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1.
Artigo em Inglês | MEDLINE | ID: mdl-33046497

RESUMO

New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.


Assuntos
Antibacterianos , Proteômica , Antibacterianos/farmacologia , Bacillus subtilis , Proteínas de Bactérias/genética , Tetraciclinas
2.
Chembiochem ; 16(7): 1101-8, 2015 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-25821129

RESUMO

The mechanism of action of aurein 2.2 and aurein 2.3, antimicrobial peptides from the frog Litoria aurea, was investigated. Proteomic profiling of the Bacillus subtilis stress response indicates that the cell envelope is the main target for both aureins. Upon treatment, the cytoplasmic membrane depolarizes and cellular ATP levels decrease. Global element analysis shows that intracellular concentrations of certain metal ions (potassium, magnesium, iron, and manganese) strongly decrease. Selective translocation of some ions over others was demonstrated in vitro. The same set of ions also leaks from B. subtilis cells treated with sublethal concentrations of gramicidin S, MP196, and nisin. Aureins do not permeabilize the cell membrane for propidium iodide thus excluding formation of large, unspecific pores. Our data suggest that the aureins acts by forming small pores thereby causing membrane depolarization, and by triggering the release of certain metal ions thus disturbing cellular ion homeostasis.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Anuros , Bacillus subtilis/citologia , Membrana Celular/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Dados de Sequência Molecular , Porosidade , Proteômica
3.
Proteomics ; 13(8): 1358-70, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23412951

RESUMO

To maintain their metal ion homeostasis, bacteria critically depend on membrane integrity and controlled ion translocation. Terrestrial Streptomyces species undermine the function of the cytoplasmic membrane as diffusion barrier for metal cations in competitors using ionophores. Although the properties of the divalent cation ionophores calcimycin and ionomycin have been characterized to some extent in vitro, their effects on bacterial ion homeostasis, the factors leading to bacterial cell death, and their ecological role are poorly understood. To gain insight into their antibacterial mechanism, we determined the metal ion composition of the soil bacterium Bacillus subtilis after treatment with calcimycin and ionomycin. Within 15 min the cells lost approximately half of their cellular iron and manganese content whereas calcium levels increased. The proteomic response of B. subtilis provided evidence that disturbance of metal cation homeostasis is accompanied by intracellular oxidative stress, which was confirmed with a ROS-specific fluorescent probe. B. subtilis showed enhanced sensitivity to the ionophores in medium lacking iron or manganese. Furthermore, in the presence of ionophores bacteria were sensitive to high calcium levels. These findings suggest that divalent cation ionophores are particularly effective against competing microorganisms in soils rich in available calcium and low in available iron and manganese.


Assuntos
Bacillus subtilis/metabolismo , Ionóforos/farmacologia , Ferro/metabolismo , Manganês/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/química , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Calcimicina/química , Cálcio/metabolismo , Ionóforos de Cálcio/farmacologia , Ecologia , Homeostase , Ionomicina/química , Ionóforos/química , Ferro/isolamento & purificação , Ferro/farmacologia , Manganês/isolamento & purificação , Manganês/farmacologia , Micronutrientes/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Microbiologia do Solo
4.
Curr Protoc Microbiol ; Chapter 1: Unit1F.2, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22875564

RESUMO

The global analysis of changes in the protein composition of bacterial cells in response to treatment with antibiotic agents grants insights into the physiological response of cells to inhibition of vital cellular functions. This unit gives an overview of how global proteomic studies can impact antibacterial drug discovery by identifying or validating compound mechanism of action and by increasing the confidence in the value of genes with unknown function as potential new targets. It describes the design and function of a reference compendium of proteomic responses to inhibition of vital cellular functions through antibacterial agents or genetic down-regulation of potential target genes. An overview of the workflow for two-dimensional gel electrophoresis-based experiments is also presented.


Assuntos
Antibacterianos/farmacologia , Bactérias/química , Descoberta de Drogas , Eletroforese em Gel Bidimensional/métodos , Proteômica/métodos , Antibacterianos/análise , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
5.
Antimicrob Agents Chemother ; 56(11): 5749-57, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22926563

RESUMO

Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane.


Assuntos
Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Bacteriocinas/farmacologia , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Nisina/farmacologia , Peptídeos/farmacologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Transporte Biológico/efeitos dos fármacos , Biomarcadores/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Eletroforese em Gel Bidimensional , Potenciais da Membrana/efeitos dos fármacos , Potássio/metabolismo , Proteoma/antagonistas & inibidores , Proteoma/genética , Proteoma/metabolismo , Relação Estrutura-Atividade , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
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