RESUMEN
The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Triazinas/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico/fisiología , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Evaluación Preclínica de Medicamentos , Farmacorresistencia Bacteriana/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a ß-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Endopeptidasas , Proteínas de Escherichia coli , Escherichia coli , Proteínas de la Membrana , Desplegamiento Proteico , Factores de Transcripción , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismoRESUMEN
A riboflavin biosynthesis pathway-specific phenotypic screen using a library of compounds, all with unspecified antibiotic activity, identified one small molecule later named ribocil, for which intrinsic antibacterial activity against Escherichia coli was completely suppressed by addition of exogenous riboflavin to the bacterial growth medium. The ability of riboflavin to suppress the activity of ribocil, and further demonstration that ribocil inhibited riboflavin synthesis (IC50 = 0.3 µM), supported that a component of the riboflavin synthesis pathway was the molecular target. Remarkably, resistance mutation selection and whole-genome sequencing showed that the target of ribocil was not an enzyme in the riboflavin biosynthesis pathway, but instead the flavin mononucleotide riboswitch, a noncoding structural RNA element in the ribB gene that encodes a key riboflavin synthesis enzyme. Although ribocil is structurally distinct from the natural riboswitch regulatory ligand flavin mononucleotide, ribocil binding to the riboswitch results in efficient repression of ribB expression and inhibition of riboflavin biosynthesis and bacterial growth. A cell-based riboswitch regulated gene reporter assay as well as an in vitro riboswitch RNA aptamer-binding assay, both of which are described in detail here along with the riboflavin pathway-specific screen, were developed to further validate the mechanism of action of ribocil and to facilitate the discovery of more potent analogues.
Asunto(s)
Descubrimiento de Drogas , Metabolismo Energético/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Mononucleótido de Flavina/metabolismo , Riboflavina/metabolismo , Riboswitch/efectos de los fármacos , Antibacterianos/farmacología , Clonación Molecular , Genes Reporteros , Ligandos , Mutación , Fenotipo , Plásmidos , Técnica SELEX de Producción de Aptámeros , Bibliotecas de Moléculas PequeñasRESUMEN
The first-in-class lipopeptide antibiotic daptomycin (DAP) is highly active against Gram-positive pathogens including ß-lactam and glycopeptide resistant strains. Its molecular mode of action remains enigmatic, since a defined target has not been identified so far and multiple effects, primarily on the cell envelope have been observed. Reduced DAP susceptibility has been described in S. aureus and enterococci after prolonged treatment courses. In line with its pleiotropic antibiotic activities, a unique, defined molecular mechanism of resistance has not emerged, instead non-susceptibility appears often accompanied by alterations in membrane composition and changes in cell wall homeostasis. We compared S. aureus strains HG001 and SG511, which differ primarily in the functionality of the histidine kinase GraS, to evaluate the impact of the GraRS regulatory system on the development of DAP non-susceptibility. After extensive serial passing, both DAPR variants reached a minimal inhibitory concentration of 31⯵g/ml and shared some phenotypic characteristics (e.g. thicker cell wall, reduced autolysis). However, based on comprehensive analysis of the underlying genetic, transcriptomic and proteomic changes, we found that both strains took different routes to achieve DAP resistance. Our study highlights the impressive genetic and physiological capacity of S. aureus to counteract pleiotropic activities of cell wall- and membrane-active compounds even when a major cell wall regulatory system is dysfunctional.
Asunto(s)
Proteínas Bacterianas/genética , Daptomicina/farmacología , Regulación Bacteriana de la Expresión Génica , Mutación , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Membrana Celular/metabolismo , Farmacorresistencia Bacteriana/genética , Genotipo , Histidina Quinasa/genética , Pruebas de Sensibilidad Microbiana , Proteómica , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/enzimología , Staphylococcus aureus/fisiologíaRESUMEN
Systemic fungal infections represent an important public health concern, and new antifungal agents are highly desirable. Herein, we describe the design, synthesis, and biological evaluation of a novel class of antifungal compounds called antibody-recruiting molecules targeting fungi (ARM-Fs). Our approach relies on the use of non-peptidic small molecules, which selectively bind fungal cells and recruit endogenous antibodies to their surfaces, resulting in immune-mediated clearance. Using the opportunistic fungal pathogen Candida albicans as a model, we identified a highly specific bifunctional molecule able to mediate the engulfment and phagocytosis of C. albicans cells by human immune cells in biologically relevant functional assays. This work represents a novel therapeutic approach to treating fungal illness with significant potential to complement and/or combine with existing treatment strategies.
Asunto(s)
Anticuerpos/inmunología , Subgrupos de Linfocitos B/inmunología , Candida albicans/inmunología , Acetilglucosamina/química , Anticuerpos/metabolismo , Subgrupos de Linfocitos B/citología , Candida albicans/metabolismo , Candida albicans/patogenicidad , Quitina/química , Quitina/metabolismo , Dinitrobencenos/química , Dinitrobencenos/inmunología , Diseño de Fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Células HL-60 , Interacciones Huésped-Patógeno , Humanos , FagocitosisRESUMEN
To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient Escherichia coli and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of E. coli DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of E. coli and Pseudomonas aeruginosa DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.
Asunto(s)
Antibacterianos/farmacología , Girasa de ADN/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Fluoroquinolonas/farmacología , Pruebas de Sensibilidad Microbiana , Dominios Proteicos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimologíaRESUMEN
Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide a mechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism.
Asunto(s)
Mononucleótido de Flavina/genética , Homeostasis/efectos de los fármacos , Pirimidinas/farmacología , Riboflavina/análogos & derivados , Riboflavina/metabolismo , Riboswitch/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Animales , Antibacterianos/farmacología , Secuencia de Bases , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/metabolismo , Staphylococcus aureus Resistente a Meticilina/fisiología , Ratones , Modelos Moleculares , Terapia Molecular Dirigida , Conformación Proteica , Riboflavina/farmacología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/fisiologíaRESUMEN
We describe our optimization efforts to improve the physicochemical properties, solubility, and off-target profile of 1, an inhibitor of TarO, an early stage enzyme in the biosynthetic pathway for wall teichoic acid (WTA) synthesis. Compound 1 displayed a TarO IC50 of 125 nM in an enzyme assay and possessed very high lipophilicity (clogP = 7.1) with no measurable solubility in PBS buffer. Structure-activity relationship (SAR) studies resulted in a series of compounds with improved lipophilic ligand efficiency (LLE) consistent with the reduction of clogP. From these efforts, analog 9 was selected for our initial in vivo study, which in combination with subefficacious dose of imipenem (IPM) robustly lowered the bacterial burden in a neutropenic Staphylococci murine infection model. Concurrent with our in vivo optimization effort using 9, we further improved LLE as exemplified by a much more druglike analog 26.
Asunto(s)
Lípidos/química , Bibliotecas de Moléculas Pequeñas , Animales , Femenino , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Solubilidad , Relación Estructura-ActividadRESUMEN
UNLABELLED: A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ(G193D) allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ(G193D) filaments are more twisted and shorter than wild-type filaments. In vivo, M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE: The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Staphylococcus aureus/citología , Staphylococcus aureus/genética , Microscopía , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Bacterial riboswitches are non-coding RNA structural elements that direct gene expression in numerous metabolic pathways. The key regulatory roles of riboswitches, and the urgent need for new classes of antibiotics to treat multi-drug resistant bacteria, has led to efforts to develop small-molecules that mimic natural riboswitch ligands to inhibit metabolic pathways and bacterial growth. Recently, we reported the results of a phenotypic screen targeting the riboflavin biosynthesis pathway in the Gram-negative bacteria Escherichia coli that led to the identification of ribocil, a small molecule inhibitor of the flavin mononucleotide (FMN) riboswitch controlling expression of this biosynthetic pathway. Although ribocil is structurally distinct from FMN, ribocil functions as a potent and highly selective synthetic mimic of the natural ligand to repress riboswitch-mediated ribB gene expression and inhibit bacterial growth both in vitro and in vivo. Herein, we expand our analysis of ribocil; including mode of binding in the FMN binding pocket of the riboswitch, mechanisms of resistance and structure-activity relationship guided efforts to generate more potent analogs.
Asunto(s)
Antifúngicos/química , Glicosilfosfatidilinositoles/biosíntesis , Aminopiridinas/química , Aminopiridinas/metabolismo , Aminopiridinas/farmacología , Aminopiridinas/uso terapéutico , Antifúngicos/metabolismo , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Humanos , Isoxazoles/química , Isoxazoles/metabolismo , Isoxazoles/farmacología , Isoxazoles/uso terapéutico , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Micosis/tratamiento farmacológico , Micosis/microbiología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
A series of benzimidazole analogs have been synthesized to improve the profile of the previous lead compounds tarocin B and 1. The syntheses, structure-activity relationships, and selected biochemical data of these analogs are described. The optimization efforts allowed the identification of 21, a fluoro-substituted benzimidazole, exhibiting potent TarO inhibitory activity and typical profile for a wall teichoic acid (WTA) biosynthesis inhibitor. Compound 21 displayed a potent synergistic and bactericidal effect in combination with imipenem against diverse methicillin-resistant Staphylococci.
Asunto(s)
Antibacterianos/farmacología , Bencimidazoles/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ácidos Teicoicos/antagonistas & inhibidores , Animales , Antibacterianos/química , Bencimidazoles/química , Pruebas de Sensibilidad Microbiana , Ratas , Relación Estructura-Actividad , Ácidos Teicoicos/biosíntesisRESUMEN
The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current ß-lactam antibiotics and created an urgent need for novel treatment options. Using an S. aureus phenotypic screening strategy, we have identified small molecule early stage wall teichoic acid (WTA) pathway-specific inhibitors predicted to be chemically synergistic with ß-lactams. These previously disclosed inhibitors, termed tarocins, demonstrate by genetic and biochemical means inhibition of TarO, the first step in WTA biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad spectrum ß-lactam antibiotics across diverse clinical isolates of methicillin-resistant Staphylococci. The synthesis and structure-activity relationships (SAR) of a tarocin series will be detailed. Tarocins and other WTA inhibitors may provide a rational strategy to develop Gram-positive bactericidal ß-lactam combination agents active against methicillin-resistant Staphylococci.
Asunto(s)
Antibacterianos/química , Ácidos Teicoicos/metabolismo , beta-Lactamas/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/farmacología , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Evaluación Preclínica de Medicamentos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , beta-Lactamas/metabolismoRESUMEN
Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro ß-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the ß-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore ß-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Racemasas y Epimerasas/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus epidermidis/metabolismo , Ácidos Teicoicos/biosíntesis , Animales , Proteínas Bacterianas/química , Biopelículas/crecimiento & desarrollo , Pared Celular/metabolismo , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Staphylococcus aureus Resistente a Meticilina , Ratones , Pruebas de Sensibilidad Microbiana , Microscopía Fluorescente , Resonancia Magnética Nuclear Biomolecular , Racemasas y Epimerasas/química , Infecciones Estafilocócicas/metabolismoRESUMEN
The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current ß-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with ß-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum ß-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal ß-lactam combination agents active against methicillin-resistant staphylococci.
Asunto(s)
Proteínas Bacterianas/metabolismo , Vías Biosintéticas/efectos de los fármacos , Pared Celular/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ácidos Teicoicos/biosíntesis , beta-Lactamas/farmacología , Animales , Pared Celular/efectos de los fármacos , Dicloxacilina/farmacología , Dicloxacilina/uso terapéutico , Femenino , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Fenotipo , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Resultado del TratamientoRESUMEN
Nonessential enzymes in the staphylococcal wall teichoic acid (WTA) pathway serve as highly validated ß-lactam potentiation targets. MnaA (UDP-GlcNAc 2-epimerase) plays an important role in an early step of WTA biosynthesis by providing an activated form of ManNAc. Identification of a selective MnaA inhibitor would provide a tool to interrogate the contribution of the MnaA enzyme in the WTA pathway as well as serve as an adjuvant to restore ß-lactam activity against methicillin-resistant Staphylococcus aureus (MRSA). However, development of an epimerase functional assay can be challenging since both MnaA substrate and product (UDP-GlcNAc/UDP-ManNAc) share an identical molecular weight. Herein, we developed a nuclear magnetic resonance (NMR) functional assay that can be combined with other NMR approaches to triage putative MnaA inhibitors from phenotypic cell-based screening campaigns. In addition, we determined that tunicamycin, a potent WTA pathway inhibitor, inhibits both S. aureus MnaA and a functionally redundant epimerase, Cap5P.
Asunto(s)
Pared Celular/efectos de los fármacos , Espectroscopía de Resonancia Magnética/métodos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Carbohidrato Epimerasas/antagonistas & inhibidores , Carbohidrato Epimerasas/química , Pared Celular/química , Humanos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ácidos Teicoicos/química , Ácidos Teicoicos/metabolismo , Azúcares de Uridina Difosfato/química , Azúcares de Uridina Difosfato/metabolismo , Resistencia betalactámica/efectos de los fármacos , beta-Lactamasas/química , beta-Lactamasas/efectos de los fármacosRESUMEN
Paramount to any rational discovery of new antibiotics displaying novel mechanisms of action is a deep knowledge of the genetic basis of microbial growth, division and virulence. The bakers' yeast,Saccharomyces cerevisiae, illustrates the highest understanding of the genetic underpinnings of microbial life, and from this framework, a systems biology paradigm has evolved, begging to be emulated in antibacterial discovery. Here, we review landmark events in the history of yeast genomics that provide this new foundation for antibacterial drug discovery.
Asunto(s)
Descubrimiento de Drogas , Genómica , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae/genética , Genoma Bacteriano , Genoma Fúngico , Haploinsuficiencia , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.
Asunto(s)
Pirimidinas/química , Pirimidinas/farmacología , ARN Bacteriano/química , ARN Bacteriano/efectos de los fármacos , Riboswitch/efectos de los fármacos , Animales , Aptámeros de Nucleótidos/química , Bacterias/citología , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Secuencia de Bases , Cristalografía por Rayos X , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Femenino , Mononucleótido de Flavina/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Transferasas Intramoleculares/genética , Ligandos , Ratones , Ratones Endogámicos DBA , Modelos Moleculares , Datos de Secuencia Molecular , Pirimidinas/aislamiento & purificación , Pirimidinas/uso terapéutico , ARN Bacteriano/genética , Reproducibilidad de los Resultados , Riboflavina/biosíntesis , Riboswitch/genética , Especificidad por SustratoRESUMEN
Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG) synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins), when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus.
Asunto(s)
Antibacterianos/farmacología , Pared Celular/metabolismo , Farmacorresistencia Bacteriana Múltiple , Staphylococcus aureus Resistente a Meticilina/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/biosíntesis , Peptidil Transferasas/metabolismo , Animales , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/microbiología , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Staphylococcus aureus Resistente a Meticilina/ultraestructura , Viabilidad Microbiana/efectos de los fármacos , Mutación , Proteínas de Unión a las Penicilinas/genética , Peptidoglicano Glicosiltransferasa/genética , Peptidoglicano Glicosiltransferasa/metabolismo , Peptidil Transferasas/genética , Filogenia , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/metabolismo , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Virulencia/efectos de los fármacosRESUMEN
Developmental transitions between single-cell yeast and multicellular filaments underpin virulence of diverse fungal pathogens. For the leading human fungal pathogen Candida albicans, filamentation is thought to be required for immune cell escape via induction of an inflammatory programmed cell death. Here we perform a genome-scale analysis of C. albicans morphogenesis and identify 102 negative morphogenetic regulators and 872 positive regulators, highlighting key roles for ergosterol biosynthesis and N-linked glycosylation. We demonstrate that C. albicans filamentation is not required for escape from host immune cells; instead, macrophage pyroptosis is driven by fungal cell-wall remodelling and exposure of glycosylated proteins in response to the macrophage phagosome. The capacity of killed, previously phagocytized cells to drive macrophage lysis is also observed with the distantly related fungal pathogen Cryptococcus neoformans. This study provides a global view of morphogenetic circuitry governing a key virulence trait, and illuminates a new mechanism by which fungi trigger host cell death.