RESUMEN
Bacterial infections continue to represent a major worldwide health hazard following the emergence of drug-resistant pathogenic strains. Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections with increased morbidity and mortality. The increasing antibiotic resistance in P. aeruginosa has led to an unmet need for discovery of new antibiotic candidates. Bacterial protein synthesis is an essential metabolic process and a validated target for antibiotic development; however, the precise structural mechanism in P. aeruginosa remains unknown. In this work, the interaction of P. aeruginosa initiation factor 1 (IF1) with the 30S ribosomal subunit was studied by NMR, which enabled us to construct a structure of IF1-bound 30S complex. A short α-helix in IF1 was found to be critical for IF1 ribosomal binding and function. A peptide derived from this α-helix was tested and displayed a high ability to inhibit bacterial growth. These results provide a clue for rational design of new antimicrobials.
Asunto(s)
Péptidos Antimicrobianos , Pseudomonas aeruginosa , Factores de Iniciación de Péptidos , Subunidades Ribosómicas , RibosomasRESUMEN
Translation initiation factor 3 (IF3) is one of the three protein factors that bind to the small ribosomal subunit and it is required for the initiation of protein biosynthesis in bacteria. IF3 contains two independent domains, N- and C-terminal domains, which are connected by a lysine-rich interdomain linker. IF3 undergoes large-scale movements and conformational changes upon binding to the 30S subunit and also during the functional regulation of initiation. However, the precise dynamic interplay of the two domains and the molecular mechanism of IF3 is not well understood. A high-resolution 3D structure of a complete IF3 in bacteria has not been solved. Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is a primary cause of nosocomial infections in humans. Here we report the NMR chemical shift assignments of IF3 from P. aeruginosa as the first step toward NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data identified nine ß-strands and four α-helices arranged in the sequential order ß1-ß2-α1-ß3-ß4-α2-ß5-α3-ß6-α4-ß7-ß8-ß9.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Resonancia Magnética Nuclear Biomolecular , Factor 3 Procariótico de Iniciación/química , Espectroscopía de Protones por Resonancia Magnética , Pseudomonas aeruginosa/metabolismo , Isótopos de Nitrógeno , Estructura Secundaria de ProteínaRESUMEN
Pseudomonas aeruginosa, an opportunistic pathogen, is highly susceptible to developing resistance to multiple antibiotics. The gene encoding aspartyl-tRNA synthetase (AspRS) from P. aeruginosa was cloned and the resulting protein characterized. AspRS was kinetically evaluated, and the KM values for aspartic acid, ATP, and tRNA were 170, 495, and 0.5 µM, respectively. AspRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 1690 chemical compounds, resulting in the identification of two inhibitory compounds, BT02A02 and BT02C05. The minimum inhibitory concentrations (MICs) were determined against nine clinically relevant bacterial strains, including efflux pump mutant and hypersensitive strains of P. aeruginosa. The compounds displayed broad-spectrum antibacterial activity and inhibited growth of the efflux and hypersensitive strains with MICs of 16 µg/mL. Growth of wild-type strains were unaffected, indicating that efflux was likely responsible for this lack of activity. BT02A02 did not inhibit growth of human cell cultures at any concentration. However, BT02C05 did inhibit human cell cultures with a cytotoxicity concentration (CC50) of 61.6 µg/mL. The compounds did not compete with either aspartic acid or ATP for binding AspRS, indicating that the mechanism of action of the compound occurs outside the active site of aminoacylation.
Asunto(s)
Antibacterianos/farmacología , Aspartato-ARNt Ligasa/genética , Pseudomonas aeruginosa/efectos de los fármacos , ARN de Transferencia de Asparagina/genética , ARN de Transferencia/genética , Proteínas Bacterianas/genética , Dominio Catalítico/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Citotoxinas/farmacología , Células HEK293 , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana/métodos , Pseudomonas aeruginosa/genéticaRESUMEN
Pseudomonas aeruginosa histidyl-tRNA synthetase (HisRS) was selected as a target for antibiotic drug development. The HisRS protein was overexpressed in Escherichia coli and kinetically evaluated. The KM values for interaction of HisRS with its three substrates, histidine, ATP, and tRNAHis, were 37.6, 298.5, and 1.5 µM, while the turnover numbers were 8.32, 16.8, and 0.57 s-1, respectively. A robust screening assay was developed, and 800 natural products and 890 synthetic compounds were screened for inhibition of activity. Fifteen compounds with inhibitory activity were identified, and the minimum inhibitory concentration (MIC) was determined for each against a panel of nine pathogenic bacteria. Each compound exhibited broad-spectrum activity. Based on structural similarity and MIC results, four compounds, BT02C02, BT02D04, BT08E04, and BT09C11, were selected for additional analysis. These compounds inhibited the activity of HisRS with IC50 values of 4.4, 9.7, 14.1, and 11.3 µM, respectively. Time-kill studies indicated a bacteriostatic mode of inhibition for each compound. BT02D04 and BT08E04 were noncompetitive with both histidine and ATP, BT02C02 was competitive with histidine but noncompetitive with ATP, and BT09C11 was uncompetitive with histidine and noncompetitive with ATP. These compounds were not observed to be toxic to human cell cultures.
Asunto(s)
Antibacterianos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Histidina-ARNt Ligasa/antagonistas & inhibidores , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Secuencia de Aminoácidos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Expresión Génica , Histidina-ARNt Ligasa/genética , Histidina-ARNt Ligasa/metabolismo , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Pseudomonas aeruginosa/genéticaRESUMEN
Four inhibitory compounds were identified using a poly-uridylic acid (polyU) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa in an in vitro screen of a synthetic compound library. The compounds were specific for inhibition of bacterial protein synthesis. In enzymatic assays, the compounds inhibited protein synthesis with IC50 values ranging from 20 to 60 µM. Minimum inhibitory concentrations (MICs) were determined in cultures for a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. All the compounds were observed to have broad-spectrum activity and inhibited an efflux pump mutant strain of P. aeruginosa with MICs of 0.5-16 µg/mL. The molecular target of two compounds was determined to be PheRS. These two compounds were bacteriostatic against both Gram-positive and Gram-negative pathogens. In competition assays, they were not observed to compete with the natural substrates ATP or phenylalanine for active site binding. The other two compounds directly inhibited the ribosome and were bactericidal against both Gram-positive and Gram-negative pathogens. In cytotoxicity MTT testing in human cell lines, the compounds were shown to be from 2500- to 30,000-fold less active than the control staurosporine.
Asunto(s)
Antibacterianos/farmacología , Descubrimiento de Drogas , Biosíntesis de Proteínas/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Antibacterianos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Bibliotecas de Moléculas PequeñasRESUMEN
Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa This system has been used for high-throughput screening of a natural-compound library. Assays were developed for each component of the system to ascertain the specific target of inhibitory compounds. In high-throughput screens, 13 compounds were identified that inhibit protein synthesis with 50% inhibitory concentrations ranging from 0.3 to >80 µM. MICs were determined for the compounds against the growth of a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Moraxella catarrhalis, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae Three of the compounds were observed to have broad-spectrum activity and inhibited a hypersensitive strain of P. aeruginosa with MICs of 8 to 16 µg/ml. The molecular target of each of the three compounds was determined to be PheRS. One compound was found to be bacteriostatic, and one compound was bactericidal against both Gram-positive and Gram-negative pathogens. The third compound was observed to be bacteriostatic against Gram-positive and bactericidal against Gram-negative bacteria. All three compounds were competitive with the substrate ATP; however, one compound was competitive, one was uncompetitive, and one noncompetitive with the amino acid substrate. Macromolecular synthesis assays confirm the compounds inhibit protein synthesis. The compounds were shown to be more than 25,000-fold less active than the control staurosporine in cytotoxicity MTT testing in human cell lines.
Asunto(s)
Antibacterianos/farmacología , Productos Biológicos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Línea Celular , Bacterias Grampositivas/efectos de los fármacos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/metabolismo , Ribosomas/efectos de los fármacosRESUMEN
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of infection in humans. P. aeruginosa can acquire resistance against multiple groups of antimicrobial agents, including ß-lactams, aminoglycosides and fluoroquinolones, and multidrug resistance is increasing in this organism which makes treatment of the infections difficult and expensive. This has led to the unmet need for discovery of new compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Translation initiation factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis, and its structure is unknown. Here we report the (1)H, (13)C and (15)N chemical shift assignments of Pa-IF1 as the basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified five ß-strands with an unusually extended ß-strand at the C-terminal end of the protein and one short α-helix arranged in the sequential order ß1-ß2-ß3-α1-ß4-ß5. This is further supported by (15)N-{(1)H} hetero NOEs. These secondary structure elements suggest the Pa-IF1 adopts the typical ß-barrel structure and is composed of an oligomer-binding motif.
Asunto(s)
Proteínas Bacterianas/química , Resonancia Magnética Nuclear Biomolecular , Factores Procarióticos de Iniciación/química , Pseudomonas aeruginosa , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/genéticaRESUMEN
Pseudomonas aeruginosa and Streptococcus pneumoniae are causative agents in a wide range of infections. Genes encoding proteins corresponding to phenylalanyl-tRNA synthetase (PheRS) were cloned from both bacteria. The two forms of PheRS were kinetically evaluated and the K(m)'s for P. aeruginosa PheRS with its three substrates, phenylalanine, ATP and tRNA(Phe) were determined to be 48, 200, and 1.2 µM, respectively, while the K(m)'s for S. pneumoniae PheRS with respect to phenylalanine, ATP and tRNA(Phe) were 21, 225 and 0.94 µM, respectively. P. aeruginosa and S. pneumoniae PheRS were used to screen a natural compound library and a single compound was identified that inhibited the function of both enzymes. The compound inhibited P. aeruginosa and S. pneumoniae PheRS with IC50's of 2.3 and 4.9 µM, respectively. The compound had a K(I) of 0.83 and 0.98 µM against P. aeruginosa and S. pneumoniae PheRS, respectively. The minimum inhibitory concentration (MIC) of the compound was determined against a panel of Gram positive and negative bacteria including efflux pump mutants and hyper-sensitive strains. MICs against wild-type P. aeruginosa and S. pneumoniae cells in culture were determined to be 16 and 32 µg/ml, respectively. The mechanism of action of the compound was determined to be competitive with the amino acid, phenylalanine, and uncompetitive with ATP. There was no inhibition of cytoplasmic protein synthesis, however, partial inhibition of the human mitochondrial PheRS was observed.
Asunto(s)
Productos Biológicos/farmacología , Ensayos Analíticos de Alto Rendimiento , Naftoles/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Pseudomonas aeruginosa/enzimología , Streptococcus pneumoniae/enzimología , Secuencia de Aminoácidos , Productos Biológicos/análisis , Células Cultivadas , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Mitocondrias/efectos de los fármacos , Datos de Secuencia Molecular , Fenilalanina-ARNt Ligasa/genética , Fenilalanina-ARNt Ligasa/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Pseudomonas aeruginosa/genética , Streptococcus pneumoniae/genéticaRESUMEN
Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (K M) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 µM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed k cat) for the hydrolysis of GTP than EF-G1A; 0.2 s(-1) vs. 0.04 s(-1). These values resulted in specificity constants (k cat (obs)/K M) for EF-G1A and EF-G1B of 0.5 x 10(3) s(-1) M(-1) and 3.0 x 10(3) s(-1) M(-1), respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factor G de Elongación Peptídica/genética , Factor G de Elongación Peptídica/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ácido Fusídico/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Hidrólisis , Cinética , Poli U/genética , Poli U/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Análisis de SecuenciaRESUMEN
We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be K M = 33 µM, k cat (obs) = 0.003 s(-1), and the specificity constant k cat (obs)/K M was 0.1 × 10(-3) s(-1) µM(-1). In the presence of EF-Ts, these values were shifted to K M = 2 µM, k cat (obs) = 0.005 s(-1), and the specificity constant k(cat)(obs)/K M was 2.5 × 10(-3) s(-1) µM(-1). The equilibrium dissociation constants governing the binding of EF-Tu to GDP (K GDP) were 30-75 nM and to GTP (K GTP) were 125-200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U)-dependent binding of Phe-tRNA(Phe) at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U)-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.