RESUMEN
Peptidoglycan (PG) composition in intact cells of methicillin-resistant Staphylococcus aureus (MRSA) and its isogenic Fem mutants has been characterized by measuring the glycine content of PG bridge structures by solid-state nuclear magnetic resonance (NMR). The glycine content estimated from integrated intensities (rather than peak heights) in the cell walls of whole cells was increased by approximately 30% for the FemA mutant and was reduced by 25% for the FemB mutant relative to expected values for homogeneous structures. In contrast, the expected compositions were observed in isolated cell walls of the same mutants. For FemA mutant whole cells, the increase was due to the presence of triglycyl bridge PG units (confirmed directly by mass spectrometric analysis), which constituted 10% of the total PG. These species were coalesced in some sort of a lattice or aggregate with spatial proximity to other PG bridges. This result suggests that the triglycyl-bridged PG units form a PG-like structure that is not incorporated into the mature cell wall.
Asunto(s)
Proteínas Bacterianas/genética , Pared Celular/química , Glicina/análisis , Staphylococcus aureus Resistente a Meticilina/química , Oligopéptidos/análisis , Peptidoglicano/química , Técnicas de Inactivación de Genes , Espectroscopía de Resonancia MagnéticaRESUMEN
Here we show that a new class of antibiotics-acyldepsipeptides-has antibacterial activity against Gram-positive bacteria in vitro and in several rodent models of bacterial infection. The acyldepsipeptides are active against isolates that are resistant to antibiotics in clinical application, implying a new target, which we identify as ClpP, the core unit of a major bacterial protease complex. ClpP is usually tightly regulated and strictly requires a member of the family of Clp-ATPases and often further accessory proteins for proteolytic activation. Binding of acyldepsipeptides to ClpP eliminates these safeguards. The acyldepsipeptide-activated ClpP core is capable of proteolytic degradation in the absence of the regulatory Clp-ATPases. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death.
Asunto(s)
Antibacterianos/clasificación , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Depsipéptidos/farmacología , Endopeptidasa Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Animales , Antibacterianos/farmacocinética , Antibacterianos/toxicidad , Bacillus subtilis/efectos de los fármacos , Bacterias/enzimología , Depsipéptidos/metabolismo , Depsipéptidos/farmacocinética , Depsipéptidos/toxicidad , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Femenino , Ratones , Estructura Molecular , Mutación , Infecciones Neumocócicas/tratamiento farmacológico , Infecciones Neumocócicas/microbiología , Unión Proteica , Procesamiento Proteico-Postraduccional , Ratas , Ratas Wistar , Sepsis/tratamiento farmacológico , Sepsis/microbiologíaRESUMEN
Finafloxacin is a new fluoroquinolone antibiotic with the unique property of increasing antibacterial activity at pH values lower than neutral. Whereas its antibacterial activity at neutral pH matches that of other quinolones in clinical use, it is expected to surpass this activity in tissues and body fluids acidified by the infection or inflammation processes. Pharmacokinetic parameters of oral single and multiple doses of up to 800 mg of finafloxacin and safety/tolerability observations were assessed in a phase I study including 95 healthy volunteers. Finafloxacin is well absorbed after oral administration, generating maximum concentrations (C(max)s) in plasma at least comparable to those of other fluoroquinolones, with a half-life of around 10 h. About one-third of the dose is excreted unchanged in the urine. Renal elimination appears to be a saturable process leading to slight increases of the area under the concentration-time curve extrapolated to infinity and dose normalized (AUC(∞,norm)) at dosages of 400 mg and above. Safety and tolerability data characterize finafloxacin as a drug with a favorable safety profile. In particular, adverse reactions regarded as class-typical of fluoroquinolones, such as, e.g., electrocardiogram (ECG) changes, neurotoxic effects, or hypoglycemia, were not observed in the study population.
Asunto(s)
Fluoroquinolonas/efectos adversos , Fluoroquinolonas/farmacocinética , Administración Oral , Adulto , Método Doble Ciego , Femenino , Fluoroquinolonas/sangre , Fluoroquinolonas/orina , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Finafloxacin is a novel fluoroquinolone that exhibits enhanced antibacterial activity under acidic conditions. The aim of this study was to define the in vitro pH-activity relationship. Finafloxacin exhibited optimal antibacterial activity between pH 5.0 and 6.0 at which MICs were 4- to 8-fold lower than those determined at neutral pH. These observations were then confirmed against a larger collection of bacteria. These data suggest that finafloxacin could potentially offer a therapeutic advantage within acidic foci of infection.
Asunto(s)
Antibacterianos/farmacología , Fluoroquinolonas/farmacología , Ciprofloxacina/farmacología , Escherichia coli/efectos de los fármacos , Concentración de Iones de Hidrógeno , Klebsiella pneumoniae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Ofloxacino/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacosRESUMEN
OBJECTIVES: Finafloxacin is an investigational fluoroquinolone exhibiting broad-spectrum activity that is enhanced under slightly acidic conditions (pH 5.0-6.5). The impact of individual and combinations of chromosomal mutations (gyrA, parC and marR) and the plasmid-mediated fluoroquinolone resistance mechanisms QepA1, QnrA1, QnrB1, QnrS1 and AAC(6')-Ib-cr were investigated. METHODS: The MICs of finafloxacin, compared with those of ciprofloxacin, levofloxacin and moxifloxacin, were determined at pH 5.8 and 7.2. RESULTS: MICs of finafloxacin compared with other fluoroquinolones at pH 5.8 were lower by a factor of 2-256. MICs of finafloxacin were unaffected by QepA1. Moreover, finafloxacin appeared not to be a substrate for AAC(6')-Ib-cr. CONCLUSIONS: Compared with ciprofloxacin, levofloxacin and moxifloxacin, finafloxacin shows higher activity especially at pH 5.8 against Escherichia coli mutants expressing known fluoroquinolone resistance determinants alone and in combinations.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Fluoroquinolonas/farmacología , Plásmidos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/genética , Girasa de ADN/química , Girasa de ADN/genética , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/química , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/metabolismo , ADN Bacteriano/genética , Escherichia coli/genética , Humanos , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad MicrobianaRESUMEN
Compositional analysis of the peptidoglycan (PG) of a wild-type methicillin-resistant Staphylococcus aureus and its fem-deletion mutants has been performed on whole cells and cell walls using stable-isotope labeling and rotational-echo double-resonance NMR. The labels included [1-(13)C,(15)N]glycine and l-[epsilon-(15)N]lysine (for a direct measure of the number of glycyl residues in the bridging segment), [1-(13)C]glycine and l-[epsilon-(15)N]lysine (concentration of bridge links), and d-[1-(13)C]alanine and [(15)N]glycine (concentrations of cross-links and wall teichoic acids). The bridging segment length changed from 5.0 glycyl residues (wild-type strain) to 2.5 +/- 0.1 (FemB) with modest changes in cross-link and bridge-link concentrations. This accurate in situ measurement for the FemB mutant indicates a heterogeneous PG structure with 25% monoglycyl and 75% triglycyl bridges. When the bridging segment was reduced to a single glycyl residue 1.0 +/- 0.1 (FemA), the level of cross-linking decreased by more than 20%, resulting in a high concentration of open N-terminal glycyl segments.
Asunto(s)
Proteínas Bacterianas/química , Espectroscopía de Resonancia Magnética/métodos , Staphylococcus aureus Resistente a Meticilina/química , Mutación , Peptidoglicano/química , Isótopos de Carbono , Staphylococcus aureus Resistente a Meticilina/genética , Isótopos de NitrógenoRESUMEN
The related lipo(depsi)peptide antibiotics daptomycin and friulimicin B show great potential in the treatment of multiply resistant gram-positive pathogens. Applying genome-wide in-depth expression profiling, we compared the respective stress responses of Bacillus subtilis. Both antibiotics target envelope integrity, based on the strong induction of extracytoplasmic function sigma factor-dependent gene expression. The cell envelope stress-sensing two-component system LiaRS is exclusively and strongly induced by daptomycin, indicative of different mechanisms of action in the two compounds.
Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Daptomicina/farmacología , Perfilación de la Expresión Génica , Péptidos/farmacología , Bacillus subtilis/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismoRESUMEN
A conditional expression system has been developed using the isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible Pspac promoter to validate essential genes of Staphylococcus aureus in vivo. The system has been applied to prove the essentiality of ligA and to evaluate the function of tarI, which was found to be essential in vitro but not in vivo.
Asunto(s)
Proteínas Bacterianas/genética , ADN Ligasas/genética , Genes Esenciales , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/genética , Absceso/microbiología , Animales , Medios de Cultivo , Modelos Animales de Enfermedad , Femenino , Regulación Bacteriana de la Expresión Génica , Humanos , Isopropil Tiogalactósido/farmacología , Ratones , Mutación , Staphylococcus aureus/efectos de los fármacosRESUMEN
Recent scientific publications demonstrate the increasing interest in measurement of genome-wide gene expression on transcript and protein level in response to treatment with antibacterial agents. Nevertheless, the number of large bacterial transcriptome and proteome datasets available so far is limited, although a high number and diversity of antibiotic-triggered expression profiles aid to optimally exploit these technologies. The first published examples substantiate the need to establish these so-called reference compendia of bacterial expression profiles, to discover the molecular mechanism-of-action of uncharacterized bioactive substances. In addition, such compendia open up ways for novel cell-based drug screening approaches.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Proteoma/análisis , Bacterias/genética , Genes Bacterianos , Mutación , Transcripción GenéticaRESUMEN
The continued evolution of resistance to antibiotics has led to wide ranging consultation at National and International levels as to how to address this issue. In addition to attempting to limit the spread of resistance there is growing consensus that a cornerstone requirement is the development of new antibiotics to help redress the balance of resistance versus available antibiotics. The availability of new technologies such as genomics has opened up new approaches for antibacterial research. It would appear that from an industry perspective, the research and development of antibiotics should be an attractive option. However, this is not the current perception at the majority of large pharmaceutical companies. In addition, the perceived failure of new technologies to create another golden age of new antibacterial classes has led many companies to prioritise other areas of research and, in some cases, to exit antibacterial research. In response, a plethora of small biotech companies have emerged with an interest in antibacterial discovery and large pharmaceutical companies may look to these as a source of development candidates although, to date, these have contributed a very low number of truly novel antibiotic lead compounds. As a reaction to these changes several initiatives are ongoing to examine ways to incentivise antibacterial research and development and ensure a healthy pipeline of compounds in the 21st Century.
Asunto(s)
Antibacterianos/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/tendencias , Industria Farmacéutica , Investigación , Evaluación Preclínica de Medicamentos/economía , Industria Farmacéutica/economía , Industria Farmacéutica/tendencias , Farmacorresistencia Bacteriana , Humanos , Investigación/tendenciasRESUMEN
Multiresistant staphylococci pose an alarmingly growing problem, especially in serious hospital infections. The recent emergence of strains with reduced susceptibility against vancomycin, the last remaining drug effective against methicillin (multi) resistant Staphylococcus aureus, highlights the urgent need for new antimicrobial agents and new therapeutic regimen. Previously, new drugs were discovered exclusively in bacterial whole cell growth assays. Today's more rational approach depends on the identification of suitable target genes and proteins. These should be bacteria-specific and essential for growth either in vitro or in vivo. Targets within cell wall synthesis and remodeling pathways might be particularly attractive because the bacterial cell wall is a unique structure occurring only in prokaryots; many of the antibiotics in use today have confirmed its 'drugability'. However, several potential targets within this field have not yet been exploited successfully for anti-staphylococcal therapy and some were discovered only recently. After a short summary of known potential targets a set of genes involved in the pentaglycine interpeptide bridge formation of the staphylococcal cell wall will be introduced as interesting targets to combat multiresistant staphylococcal infections. Copyright 1999 Harcourt Publishers LtdCopyright DUMMY.
Asunto(s)
Antiinfecciosos/farmacología , Antiinfecciosos/uso terapéutico , Infecciones/tratamiento farmacológico , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/epidemiología , Biomarcadores , Industria Farmacéutica , Infecciones por VIH/complicaciones , Hepatitis B/tratamiento farmacológico , Hepatitis C/tratamiento farmacológico , Humanos , Infecciones/epidemiología , InvestigaciónRESUMEN
The need for new antibiotics to effectively treat antibiotic-resistant infections remains unfulfilled. Despite the well-publicised concern over this issue, only two novel antibiotic classes have been introduced in the past 20 years alongside several new agents of existing classes. Accordingly, the current antibiotic armoury remains inadequate to meet the challenges posed by resistance today. More worryingly, there are very few new agents being developed that can be expected to replace existing antibiotics that succumb to the rising tide of resistance.
RESUMEN
We analyzed the emergence of daptomycin nonsusceptibility in a patient with persistent vancomycin-intermediate Staphylococcus aureus (VISA) bacteremia. The daptomycin-nonsusceptible VISA's cell wall demonstrated a reduction in muramic acid O-acetylation, a phenotypic parameter not previously reported for VISA; some isolates also contained a single point mutation in the mprF gene.
Asunto(s)
Antibacterianos/farmacología , Daptomicina/farmacología , Farmacorresistencia Bacteriana/genética , Endocarditis Bacteriana/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Resistencia a la Vancomicina/genética , Acetilación , Aminoaciltransferasas , Bacteriemia/microbiología , Proteínas Bacterianas/genética , Pared Celular/química , Electroforesis en Gel de Campo Pulsado , Humanos , Ácidos Murámicos/metabolismo , Mutación/genética , FenotipoRESUMEN
Gene-expression profiling technologies in general, and proteomic technologies in particular have proven extremely useful to study the physiological response of bacterial cells to various environmental stress conditions. Complex protein toolkits coordinated by sophisticated regulatory networks have evolved to accommodate bacterial survival under ever-present stress conditions such as varying temperatures, nutrient availability, or antibiotics produced by other microorganisms that compete for habitat. In the last decades, application of man-made antibacterial agents resulted in additional bacterial exposure to antibiotic stress. Whereas the targeted use of antibiotics has remarkably reduced human suffering from infectious diseases, the ever-increasing emergence of bacteria that are resistant to antibiotics has led to an urgent need for novel antibiotic strategies. The intent of this review is to present an overview of the major achievements of proteomic approaches to study adaptation networks that are crucial for bacterial survival with a special emphasis on the stress induced by antibiotic treatment. A further focus will be the review of the, so far few, published efforts to exploit the knowledge derived from bacterial proteomic studies directly for the antibacterial drug-discovery process.
Asunto(s)
Antibacterianos/administración & dosificación , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Diseño de Fármacos , Electroforesis en Gel Bidimensional/métodos , Proteómica/métodos , Antibacterianos/química , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Electroforesis en Gel Bidimensional/tendencias , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/tendencias , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/fisiología , Espectrometría de Masas/métodos , Espectrometría de Masas/tendencias , Proteómica/tendenciasRESUMEN
The NfrA protein, a putative essential oxidoreductase in the soil bacterium Bacillus subtilis, is induced under heat shock and oxidative stress conditions. In order to characterize the function of an homologous NfrA protein in Staphylococcus aureus, an nfrA deletion strain was constructed, the protein was purified, the enzymatic activity was determined, and the transcriptional regulation was investigated. The experiments revealed that NfrA is not essential in S. aureus. The purified protein oxidized NADPH but not NADH, producing NADP in the presence of flavin mononucleotide, suggesting that NfrA is an NADPH oxidase in S. aureus. In addition, the NfrA enzyme showed nitroreductase activity and weak disulfide reductase activity. Transcription was strongly induced by ethanol, diamide, and nitrofurantoin. Hydrogen peroxide induced nfrA transcription only at high concentrations. The expression of nfrA was independent of the alternative sigma factor sigma(B). Furthermore, the transcriptional start site was determined, which allowed identification of a PerR box homologous sequence upstream of the nfrA promoter. The observations presented here suggest that NfrA is a nonessential NADPH oxidoreductase which may play a role in the oxidative stress response of S. aureus, especially in keeping thiol-disulfide stress in balance.
Asunto(s)
Mononucleótido de Flavina/fisiología , NADPH Oxidasas/metabolismo , Staphylococcus aureus/enzimología , Mapeo Cromosómico , Diamida , Etanol , Regulación Bacteriana de la Expresión Génica , NADP/fisiología , NADPH Oxidasas/química , NADPH Oxidasas/genética , Nitrofurantoína , Estrés Oxidativo , Staphylococcus aureus/genética , Transcripción Genética/efectos de los fármacosRESUMEN
We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.
Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Proteómica , Antiinfecciosos Urinarios/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de los fármacos , Dermatoglifia del ADN , Electroforesis en Gel de Poliacrilamida , Procesamiento de Imagen Asistido por Computador , Marcaje Isotópico , Metionina/metabolismo , Nitrofurantoína/farmacología , Pirimidinonas/farmacología , Radioisótopos de AzufreRESUMEN
The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility. GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains. The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains. Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype. This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains. Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain. In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin. We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells.
Asunto(s)
Antibacterianos/farmacología , Bacteriólisis/efectos de los fármacos , Pared Celular/química , Glicopéptidos/farmacología , Lisostafina/farmacología , Staphylococcus aureus/efectos de los fármacos , Northern Blotting , Cromatografía Líquida de Alta Presión , Citoplasma/química , Citoplasma/metabolismo , Peptidoglicano/farmacología , Fósforo/química , Ácidos Teicoicos/metabolismo , Resistencia a la VancomicinaRESUMEN
Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.