RESUMEN
BACKGROUND: Staphylococcus aureus is a common pathogen with strains that are resistant to existing antibiotics. MurJ from S. aureus (SaMurJ), an integral membrane protein functioning as Lipid II flippase, is a potential target for developing new antibacterial agents against this pathogen. Successful expression and purification of this protein shall be useful in the development of drugs against this target. OBJECTIVE: In this study, we demonstrated the optimized expression and purification procedures of SaMurJ, identified suitable detergent for extracting and solubilizing the protein, and examined the peptidisc system to generate a detergent-free environment. METHODS: SaMurJ fused with N-terminal ten-His tag was expressed without induction. Six detergents were selected for screening the most efficient candidate for extraction and solubilization of the protein. The thermostability of the detergent-solubilized protein was assessed by evaluated temperature incubation. Different ratios of peptidisc bi-helical peptide (NSPr) to SaMurJ were mixed and the on-bead peptidisc assembly method was applied. RESULTS: SaMurJ expressed in BL21(DE3) was confirmed by peptide fingerprinting, with a yield of 1 mg SaMurJ per liter culture. DDM was identified as the optimum detergent for solubilization and the nickel affinity column enabled SaMurJ purification with a purity of ~88%. However, NSPr could not stabilize SaMurJ. CONCLUSION: The expression and purification of SaMurJ were successful, with high purity and good yield. SaMurJ can be solubilized and stabilized by a DDM-containing buffer.
Asunto(s)
Proteínas Bacterianas , Staphylococcus aureus , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Detergentes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Solubilidad , Expresión Génica , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivadosRESUMEN
The Filamenting temperature-sensitive mutant Z (FtsZ), an essential GTPase in bacterial cell division, is highly conserved among Gram-positive and Gram-negative bacteria and thus considered an attractive target to treat antibiotic-resistant bacterial infections. In this study, a new class of FtsZ inhibitors bearing the pyrimidine-quinuclidine scaffold was identified from structure-based virtual screening of natural product libraries. Iterative rounds of in silico studies and biological evaluation established the preliminary structure-activity relationships of the new compounds. Potent FtsZ inhibitors with low micromolar IC50 and antibacterial activity against S. aureus and E. coli were found. These findings support the use of virtual screening and structure-based design for the rational development of new antibacterial agents with innovative mechanisms of action.
Asunto(s)
Antibacterianos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , GTP Fosfohidrolasas/antagonistas & inhibidores , Animales , Antibacterianos/química , Sitios de Unión , Bovinos , Evaluación Preclínica de Medicamentos , Escherichia coli/efectos de los fármacos , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Conformación Proteica , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína , Pirimidinas/química , Quinuclidinas/química , Homología de Secuencia de Aminoácido , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Relación Estructura-Actividad , Tubulina (Proteína)/químicaRESUMEN
Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for â¼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.
Asunto(s)
Antineoplásicos , Dickeya chrysanthemi , Neoplasias Gástricas , Humanos , Animales , Ratones , Asparaginasa/genética , Asparaginasa/farmacología , Asparaginasa/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/metabolismo , Asparagina , Glutamina , Neoplasias Gástricas/tratamiento farmacológico , Enterobacteriaceae/metabolismo , Albúmina SéricaRESUMEN
The Ω-loop at the active site of ß-lactamases exerts significant impact on the kinetics and substrate profile of these enzymes by forming part of the substrate binding site and posing as steric hindrance toward bulky substrates. Mutating certain residues on the Ω-loop has been a general strategy for molecular evolution of ß-lactamases to expand their hydrolytic activity toward extended-spectrum antibiotics through a mechanism believed to involve enhanced structural flexibility of the Ω-loop. Yet no structural information is available that demonstrates such flexibility or its relation to substrate profile and enzyme kinetics. Here we report an engineered ß-lactamase that contains an environment-sensitive fluorophore conjugated near its active site to probe the structural dynamics of the Ω-loop and to detect the binding of diverse substrates. Our results show that this engineered ß-lactamase has improved binding kinetics and positive fluorescence signal toward oxyimino-cephalosporins, but shows little such effect to non-oxyimino-cephalosporins. Structural studies reveal that the Ω-loop adopts a less stabilized structure, and readily undergoes conformational change to accommodate the binding of bulky oxyimino-cephalosporins while no such change is observed for non-oxyimino-cephalosporins. Mutational studies further confirm that this substrate-induced structural change is directly responsible for the positive fluorescence signal specific to oxyimino-cephalosporins. Our data provide mechanistic evidence to support the long-standing model that the evolutionary strategy of mutating the Ω-loop leads to increased structural flexibility of this region, which in turn facilitates the binding of extended spectrum ß-lactam antibiotics. The oxyimino-cephalosporin-specific fluorescence profile of our engineered ß-lactamase also demonstrates the possibility of designing substrate-selective biosensing systems.
Asunto(s)
Cefalosporinas/metabolismo , beta-Lactamasas/metabolismo , Sitios de Unión , Dominio Catalítico , Colorantes Fluorescentes , Cinética , Conformación Molecular , Sondas Moleculares , Mutagénesis Sitio-Dirigida , Docilidad , Ingeniería de Proteínas , Especificidad por Sustrato , beta-Lactamasas/químicaRESUMEN
The protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS(+) wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH(2)O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log(10) in sterile ddH(2)O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log(10) in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS(+) and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.
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Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Escherichia coli O157/fisiología , Regulación Bacteriana de la Expresión Génica , Viabilidad Microbiana , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Medios de Cultivo/química , Escherichia coli O157/genética , Escherichia coli O157/crecimiento & desarrollo , Eliminación de Gen , Factor sigma/genética , Microbiología del AguaRESUMEN
Herein is described the development of a novel switch-on fluorescence assay for detecting ß-lactamases. The fluorescence assay comprises two components: solid beads coated with a ß-lactam antibiotic, which is linked to an environment-sensitive fluorophore (dansylaminothiophenol, DTA), and amyloid fibrils of hen lysozyme (acting as fluorescence enhancer and visual tool). In the presence of the clinically significant TEM-1 ß-lactamase, the DTA-antibiotic complex on the solid beads is hydrolyzed, thus releasing the DTA dye into solution. The DTA dye is only weakly fluorescent in solution but gives strong green fluorescence upon binding to lysozyme fibrils. These strongly fluorescent DTA-bound fibrils can be easily visualized by the naked eye upon illumination of the sample with a simple UV lamp. The fluorescence assay can detect TEM-1 at low concentration (0.01 nM). In contrast, no observable fluorescence appears when the fluorescence assay is performed on samples without the TEM-1 ß-lactamase.
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Amiloide/metabolismo , Antibacterianos/química , Escherichia coli/enzimología , Lactamas/química , beta-Lactamasas/análisis , Fluorescencia , Estructura Molecular , Estereoisomerismo , beta-Lactamasas/metabolismoRESUMEN
The fluorescein-labeled E166C mutant of the PenPC beta-lactamase (E166Cf) represents a successful model in the construction of "switch-on" fluorescent biosensors from nonallosteric proteins (Chan P.-H. et al.; J. Am Chem. Soc., 2004, 126, 4074). This paper focuses on the study of the biosensing mechanism by which the E166Cf biosensor changes its fluorescence upon beta-lactam binding and hydrolysis. Mass spectrometric and stopped-flow fluorescence studies of E166Cf with cefuroxime, penicillin G, and 6-aminopenicillanic acid reveal that the formation of enzyme-substrate complex enhances the fluorescence of E166Cf, and the subsequent regeneration of the free enzyme restores the weak fluorescence of E166Cf. Molecular modeling studies of E166Cf with penicillin G show that the fluorescein label is likely to share a common space with the beta-lactam and thiazolidine rings of the antibiotic in the active site. This spatial clash appears to cause the fluorescein label to move from the active site to the external aqueous environment upon substrate binding and hence experience higher water exposure. Steady-state fluorescence measurements indicate that the fluorescence of E166Cf can be enhanced by 6-aminopenicillanic acid, which consists of the beta-lactam and thiazolidine rings only. Thermal denaturation experiments of the wild-type enzyme, E166C, and E166Cf reveal that the E166C mutation is likely to increase the flexibility of the Omega-loop. This "modified" structural property might compensate for the possible steric effect of the fluorescein label on substrate binding.
Asunto(s)
Antibacterianos/análisis , Técnicas Biosensibles/métodos , Fluoresceínas/química , Colorantes Fluorescentes/química , beta-Lactamasas/química , beta-Lactamas/análisis , Antibacterianos/química , Cefuroxima/análisis , Cefuroxima/química , Dicroismo Circular , Simulación por Computador , Cinética , Espectrometría de Masas , Modelos Moleculares , Ácido Penicilánico/análogos & derivados , Ácido Penicilánico/análisis , Ácido Penicilánico/química , Penicilina G/análisis , Penicilina G/química , Desnaturalización Proteica , Espectrometría de Fluorescencia/métodos , beta-Lactamasas/metabolismo , beta-Lactamas/químicaRESUMEN
We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 ß-lactamase through the combined strategy of Val216âCys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when ß-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent ß-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216âCys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type ß-lactamase variants for in vitro drug screening.
Asunto(s)
Técnicas Biosensibles , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , beta-Lactamasas/química , beta-Lactamas/análisis , Sustitución de Aminoácidos , Dominio Catalítico , Evaluación Preclínica de Medicamentos/métodos , Proteínas de Escherichia coli/genética , Mutación Missense , beta-Lactamasas/genética , beta-Lactamas/químicaRESUMEN
A new switch-on fluorescent probe containing the natural product cryptolepine analogue benzofuroquinolinium moiety (binding scaffold) and a benzothiazole moiety (signalling unit) shows a remarkable fluorescence enhancement selective for the G-quadruplex nucleic acid structure. Binding studies revealed that the highly selective response of the fluorescent probe arises from end-stack binding to G-quadruplex.
Asunto(s)
Colorantes Fluorescentes/metabolismo , G-Cuádruplex , Sustancias Intercalantes/metabolismo , Compuestos de Quinolinio/metabolismo , Benzotiazoles/química , Sitios de Unión , Línea Celular Tumoral , ADN/metabolismo , Fluorescencia , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Humanos , Alcaloides Indólicos/química , Sustancias Intercalantes/análisis , Sustancias Intercalantes/química , Microscopía Fluorescente , Modelos Moleculares , Quinolinas/química , Compuestos de Quinolinio/análisis , Compuestos de Quinolinio/química , Espectrometría de FluorescenciaRESUMEN
The increasing emergence of new bacterial beta-lactamases that can efficiently hydrolyze beta-lactam antibiotics to clinically inactive carboxylic acids has created an intractable problem in the treatment of bacterial infections, and it is highly desirable to develop a useful tool that can rapidly screen bacteria for beta-lactamases against a variety of antibiotic candidates in a high-throughput manner. This paper describes the use of a fluorescein-labeled beta-lactamase mutant (E166Cf) as a convenient fluorescent tool to screen beta-lactamases, including the Bacillus cereus beta-lactamase I (PenPC), B. cereus beta-lactamase II, Bacillus licheniformis PenP, Escherichia coli TEM-1, and Enterobacter cloacae P99 against various beta-lactam antibiotics (penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin, moxalactam, cephaloridine), using a 96-well microplate reader. The E166Cf mutant was constructed by replacing Glu166 on the flexible Omega-loop, which is close to the enzyme's active site, with a cysteine residue on a class A beta-lactamase (B. cereus PenPC) and subsequently labeling the mutant with thiol-reactive fluorescein-5-maleimide. Such modifications significantly impaired the hydrolytic activity of the E166Cf mutant compared to that of the wild-type enzyme. The fluorescence intensity of the E166Cf mutant increases in the presence of beta-lactam antibiotics. For antibiotics that are resistant to hydrolysis by the E166Cf mutant (cefuroxime, cefoxitin, moxalactam), the fluorescence signal slowly increases until it reaches a plateau. For antibiotics that can be slowly hydrolyzed by the E166Cf mutant (penicillin G, penicillin V, ampicillin), the fluorescence signal rapidly increases to the plateau and then declines after a prolonged incubation. The E166Cf mutant retains its characteristic pattern of fluorescence signals in the presence of both bacterial beta-lactamases and beta-lactamase-resistant antibiotics. In contrast, in the presence of both bacterial beta-lactamases and beta-lactamase-sensitive antibiotics, the fluorescence signals of the E166Cf mutant were decreased. The fluorescence signals from the E166Cf mutant allow an unambiguous differentiation of beta-lactamase-resistant antibiotics from beta-lactamase-sensitive ones in the screening of bacterial beta-lactamases against a panel of antibiotic candidates. This simple method may provide an alternative tool in choosing potent beta-lactam antibiotics for treatment of bacterial infections.