scholarly journals Rv1218c, an ABC Transporter of Mycobacterium tuberculosis with Implications in Drug Discovery

2010 ◽  
Vol 54 (12) ◽  
pp. 5167-5172 ◽  
Author(s):  
Meenakshi Balganesh ◽  
Sanjana Kuruppath ◽  
Nimi Marcel ◽  
Sreevalli Sharma ◽  
Anju Nair ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Discovery ◽  
Bacterial Infections ◽  
Fold Increase ◽  
Potential Candidate ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Experimental Drugs ◽  
Physiological Relevance ◽  
In The Wild

ABSTRACT Efflux systems are important in determining the efficacy of antibiotics used in the treatment of bacterial infections. In the last decade much attention has been paid to studying the efflux pumps of mycobacteria. New classes of compounds are under investigation for development into potential candidate drugs for the treatment of tuberculosis. Quite often, these have poor bactericidal activities but exhibit excellent target (biochemical) inhibition. Microarray studies conducted in our laboratories for deciphering the mode of action of experimental drugs revealed the presence of putative ABC transporters. Among these transporters, Rv1218c was chosen for studying its physiological relevance in mediating efflux in Mycobacterium tuberculosis. A ΔRv1218c mutant of M. tuberculosis displayed a 4- to 8-fold increase in the inhibitory and bactericidal potency for different classes of compounds. The MICs and MBCs were reversed to wild-type values when the full-length Rv1218c gene was reintroduced into the ΔRv1218c mutant on a multicopy plasmid. Most of the compound classes had significantly better bactericidal activity in the ΔRv1218c mutant than in the wild-type H37Rv, suggesting the involvement of Rv1218c gene product in effluxing these compounds from M. tuberculosis. The implication of these findings on tuberculosis drug discovery is discussed.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

scholarly journals The hetF Gene Product Is Essential to Heterocyst Differentiation and Affects HetR Function in the Cyanobacterium Nostoc punctiforme

2001 ◽  
Vol 183 (8) ◽  
pp. 2654-2661 ◽  
Author(s):  
Francis C. Y. Wong ◽  
John C. Meeks
Keyword(s):  
Fluorescent Protein ◽  
Filamentous Cyanobacterium ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Nostoc Punctiforme ◽  
Combined Nitrogen ◽  
In The Wild ◽  
Step Down ◽  
Green Fluorescent ◽  
Heterocyst Development

ABSTRACT A novel gene, hetF, was identified as essential for heterocyst development in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133. In the absence of combined nitrogen, hetF mutants were unable to differentiate heterocysts, whereas extra copies of hetF intrans induced the formation of clusters of heterocysts. Sequences hybridizing to a hetF probe were detected only in heterocyst-forming cyanobacteria. The inactivation and multicopy effects of hetF were similar to those of hetR, which encodes a self-degrading serine protease thought to be a central regulator of heterocyst development. Increased transcription ofhetR begins in developing cells 3 to 6 h after deprivation for combined nitrogen (N step-down), and the HetR protein specifically accumulates in heterocysts. In the hetFmutant, this increase in hetR transcription was delayed, and a hetR promoter::green fluorescent protein (GFP) transcriptional reporter indicated that increased transcription of hetR occurred in all cells rather than only in developing heterocysts. When a fully functional HetR-GFP fusion protein was expressed in the hetF mutant from a multicopy plasmid, HetR-GFP accumulated nonspecifically in all cells under nitrogen-replete conditions; when expressed in the wild type, HetR-GFP was observed only in heterocysts after N step-down. HetF therefore appears to cooperate with HetR in a positive regulatory pathway and may be required for the increased transcription of hetR and localization of the HetR protein in differentiating heterocysts.

scholarly journals Mycobacterium tuberculosis Dihydrofolate Reductase Is Not a Target Relevant to the Antitubercular Activity of Isoniazid

2010 ◽  
Vol 54 (9) ◽  
pp. 3776-3782 ◽  
Author(s):  
Feng Wang ◽  
Paras Jain ◽  
Gulcin Gulten ◽  
Zhen Liu ◽  
Yicheng Feng ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dihydrofolate Reductase ◽  
Fold Increase ◽  
Antitubercular Activity ◽  
Experimental Sample ◽  
Wild Type ◽  
Negative Results ◽  
Sequencing Studies ◽  
Type Strains

ABSTRACT Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro, and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia coli cell-based system was designed that allowed coexpression of both M. tuberculosis katG and dfrA genes in the presence of INH. The DHFR protein isolated from the experimental sample was not found bound with any INH-NADP adduct by enzyme inhibition assay and mass spectroscopic analysis. We also used whole-genome sequencing to determine whether polymorphisms in dfrA could be detected in six INH-resistant clinical isolates known to lack mutations in inhA and katG, but no such mutations were found. The dfrA overexpression experiments, together with the biochemical and sequencing studies, conclusively demonstrate that DHFR is not a target relevant to the antitubercular activity of INH.

scholarly journals Computational evidence of a new allosteric communication pathway between active sites and putative regulatory sites in the alanine racemase ofMycobacterium tuberculosis

2018 ◽  
Author(s):  
Jayanthy Jyothikumar ◽  
Sushil Chandani ◽  
Tangirala Ramakrishna
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Discovery ◽  
Side Effects ◽  
Active Site ◽  
Bacterial Infections ◽  
Active Sites ◽  
Pyridoxal Phosphate ◽  
Bacterial Species ◽  
Alanine Racemase ◽  
Docking Simulations

AbstractAlanine racemase, a popular drug target fromMycobacterium tuberculosis, catalyzes the biosynthesis of D-alanine, an essential component in bacterial cell walls. With the help of elastic network models of alanine racemase fromMycobacterium tuberculosis, we show that the mycobacterial enzyme fluctuates between two undiscovered states—a closed and an open state. A previous experimental screen identified several drug-like lead compounds against the mycobacterial alanine racemase, whose inhibitory mechanisms are not known. Docking simulations of the inhibitor leads onto the mycobacterial enzyme conformations obtained from the dynamics of the enzyme provide first clues to a putative regulatory role for two new pockets targeted by the leads. Further, our results implicate the movements of a short helix, behind the communication between the new pockets and the active site, indicating allosteric mechanisms for the inhibition. Based on our findings, we theorize that catalysis is feasible only in the open state. The putative regulatory pockets and the enzyme fluctuations are conserved across several alanine racemase homologs from diverse bacterial species, mostly pathogenic, pointing to a common regulatory mechanism important in drug discovery.Author summaryIn spite of the discovery of many inhibitors against the TB-causing pathogenMycobacterium tuberculosis, only a very few have reached the market as effective TB drugs. Most of the marketed TB drugs induce toxic side effects in patients, as they non-specifically target human cells in addition to pathogens. One such TB drug, D-cycloserine, targets pyridoxal phosphate moiety non-specifically regardless of whether it is present in the pathogen or the human host enzymes. D-cycloserine was developed to inactivate alanine racemase in TB causing pathogen. Alanine racemase is a bacterial enzyme essential in cell wall synthesis. Serious side effects caused by TB drugs like D-cycloserine, lead to patients’ non-compliance with treatment regimen, often causing fatal outcomes. Current drug discovery efforts focus on finding specific, non-toxic TB drugs. Through computational studies, we have identified new pockets on the mycobacterial alanine racemase and show that they can bind drug-like compounds. The location of these pockets away from the pyridoxal phosphate-containing active site, make them attractive target sites for novel, specific TB drugs. We demonstrate the presence of these pockets in alanine racemases from several pathogens and expect our findings to accelerate the discovery of non-toxic drugs against TB and other bacterial infections.

scholarly journals Function of the ςE Regulon in Dead-Cell Lysis in Stationary-Phase Escherichia coli

2000 ◽  
Vol 182 (18) ◽  
pp. 5231-5237 ◽  
Author(s):  
Takeshi Nitta ◽  
Hiroshi Nagamitsu ◽  
Masayuki Murata ◽  
Hanae Izu ◽  
Mamoru Yamada
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Cell Lysis ◽  
Dead Cell ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli ◽  
Intracellular Proteins ◽  
In The Wild ◽  
Cloned Gene

ABSTRACT Elevation of active ςE levels in Escherichia coli by either repressing the expression of rseAencoding an anti-ςE factor or cloning rpoE in a multicopy plasmid, led to a large decrease in the number of dead cells and the accumulation of cellular proteins in the medium in the stationary phase. The numbers of CFU, however, were nearly the same as those of the wild type or cells devoid of the cloned gene. In the wild-type cells, rpoE expression was increased in the stationary phase and a low-level release of intracellular proteins was observed. These results suggest that dead cell lysis in stationary-phase E. coli occurs in a ςE-dependent fashion. We propose there is a novel physiological function of the ςE regulon that may guarantee cell survival in prolonged stationary phase by providing nutrients from dead cells for the next generation.

scholarly journals Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2348
Author(s):  
Laure Tonini ◽  
Bashir Sadet ◽  
Alexandre Stella ◽  
David Bouyssié ◽  
Jérôme Nigou ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mycobacterium Tuberculosis ◽  
Large Scale ◽  
Therapeutic Targets ◽  
Multidrug Resistant ◽  
Host Cells ◽  
Bacterial Protein ◽  
Wild Type ◽  
In The Wild ◽  
Resistant Strains

To date, Mycobacterium tuberculosis (Mtb) remains the world’s greatest infectious killer. The rise of multidrug-resistant strains stresses the need to identify new therapeutic targets to fight the epidemic. We previously demonstrated that bacterial protein-O-mannosylation is crucial for Mtb infectiousness, renewing the interest of the bacterial-secreted mannoproteins as potential drug-targetable virulence factors. The difficulty of inventorying the mannoprotein repertoire expressed by Mtb led us to design a stringent multi-step workflow for the reliable identification of glycosylated peptides by large-scale mass spectrometry-based proteomics. Applied to the differential analyses of glycoproteins secreted by the wild-type Mtb strain—and by its derived mutant invalidated for the protein-O-mannosylating enzyme PMTub—this approach led to the identification of not only most already known mannoproteins, but also of yet-unknown mannosylated proteins. In addition, analysis of the glycoproteome expressed by the isogenic recombinant Mtb strain overexpressing the PMTub gene revealed an unexpected mannosylation of proteins, with predicted or demonstrated functions in Mtb growth and interaction with the host cell. Since in parallel, a transient increased expression of the PMTub gene has been observed in the wild-type bacilli when infecting macrophages, our results strongly suggest that the Mtb mannoproteome may undergo adaptive regulation during infection of the host cells. Overall, our results provide deeper insights into the complexity of the repertoire of mannosylated proteins expressed by Mtb, and open the way to novel opportunities to search for still-unexploited potential therapeutic targets.

scholarly journals Increased Production of Zeaxanthin and Other Pigments by Application of Genetic Engineering Techniques toSynechocystis sp. Strain PCC 6803

2000 ◽  
Vol 66 (1) ◽  
pp. 64-72 ◽  
Author(s):  
Delphine Lagarde ◽  
Laurent Beuf ◽  
Wim Vermaas
Keyword(s):  
Mutant Strain ◽  
Fold Increase ◽  
Carotenoid Biosynthesis ◽  
Carotenoid Content ◽  
Wild Type ◽  
Gene Encoding ◽  
Genes Encoding ◽  
In The Wild ◽  
Β Carotene ◽  
Pcc 6803

ABSTRACT The psbAII locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis inSynechocystis sp. strain PCC 6803 under the control of the strong psbAII promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (ipi) and theSynechocystis β-carotene hydroxylase (crtR) and the linked Synechocystis genes coding for phytoene desaturase and phytoene synthase (crtP andcrtB, respectively) were introduced intoSynechocystis, replacing the psbAII coding sequence. Expression of ipi, crtR, andcrtP and crtB led to a large increase in the corresponding transcript levels in the mutant strains, showing that the psbAII promoter can be used to drive transcription and to overexpress various genes in Synechocystis. Overexpression of crtP and crtB led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the β-carotene and echinenone contents remained unchanged. Overexpression of crtR induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the β-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of ipi did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of Synechocystis, the crtOgene, encoding β-carotene ketolase, which converts β-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.

scholarly journals The adapter protein Myd88 plays an important role in limiting mycobacterial growth in a zebrafish model for tuberculosis

2021 ◽  
Author(s):  
Rohola Hosseini ◽  
Gerda E. M. Lamers ◽  
Erik Bos ◽  
Pancras C. W. Hogendoorn ◽  
Abraham J. Koster ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Intracellular Bacteria ◽  
Adapter Protein ◽  
Wild Type ◽  
Zebrafish Model ◽  
Compact Structure ◽  
Electron Microscopy Analysis ◽  
In The Wild ◽  
Morphological Differences ◽  
Mycobacterial Growth

AbstractTuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells.

scholarly journals Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (7) ◽  
pp. 3255-3263 ◽  
Author(s):  
H Yashiroda ◽  
T Oguchi ◽  
Y Yasuda ◽  
A Toh-E ◽  
Y Kikuchi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ubiquitin Ligase ◽  
Gradient Centrifugation ◽  
High Dose ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
In The Wild

We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt- phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5. Furthermore, both Bul1 and Rsp5 appeared to be associated with large complexes which were separated through a sucrose gradient centrifugation, and Rsp5 was coimmunoprecipitated with Bul1. We discuss the possibility that Bul1 functions together with Rsp5 in protein ubiquitination.

scholarly journals Activation of 2,4-diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a putative dioxygenase

2018 ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Data Support ◽  
In The Wild

AbstractThe diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on wild-type and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant, but not the wild-type. A metabolite consistent with a mono-hydroxylated form was identified in the wild-type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

Sign in / Sign up

Export Citation Format

Share Document