RESUMO
Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins (Lsr2 and Lsr2-like, or LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 activity in model species and uncharacterized isolates resulted in the production of new metabolites not seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and provide global control over Streptomyces' arsenal of signaling and antagonistic compounds.
Assuntos
Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , Streptomyces/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Sítios de Ligação , Vias Biossintéticas/genética , Cromossomos Bacterianos/genética , Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal/genética , Genes Bacterianos , Metaboloma/genética , Mutação/genética , Fenótipo , Streptomyces/genética , VolatilizaçãoRESUMO
Actinomycete secondary metabolites are a renowned source of antibacterial chemical scaffolds. Herein, we present a target-specific approach that increases the detection of antimetabolites from natural sources by screening actinomycete-derived extracts against nutrient transporter deletion strains. On the basis of the growth rescue patterns of a collection of 22 Escherichia coli (E. coli) auxotrophic deletion strains representative of the major nutrient biosynthetic pathways, we demonstrate that antimetabolite detection from actinomycete-derived extracts prepared using traditional extraction platforms is masked by nutrient supplementation. In particular, we find poor sensitivity for the detection of antimetabolites targeting vitamin biosynthesis. To circumvent this and as a proof of principle, we exploit the differential activity of actinomycete extracts against E. coli ΔyigM, a biotin transporter deletion strain versus wildtype E. coli. We achieve more than a 100-fold increase in antimetabolite sensitivity using this method and demonstrate a successful bioassay-guided purification of the known biotin antimetabolite, amiclenomycin. Our findings provide a unique solution to uncover the full potential of naturally derived antibiotics.
Assuntos
Actinobacteria/metabolismo , Antimetabólitos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Descoberta de Drogas , Actinobacteria/crescimento & desenvolvimento , Antimetabólitos/farmacologia , Biotina/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms. IMPORTANCE: With the rise of antibiotic drug resistance, there is an urgent need for new antibacterial drugs. Here, we studied a group of genes that are essential for the growth of Escherichia coli under nutrient limitation, culture conditions that arguably better represent nutrient availability during an infection than rich microbiological media. Indeed, many such nutrient stress genes are essential for infection in a variety of pathogens. Thus, the respective proteins represent a pool of potential new targets for antibacterial drugs that have been largely unexplored. We have created all possible double deletion mutants through a genetic cross of nutrient stress genes and the E. coli deletion collection. An analysis of the growth of the resulting clones on rich media revealed a robust, dense, and complex network for nutrient acquisition and biosynthesis. Importantly, our data reveal new genetic connections to guide innovative approaches for the development of new antibacterial compounds targeting bacteria under nutrient stress.
Assuntos
Meios de Cultura/química , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Redes e Vias Metabólicas/genética , Estresse Fisiológico , Cruzamentos Genéticos , Escherichia coli/genética , Deleção de GenesRESUMO
Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.
Assuntos
Burkholderia cenocepacia/efeitos dos fármacos , Burkholderia cenocepacia/crescimento & desenvolvimento , Avaliação Pré-Clínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antibacterianos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Ovinos , Bibliotecas de Moléculas Pequenas/toxicidadeRESUMO
The widespread emergence of antibiotic drug resistance has resulted in a worldwide healthcare crisis. In particular, the extensive use of ß-lactams, a highly effective class of antibiotics, has been a driver for pervasive ß-lactam resistance. Among the most important resistance determinants are the metallo-ß-lactamases (MBL), which are zinc-requiring enzymes that inactivate nearly all classes of ß-lactams, including the last-resort carbapenem antibiotics. The urgent need for new compounds targeting MBL resistance mechanisms has been widely acknowledged; however, the development of certain types of compounds-namely metal chelators-is actively avoided due to host toxicity concerns. The work herein reports the identification of a series of zinc-selective spiro-indoline-thiadiazole analogues that, in vitro, potentiate ß-lactam antibiotics against an MBL-carrying pathogen by withholding zinc availability. This study demonstrates the ability of one such analogue to inhibit NDM-1 in vitro and, using a mouse model of infection, shows that combination treatment of the respective analogue with meropenem results in a significant decrease in bacterial burden in contrast to animals that received antibiotic treatment alone. These results support the therapeutic potential of these chelators in overcoming antibiotic resistance.
RESUMO
High-throughput screening (HTS) of chemical and microbial strain collections is an indispensable tool for modern chemical and systems biology; however, HTS data sets have inherent systematic and random error, which may lead to false-positive or false-negative results. Several methods of normalization of data exist; nevertheless, due to the limitations of each, no single method has been universally adopted. Here, we present a method of data visualization and normalization that is effective, intuitive, and easy to implement in a spreadsheet program. For each plate, the data are ordered by ascending values and a plot thereof yields a curve that is a signature of the plate data. Curve shape characteristics provide intuitive visualization of the frequency and strength of inhibitors, activators, and noise on the plate, allowing potentially problematic plates to be flagged. To reduce plate-to-plate variation, the data can be normalized by the mean of the middle 50% of ordered values, also called the interquartile mean (IQM) or the 50% trimmed mean of the plate. Positional effects due to bias in columns, rows, or wells can be corrected using the interquartile mean of each well position across all plates (IQMW) as a second level of normalization. We illustrate the utility of this method using data sets from biochemical and phenotypic screens.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Modelos TeóricosRESUMO
The dwindling supply of antibiotics that remain effective against drug-resistant bacterial pathogens has precipitated efforts to identify new compounds that inhibit bacterial growth using untapped mechanisms of action. Here, we report both (1) a high-throughput screening methodology designed to discover chemical perturbants of the essential, yet unexploited, process of bacterial iron homeostasis, and (2) our findings from a small-molecule screen of more than 30,000 diverse small molecules that led to the identification and characterization of two spiro-indoline-thiadiazoles that disrupt iron homeostasis in bacteria. We show that these compounds are intracellular chelators with the capacity to exist in two isomeric states. Notably, these spiroheterocyles undergo a transition to an open merocyanine chelating form with antibacterial activity that is specifically induced in the presence of its transition-metal target.
Assuntos
Escherichia coli/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Quelantes de Ferro/química , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Compostos Organometálicos/farmacologia , Elementos de Transição/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Benzopiranos/síntese química , Benzopiranos/química , Benzopiranos/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/metabolismo , Ensaios de Triagem em Larga Escala , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Quelantes de Ferro/síntese química , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Bibliotecas de Moléculas Pequenas , Compostos de Espiro/química , Estereoisomerismo , Relação Estrutura-Atividade , Tiadiazóis/química , Fatores de Transcrição/antagonistas & inibidores , Elementos de Transição/químicaRESUMO
Iron is an essential growth component in all living organisms and plays a central role in numerous biochemical processes due to its redox potential and high affinity for oxygen. The use of iron chelators has been suggested as a novel therapeutic approach towards parasitic infections, such as malaria, sleeping sickness and leishmaniasis. Known iron chelating agents such as Deferoxamine and the 3-hydroxypyridin-4-one (HPO) Deferiprone possess anti-parasitic activity but suffer from mammalian toxicity, relatively modest potency, and/or poor oral availability. In this study, we have developed novel derivatives of Deferiprone with increased anti-parasitic activity and reduced cytotoxicity against human cell lines. Of particular interest are several new derivatives in which the HPO scaffold has been conjugated, via a linker, to the 4-aminoquinoline ring system present in the known anti-malaria drug Chloroquine. We report the inhibitory activity of these novel analogues against four parasitic protozoa, Trypanosoma brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum, and, for direct comparison, against human cells lines. We also present data, which support the hypothesis that iron starvation is the major cause of growth inhibition of these new Deferiprone-Chloroquine conjugates in T. brucei.
Assuntos
Aminoquinolinas/química , Antiprotozoários/farmacologia , Quelantes de Ferro/farmacologia , Piridonas/farmacologia , Antiprotozoários/síntese química , Antiprotozoários/química , Deferiprona , Relação Dose-Resposta a Droga , Quelantes de Ferro/síntese química , Quelantes de Ferro/química , Leishmania infantum/efeitos dos fármacos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Piridonas/síntese química , Piridonas/química , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The protozoan parasite causing human African trypanosomiasis, Trypanosoma brucei, displays cysteine peptidase activity, the chemical inhibition of which is lethal to the parasite. This activity comprises a cathepsin B (TbCATB) and a cathepsin L (TbCATL). Previous RNA interference (RNAi) data suggest that TbCATB rather than TbCATL is essential to survival even though silencing of the latter was incomplete. Also, chemical evidence supporting the essentiality of either enzyme which would facilitate a target-based drug development programme is lacking. Using specific peptidyl inhibitors and substrates, we quantified the contributions of TbCATB and TbCATL to the survival of T. brucei. At 100 µM, the minimal inhibitory concentration that kills all parasites in culture, the non-specific cathepsin inhibitors, benzyloxycarbonyl-phenylalanyl-arginyl-diazomethyl ketone (Z-FA-diazomethyl ketone) and (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline methyl ester (CA-074Me) inhibited TbCATL and TbCATB by >99%. The cathepsin L (CATL)-specific inhibitor, ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) (CAA0225), killed parasites with >99% inhibition of TbCATL but only 70% inhibition of TbCATB. Conversely, the cathepsin B (CATB)-specific inhibitor, (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074), did not affect survival even though TbCATB inhibition at >95% was statistically indistinguishable from the complete inhibition by Z-FA-diazomethyl ketone and CA-074Me. The observed inhibition of TbCATL by CA-074 and CA-074Me was shown to be facilitated by the reducing intracellular environment. All inhibitors, except the CATB-specific inhibitor, CA-074, blockaded lysosomal hydrolysis prior to death. The results suggest that TbCATL, rather than TbCATB, is essential to the survival of T. brucei and an appropriate drug target.
Assuntos
Catepsina L/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma brucei brucei/fisiologia , Fatores de Virulência/fisiologia , Antiprotozoários/farmacologia , Catepsina L/genética , Sobrevivência Celular , Testes de Sensibilidade Microbiana , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/patogenicidade , Fatores de Virulência/genéticaRESUMO
Galactosyltransferases (GalT) are important molecular targets in a range of therapeutic areas, including infection, inflammation, and cancer. GalT inhibitors are therefore sought after as potential lead compounds for drug discovery. We have recently discovered a new class of GalT inhibitors with a novel mode of action. In this publication, we describe a series of analogues which provide insights, for the first time, into SAR for this new mode of GalT inhibition. We also report that a new C-glycoside, designed as a chemically stable analogue of the most potent inhibitor in this series, retains inhibitory activity against a panel of GalTs. Initial results from cellular studies suggest that despite their polarity, these sugar-nucleotides are taken up by HL-60 cells. Results from molecular modeling studies with a representative bacterial GalT provide a rationale for the differences in bioactivity observed in this series. These findings may provide a blueprint for the rational development of new GalT inhibitors with improved potency.