RESUMEN
We deleted a subset of 27 open reading frames (ORFs) from Escherichia coli which encode previously uncharacterized, probably soluble gene products homologous to proteins from a broad spectrum of bacterial pathogens such as Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis and only distantly related to eukaryotic proteins. Six novel bacteria-specific genes essential for growth in complex medium could be identified through a combination of bioinformatics-based and experimental approaches. We also compared our data to published results of gene inactivation projects with Mycoplasma genitalium and Bacillus subtilis and looked for homologs in all known prokaryotic genomes. Such analyses highlight the enormous metabolic flexibility of prokaryotes. Six of 27 studied genes have been functionally characterized up to now, amongst these four of the essential genes. The gene products YgbP, YgbB and YchB are involved in the non-mevalonate pathway of isoprenoid biosynthesis. KdtB is characterized as the posphopantetheine adenylyltransferase CoaD. There are indications that the other two essential gene products YjeE and YqgF, which we have identified, also possess enzymatic functions. These findings demonstrate the potential of such proteins to be used in screening of large chemical libraries for inhibitors which could be further developed to novel broad-spectrum antibiotics.
Asunto(s)
Enterococcus faecalis/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/patogenicidad , Genes Bacterianos , Genes Esenciales , Haemophilus influenzae/genética , Sistemas de Lectura Abierta , Liasas de Fósforo-Oxígeno , Staphylococcus aureus/genética , Streptococcus pneumoniae/genética , Bacillus subtilis/genética , Bacillus subtilis/patogenicidad , Proteínas Bacterianas/genética , Clonación Molecular , Farmacorresistencia Microbiana/genética , Enterococcus faecalis/patogenicidad , Haemophilus influenzae/patogenicidad , Datos de Secuencia Molecular , Mycoplasma/genética , Mycoplasma/patogenicidad , Nucleotidiltransferasas/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia , Staphylococcus aureus/patogenicidad , Streptococcus pneumoniae/patogenicidadRESUMEN
It is generally assumed that type A lantibiotics primarily kill bacteria by permeabilization of the cytoplasmic membrane. As previous studies had demonstrated that nisin interacts with the membrane-bound peptidoglycan precursors lipid I and lipid II, we presumed that this interaction could play a role in the pore formation process of lantibiotics. Using a thin-layer chromatography system, we found that only nisin and epidermin, but not Pep5, can form a complex with [14C]-lipid II. Lipid II was then purified from Micrococcus luteus and incorporated into carboxyfluorescein-loaded liposomes made of phosphatidylcholine and cholesterol (1:1). Liposomes supplemented with 0.05 or 0.1 mol% of lipid II did not release any marker when treated with Pep5 or epilancin K7 (peptide concentrations of up to 5 mol% were tested). In contrast, as little as 0.01 mol% of epidermin and 0.1 mol% of nisin were sufficient to induce rapid marker release; phosphatidylglycerol-containing liposomes were even more susceptible. Controls with moenomycin-, undecaprenol- or dodecaprenolphosphate-doped liposomes demonstrated the specificity of the lantibiotics for lipid II. These results were correlated with intact cells in an in vivo model. M. luteus and Staphylococcus simulans were depleted of lipid II by preincubation with the lipopeptide ramoplanin and then tested for pore formation. When applied in concentrations below the minimal inhibitory concentration (MIC) and up to 5-10 times the MIC, the pore formation by nisin and epidermin was blocked; at higher concentrations of the lantibiotics the protective effect of ramoplanin disappeared. These results demonstrate that, in vitro and in vivo, lipid II serves as a docking molecule for nisin and epidermin, but not for Pep5 and epilancin K7, and thereby facilitates the formation of pores in the cytoplasmic membrane.
Asunto(s)
Antibacterianos/farmacología , Depsipéptidos , Nisina/farmacología , Péptidos Cíclicos , Péptidos , Peptidoglicano/metabolismo , Antibacterianos/metabolismo , Bacteriocinas , Liposomas/metabolismo , Micrococcus luteus/efectos de los fármacos , Micrococcus luteus/metabolismo , Nisina/metabolismo , Peptidoglicano/efectos de los fármacos , Oligosacáridos de Poliisoprenil Fosfato/metabolismo , Staphylococcus/efectos de los fármacos , Staphylococcus/metabolismoRESUMEN
The lantibiotic mersacidin exerts its bactericidal action by inhibition of peptidoglycan biosynthesis. It interferes with the membrane-associated transglycosylation reaction; during this step the ultimate monomeric peptidoglycan precursor, undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (lipid II) is converted into polymeric nascent peptidoglycan. In the present study we demonstrate that the molecular basis of this inhibition is the interaction of mersacidin with lipid II. The adsorption of [14C]mersacidin to growing cells, as well as to isolated membranes capable of in vitro peptidoglycan synthesis, was strictly dependent on the availability of lipid II, and antibiotic inhibitors of lipid II formation strongly interfered with this binding. Direct evidence for the interaction was provided by studies with isolated lipid II. [14C]mersacidin associated tightly with [14C]lipid II micelles; the complex was stable even in the presence of 1% sodium dodecyl sulfate. Furthermore, the addition of isolated lipid II to the culture broth efficiently antagonized the bactericidal activity of mersacidin. In contrast to the glycopeptide antibiotics, complex formation does not involve the C-terminal D-alanyl-D-alanine moiety of the lipid intermediate. Thus, the interaction of mersacidin with lipid II apparently occurs via a binding site which is not targeted by any antibiotic currently in use.
Asunto(s)
Antibacterianos/farmacología , Péptidos , Peptidoglicano/metabolismo , Oligosacáridos de Poliisoprenil Fosfato/metabolismo , Radiofármacos/metabolismo , Antibacterianos/metabolismo , Antiinfecciosos Locales/farmacología , Bacitracina/farmacología , Bacteriocinas , Sitios de Unión , Radioisótopos de Carbono , Micrococcus luteus/efectos de los fármacos , Peptidoglicano/efectos de los fármacos , Vancomicina/farmacologíaRESUMEN
The lantibiotic mersacidin has been previously reported to interfere with bacterial peptidoglycan biosynthesis, [Brötz, H., Bierbaum, G., Markus, A., Molitor, E. & Sahl, H.-G. (1995) Antimicrob. Agents Chemother. 39, 714-719]. Here, we focus on the target reaction and describe a mersacidin-induced accumulation of UDP-N-acetylmuramoyl-pentapeptide, indicating that inhibition of peptidoglycan synthesis occurs after the formation of cytoplasmic precursors. In vitro studies involving a wall-membrane particulate fraction of Bacillus megaterium KM demonstrated that mersacidin did not prevent the synthesis of lipid II [undecaprenyl-diphosphoryl-N-acetylmuramoyl-(pentapeptide)-N-ac ety lglucosamine] but specifically the subsequent conversion of this intermediate into polymeric nascent glycan strands by transglycosylation. Comparison with other inhibitors of transglycosylation shows that the effective concentration of mersacidin in vitro is in the range of that of the glycopeptide antibiotic vancomycin but 2-3 orders of magnitude higher than that of the competitive enzyme inhibitor moenomycin. The analogy to the glycopeptides may hint at an interaction of mersacidin with the peptidoglycan precursor rather than with the enzyme. Unlike vancomycin however, mersacidin inhibits peptidoglycan formation from UDP-N-acetylmuramoyl-tripeptide and is active against Enterococcus faecium expressing the vanA resistance gene cluster. This indicates that the molecular target site of mersacidin differs from that of vancomycin and that no cross-resistance exists between the two antibiotics.
Asunto(s)
Antibacterianos/farmacología , Péptidos , Peptidoglicano/biosíntesis , Secuencia de Aminoácidos , Antibacterianos/uso terapéutico , Bacillus megaterium/efectos de los fármacos , Bacillus megaterium/metabolismo , Proteínas Bacterianas/genética , Bacteriocinas , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Cromatografía Líquida de Alta Presión , Enterococcus/efectos de los fármacos , Glicosilación/efectos de los fármacos , Lípidos de la Membrana/análisis , Datos de Secuencia Molecular , Ácidos Murámicos/metabolismo , Mutación/genética , Uridina Difosfato N-Acetilglucosamina/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/metabolismo , Vancomicina/farmacología , Vancomicina/uso terapéuticoRESUMEN
Mersacidin is a lanthionine-containing peptide antibiotic (lantibiotic), able to inhibit the growth of a number of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) in a manner similar to, but distinct from, vancomycin. In order to further understand the mode of action of this lantibiotic, Staphylococcus simulans 22 cells were treated either with the antibiotics penicillin, tunicamycin or vancomycin or with mersacidin and then compared with untreated cells after electron microscopic examination. Mersacidin treatment brought about a time-dependent, generalised decrease in the thickness of the bacterial cell wall. In addition, mersacidin treatment caused a roughening of the cell wall surface layer and also reduced the thickness and frequency of formation of dividing cell septa. Reduction of cell wall thickness appears to result from inhibition of new wall biosynthesis combined with cell wall turnover. These features of mersacidin-induced effects on cell morphology confirm that it has a novel mode of action (Brötz, H., G. Bierbaum, A. Markus, E. Molitor, and H.-G. Sahl: Antimicrob. Agents Chemother. 39 [1995] 714-719), probably directed towards a membrane-bound biosynthetic step but not towards a specific penicillin-binding-protein.
Asunto(s)
Alanina/análogos & derivados , Antibacterianos/farmacología , Péptidos , Staphylococcus/efectos de los fármacos , Staphylococcus/ultraestructura , Alanina/farmacología , Bacteriocinas , Penicilinas/farmacología , Sulfuros , Tunicamicina/farmacología , Vancomicina/farmacologíaRESUMEN
Mersacidin is a lanthionine-containing peptide antibiotic that shows a good in vivo efficiency against methicillin-resistant Staphylococcus aureus. It is excreted during early stationary phase and could be purified from culture supernatant in a one-step procedure by reversed phase HPLC. Its structural gene was cloned from chromosomal DNA of the producer strain Bacillus subtilis HIL Y-85,54728. Sequencing revealed that pre-mersacidin consists of an unusually long 48 amino acid leader sequence and a 20 amino acid propeptide part which is modified during biosynthesis to the mature lantibiotic. The comparison of the mersacidin prepeptide with those of hitherto known lantibiotics demonstrates that mersacidin is more closely related to type B lantibiotic cinnamycin than to type A lantibiotics.
Asunto(s)
Antibacterianos/biosíntesis , Péptidos , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/clasificación , Antibacterianos/farmacología , Bacillus subtilis/genética , Bacteriocinas , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Genes Bacterianos , Resistencia a la Meticilina/genética , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genéticaRESUMEN
Mersacidin is an antibiotic peptide produced by Bacillus sp. strain HIL Y-85,54728 that belongs to the group of lantibiotics. Its activity in vivo against methicillin-resistant Staphylococcus aureus strains compares with that of the glycopeptide antibiotic vancomycin (S. Chatterjee, D. K. Chatterjee, R. H. Jani, J. Blumbach, B. N. Ganguli, N. Klesel, M. Limbert, and G. Seibert, J. Antibiot. 45:839-845, 1992). Incubation of Staphylococcus simulans 22 with mersacidin resulted in the cessation of growth and slow lysis. Biosyntheses of DNA, RNA, and protein were not affected, whereas incorporation of glucose and D-alanine was inhibited and a regular reduction in the level of cell wall thickness was observed. Thus, unlike type A lantibiotics, mersacidin does not form pores in the cytoplasmic membrane but rather inhibits cell wall biosynthesis. Comparison with tunicamycin-treated cells indicated that peptidoglycan rather than teichoic acid metabolism is primarily affected. Mersacidin caused the excretion of a putative cell wall precursor into the culture supernatant. The formation of polymeric peptidoglycan was effectively inhibited in an in vitro assay, probably on the level of transglycosylation. In contrast to vancomycin, the activity of mersacidin was not antagonized by the tripeptide diacetyl-L-Lys-D-Ala-D-Ala, indicating that on the molecular level its mode of action differs from those of glycopeptide antibiotics. These data together with electron microscopy suggest that mersacidin acts on a novel target, which opens new perspectives for the treatment of methicillin-resistant S. aureus.