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1.
Antimicrob Agents Chemother ; : AAC0086421, 2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34606341

RESUMO

Rifamycins, such as rifampicin, are potent inhibitors of bacterial RNA polymerases and widely used antibiotics. Usually rifamycin-resistance is associated with mutations in RNAP that preclude rifamycins binding. However, some bacteria have ADP-ribosyl transferases Arr that ADP-ribosylate rifamycin molecules, thus inactivating their antimicrobial activity. Here we directly show that ADP-ribosylation abolishes inhibition of transcription by rifampicin, the most widely used rifamycin antibiotic. We also show that natural rifamycin, Kanglemycin A, which has a unique sugar moiety at the ansa-chain close to the Arr-modification site, does not bind to Arr from M. smegmatis, and thus is not susceptible to inactivation. We, however, found that Kanglemycin A can still be ADP-ribosylated by Arr of an emerging pathogen M. abscessus. Interestingly, the only part of Arr which exhibits no homology between the species is the part that sterically clashes with sugar moiety of Kanglemycin A in M. smegmatis Arr. This suggests that M. abscessus has encountered KglA or rifamycin with similar sugar modification in the course of evolution. The results show that KglA could be effective antimicrobial against some of the Arr encoding bacteria.

2.
Nucleic Acids Res ; 49(15): 8777-8784, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365509

RESUMO

Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3' end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3' end of RNA within the active center. We show that the TL is required to transfer the 3' end of misincorporated transcript from cleavage-inefficient 'misincorporation site' into the cleavage-efficient 'backtracked site', where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Clivagem do RNA
3.
PLoS Pathog ; 16(7): e1008672, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32706832

RESUMO

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level ß-lactam resistance (oxacillin MIC 2-4 µg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 µg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit ß) or rpoC (RNA polymerase subunit ß') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.


Assuntos
Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/genética , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos
4.
EcoSal Plus ; 9(1)2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32342856

RESUMO

RNA polymerases (RNAPs) accomplish the first step of gene expression in all living organisms. However, the sequence divergence between bacterial and human RNAPs makes the bacterial RNAP a promising target for antibiotic development. The most clinically important and extensively studied class of antibiotics known to inhibit bacterial RNAP are the rifamycins. For example, rifamycins are a vital element of the current combination therapy for treatment of tuberculosis. Here, we provide an overview of the history of the discovery of rifamycins, their mechanisms of action, the mechanisms of bacterial resistance against them, and progress in their further development.

5.
Biochem Soc Trans ; 47(1): 339-350, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30647141

RESUMO

Transcription, the first phase of gene expression, is performed by the multi-subunit RNA polymerase (RNAP). Bacterial RNAP is a validated target for clinical antibiotics. Many natural and synthetic compounds are now known to target RNAP, inhibiting various stages of the transcription cycle. However, very few RNAP inhibitors are used clinically. A detailed knowledge of inhibitors and their mechanisms of action (MOA) is vital for the future development of efficacious antibiotics. Moreover, inhibitors of RNAP are often useful tools with which to dissect RNAP function. Here, we review the MOA of antimicrobial transcription inhibitors.


Assuntos
Antibacterianos/farmacologia , Bactérias/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química
6.
Mol Cell ; 72(2): 263-274.e5, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30244835

RESUMO

Antibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered-compared with rifampicin-conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance.


Assuntos
Produtos Biológicos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Rifabutina/farmacologia , Rifampina/farmacologia , Rifamicinas/farmacologia , Antituberculosos/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação/efeitos dos fármacos , Mutação/genética , Mycobacterium tuberculosis/genética , Thermus thermophilus/efeitos dos fármacos , Thermus thermophilus/genética
7.
J Nat Prod ; 80(5): 1558-1562, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28398740

RESUMO

The madurastatins are pentapeptide siderophores originally described as containing an unusual salicylate-capped N-terminal aziridine ring. Isolation of madurastatin C1 (1) (also designated MBJ-0034), from Actinomadura sp. DEM31376 (itself isolated from a deep sea sediment), prompted structural reevaluation of the madurastatin siderophores, in line with the recent work of Thorson and Shaaban. NMR spectroscopy in combination with partial synthesis allowed confirmation of the structure of madurastatin C1 (1) as containing an N-terminal 2-(2-hydroxyphenyl)oxazoline in place of the originally postulated aziridine, while absolute stereochemistry was determined via Harada's advanced Marfey's method. Therefore, this work further supports Thorson and Shaaban's proposed structural revision of the madurastatin class of siderophores (madurastatins A1 (2), B1 (3), C1 (1), and MBJ-0036 (4)) as N-terminal 2-(2-hydroxyphenyl)oxazolines.


Assuntos
Aziridinas/química , Oligopeptídeos/química , Peptídeos/química , Piperidonas/química , Sideróforos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Estereoisomerismo
8.
Am J Ther ; 24(2): e144-e149, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-26057141

RESUMO

Drug-induced hepatitis (DIH) is one of the major complications among the treatment of patients with tuberculosis (TB); it might even be fatal. This study tries to address the recurrence of DIH with 2 anti-TB regimens. In the retrospective study from 2007 to 2010, 135 TB patients with DIH who were older than 16 years were entered to study. The patients with DIH were randomly treated with a regimen, including isoniazid, rifampin, and ethambutol, plus either ofloxacin or pyrazinamide. The patients were reviewed for occurrence of recurrent DIH. Cure and completed treatment were considered as acceptable treatment outcomes, whereas default of treatment, treatment failure, and death were considered to be unacceptable outcomes. Therefore, 135 subjects with DIH were reviewed, and 23 patients (17%) experienced recurrence of hepatitis (19 cases in the ofloxacin group and 4 cases in the pyrazinamide group). There is no significant difference in recurrence of hepatitis between these 2 groups (P = 0.803). An acceptable outcome was observed in 95 patients (70.4%), and an unacceptable outcome was seen in 14 cases (10.3%). There was no significant difference in outcomes between these 2 regimens (P = 0.400, odds ratio = 1.62, 95% confidence interval, 0.524-4.98). The results of our study suggest that ofloxacin-based anti-TB regimen does not decrease the risk of recurrent DIH. Therefore, adding ofloxacin in the case of DIH is not recommended.


Assuntos
Antibacterianos/efeitos adversos , Antituberculosos/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Ofloxacino/efeitos adversos , Pirazinamida/efeitos adversos , Tuberculose Pulmonar/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antituberculosos/uso terapêutico , Quimioterapia Combinada , Etambutol/uso terapêutico , Feminino , Hepatite/etiologia , Humanos , Isoniazida/uso terapêutico , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Rifampina/uso terapêutico , Adulto Jovem
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