concentration in 15,211 Mycobacterium tuberculosis patient isolates from 27 countries across five continents.This work identifies 449 unique MIC-elevating genetic determinants across thirteen drugs, as well as 91 mutations resulting in hypersensitivity for eleven drugs. Our results provide a guide for further implementation of personalized medicine for the treatment of tuberculosis using genetics-based diagnostics and can serve as a training set for novel approaches to predict drug resistance.

scholarly journals Comparative Transcriptional Study of the Putative Mannose Donor Biosynthesis Genes in Virulent Mycobacterium tuberculosis and Attenuated Mycobacterium bovis BCG Strains

2011 ◽  
Vol 79 (11) ◽  
pp. 4668-4673 ◽  
Author(s):  
Tracy L. Keiser ◽  
Abul K. Azad ◽  
Evelina Guirado ◽  
Robert Bonacci ◽  
Larry S. Schlesinger
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Broth Culture ◽  
Biosynthesis Pathway ◽  
Regulation Of Expression ◽  
Content Type ◽  
Expression Levels ◽  
Genes Encoding ◽  
Recognition And Response

ABSTRACTMycobacterium tuberculosiscontains mannosylated cell wall components which are important in macrophage recognition and response. The building block for the mannosyl constituents of these components is GDP-mannose, which is synthesized through a series of enzymes involved in the mannose donor biosynthesis pathway. Nothing is known about the expression levels of the genes encoding these enzymes during the course of infection. To generate transcriptional profiles for the mannose donor biosynthesis genes from virulentM. tuberculosisand attenuatedMycobacterium bovisBCG, bacteria were grown in broth culture and within human macrophages. Our results with broth-grown bacteria show that there are differences in expression of the selected genes betweenM. tuberculosisand BCG, with increased expression ofmanCinM. tuberculosisandmanAin BCG during stationary-phase growth. Results forM. tuberculosisextracted from within macrophages show thatwhiB2is highly expressed andmanBandmanCare moderately expressed during infection.Rv3256c,Rv3258c, andppm1have high expression levels early and decreased expression as the infection progresses. Results with BCG show that, as inM. tuberculosis,whiB2is highly expressed throughout infection, whereas there is either low expression or little change in expression of the remaining genes studied. Overall, our results show that there is differential regulation of expression of several genes in the mannose donor biosynthesis pathway ofM. tuberculosisand BCG grown in broth and within macrophages, raising the possibility that the level of mannose donors may vary during the course of infection and thereby impact the biosynthesis of mannose-containing cell wall molecules.

scholarly journals Chitinases from Uncultured Marine Microorganisms

1999 ◽  
Vol 65 (6) ◽  
pp. 2553-2557 ◽  
Author(s):  
Matthew T. Cottrell ◽  
Jessica A. Moore ◽  
David L. Kirchman
Keyword(s):  
Coastal Waters ◽  
Bacterial Communities ◽  
Plaque Assay ◽  
Microtiter Plate ◽  
Culturable Bacteria ◽  
Marine Microorganisms ◽  
Microtiter Plate Assay ◽  
Genes Encoding ◽  
Chitinase Genes ◽  
Hydrolysis Of

ABSTRACT Our understanding of the degradation of organic matter will benefit from a greater appreciation for the genes encoding enzymes involved in the hydrolysis of biopolymers such as chitin, one of the most abundant polymers in nature. To isolate representative and abundant chitinase genes from uncultivated marine bacteria, we constructed libraries of genomic DNA isolated from coastal and estuarine waters. The libraries were screened for genes encoding proteins that hydrolyze a fluorogenic analogue of chitin, 4-methylumbelliferyl β-d-N,N′-diacetylchitobioside (MUF-diNAG). The abundance of clones capable of MUF-diNAG hydrolysis was higher in the library constructed with DNA from the estuary than in that constructed with DNA from coastal waters, although the abundance of positive clones was also dependent on the method used to screen the library. Plaque assays revealed nine MUF-diNAG-positive clones of 75,000 screened for the estuarine sample and two clones of 750,000 for the coastal sample. A microtiter plate assay revealed approximately 1 positive clone for every 500 clones screened in the coastal library. The number of clones detected with the plaque assay was consistent with estimates of the portion of culturable bacteria that degrade chitin. Our results suggest that culture-dependent methods do not greatly underestimate the portion of marine bacterial communities capable of chitin degradation.

Design, Synthesis and Antitubercular Evaluation of New Benzimidazole Scaffolds

2021 ◽  
Vol 18 (4) ◽  
pp. 375-383
Author(s):  
Smriti Yadav ◽  
Bharath Kumar Inturi ◽  
Shrinidhi B.R ◽  
Pooja H.J ◽  
Neenu Ganesh ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Lines ◽  
Normal Cell ◽  
Acyl Carrier Protein ◽  
Antitubercular Activity ◽  
Resistance Mechanisms ◽  
Docking Studies ◽  
Chemical Library ◽  
Activity Data ◽  
Mycobacterium Tuberculosis H37rv

Background: To overcome one of the resistance mechanisms of Isoniazid (INH), there is a need for an antitubercular agent that can inhibit InhA enzyme by circumventing the formation of INH-NAD+ adduct. Objective: The objective of the study is the development of novel antitubercular agents that target Mycobacterium tuberculosis InhA (Enoyl Acyl Carrier Protein Reductase). Methods: A small-molecule chemical library was used for the identification of the novel InhA inhibitors using primary screening and molecular docking studies followed by the scaffold hopping approach. The designed molecules, 2-(2-(hydroxymethyl)-1H- benzo[d] imidazole-1-yl)- N- substituted acetamides were synthesized by reacting (1H- benzo[d]imidazole -2-yl)methanol with appropriate 2-chloro-N-substituted acetamides / dialkylamino carbonyl chlorides respectively in good yields (42-65%). The antitubercular activity of synthesized compounds was determined by Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis H37Rv strain. The selected compounds were screened for cytotoxicity on normal cell lines. Results: The antitubercular activity data revealed that the 4-chlorophenyl substituted derivative (3b) showed good MIC value at 6.25 μg/mL and, dimethylacetamide substituted derivative (3i) showed MIC at 25 μg/mL among the tested compounds. The substitution of dimethylacetamide (3i) group on the 1st position of benzimidazole has good antitubercular activity (25μg/mL) in comparison to the diethyl acetamide group (3j, 100μg/mL). Conclusion: The antitubercular activity data indicated that the tested compounds exhibited well to moderate inhibition of the H37Rv strains. The compounds (3b) with electronegative substitution on the phenyl moiety exhibited better antitubercular activity than that of the other substitutions. The active compounds have displayed a good safety profile on normal cell lines.

Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 967-978 ◽  
Author(s):  
C. Viana-Niero ◽  
P. E. de Haas ◽  
D. van Soolingen ◽  
S. C. Leão
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Phospholipase C ◽  
Genetic Polymorphisms ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Clinical Isolates ◽  
Significant Similarity ◽  
Genomic Deletions ◽  
Genes Encoding ◽  
Mycobacterium Africanum

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

scholarly journals A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents

2021 ◽  
Vol 9 (6) ◽  
pp. 1323
Author(s):  
Etai Boichis ◽  
Nadejda Sigal ◽  
Ilya Borovok ◽  
Anat A. Herskovits
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Protein Pair ◽  
Growth Conditions ◽  
Wall Structure ◽  
Antibiotic Resistant ◽  
Extracellular Milieu ◽  
Virion Release ◽  
Genes Encoding

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.

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