RESUMEN
Pseudomonas aeruginosa is an important pathogen of both community and hospital acquired infections, and a major threat to public health for continuous emergence of multi-drug resistance. Current prevalence and pattern of multidrug resistance in the clinical isolates of P. aeruginosa is reported here. Samples were collected from September 2013 to January 2014 tertiary care hospital, Peshawar. Samples were subjected to phenotypic and molecular based detection of P. aeruginosa and were further processed for multidrug resistance pattern. Out of 3700 samples, 102 were identified as MDR P. aeruginosa. Prevalence of MDR isolates were found in pus (34.3%), wounds (28.4%), urine (19.6%), blood (14.7%) and sputum (2.9%) respectively. Isolates were more resistant to Sulphamethoxazole/Trimethoprim (98.04%), Amoxycillin/Clavulanic acid, Doxycycline and Chloramphenicol (95.1%) each, while least resistant to Imipenem (43.1%), Cefoperazone/Sulbactam (50.98%) and Amikacin (53.9%). Extensive MDR pattern was observed in P. aeruginosa was found as (n = 17, 16.6%) isolates were resistant to all four classes of antibiotics. Increased burden of MDR P. aeruginosa was documented in the study. Moreover, some isolates were even resistant to four classes of antibiotics. Findings of the study will be helpful to devise an appropriate antibiotic treatment strategy against MDR P. aeruginosa to cope the chances of evolving resistant pathogens.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Infecciones por Pseudomonas/epidemiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Antibacterianos/farmacología , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Pakistán/epidemiología , Prevalencia , Pseudomonas aeruginosa/efectos de los fármacos , Centros de Atención TerciariaAsunto(s)
Proteínas de la Membrana/química , Nanoestructuras/química , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiología , Membrana Dobles de Lípidos , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de ProteínaAsunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Biotecnología , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Sistema Libre de Células , Difusión , Proteínas de Escherichia coli/química , Interacciones Hidrofóbicas e Hidrofílicas , Cuerpos de Inclusión/química , Estructura Secundaria de ProteínaRESUMEN
An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a "pure" PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1:10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to "wrap" the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Polietilenglicoles/química , Agua/química , Animales , Cristalografía por Rayos X , Fluorescencia , Peso Molecular , Péptidos/química , Polímeros/química , Escifozoos/químicaRESUMEN
Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (k(m) 0,19; wild-type) to ß-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for ß-Ala (k(m) 0,07) and lowered the affinity for Gly (k(m) 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (k(m) 0,23) as a substrate, while affinity to Gly was preserved (k(m) 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event.
Asunto(s)
Glutatión Sintasa/genética , Glutatión Sintasa/metabolismo , Mutagénesis Sitio-Dirigida , Péptido Sintasas/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Activación Enzimática , Glutatión Sintasa/química , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Polymer based nanocompartments have potential applications in synthetic biology, medicine (drug release) and industrial biotechnology (chiral nanoreactors, multistep syntheses, selective product recovery). A step towards the aforementioned goals is the polymer membrane functionalization through covalent bonding of chemical anchors or insertion of proteins/peptides, to obtain specific properties like recognition, catalytic activity and facilitated diffusion, mimicking the complexity of a biological membrane. The use of genetic engineering techniques widens the possible applications of peptides and proteins specifically designed for polymer membrane interactions. A fusion protein (CecEGFP) based on the antibacterial peptide Cecropin A and the EGFP (Enhanced Green Fluorescent Protein) was designed, expressed and biophysically characterized. CecEGFP interaction with the tri-block copolymer PIB-PEG-PIB (PIB=polyisobutylene, PEG=polyethylene glycol) based polymersome membrane was analyzed by circular dichroism as well as EGFP and Trp fluorescence measurements. Results proved that Cecropin A is usable as a "membrane surface anchor" for water soluble proteins, as it inserts into the polymer membrane. The aim and novelty of this study is within the design of fusion proteins specifically developed for polymer membrane interactions. The use of amphiphilic Cecropin A "anchoring" water soluble proteins to the polymersome surface, avoids chemical coupling between polymers and proteins.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Proteínas Fluorescentes Verdes/química , Membranas Artificiales , Polienos/química , Polietilenglicoles/química , Polímeros/química , Proteínas Recombinantes de Fusión/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Clonación Molecular , Fluoresceínas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. RESULTS: The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal ß-strands, resulting in variant FhuA Δ1-159 Exp. The total number of ß-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high ß-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. CONCLUSION: In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Secuencia de Aminoácidos , Secuencia de Bases , Bencidinas/metabolismo , Dicroismo Circular , Difusión , Escherichia coli/metabolismo , Ingeniería Genética , Peroxidasa de Rábano Silvestre/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. RESULTS: To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 ß-sheets, prior to the more regular periplasmatic ß-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the ß-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB(1000)-PEG(6000)-PIB(1000) (PIB = polyisobutylene, PEG = polyethyleneglycol) has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine). Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB(1000)-PEG(6000)-PIB(1000) membrane. Furthermore labeling of the Lys-NH(2) groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. CONCLUSION: Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/síntesis química , Proteínas de Escherichia coli/síntesis química , Membranas/química , Ingeniería de Proteínas , Secuencia de Aminoácidos , Bencidinas/química , Peroxidasa de Rábano Silvestre/química , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Polienos/química , Polietilenglicoles/química , Polímeros/química , Estructura Secundaria de ProteínaRESUMEN
Membrane protein isolation is a challenging problem. In fact especially their extraction from the respective membrane is difficult and often goes along with losses in yield. Usually expensive detergents are needed to extract the target protein from the membrane. Therefore finding an efficient overexpression and extraction method and an alternative to detergents is desirable. In this study we describe a new and fast method to express, extract and purify an engineered variant of the FhuA protein (FhuA Δ1-159) that acts as passive diffusion channel, using a diblock copolymer as an alternative to detergents like octyl-POE (n-octylpolyoxyethylene). The N-terminal leader sequence, facilitating the protein's transport to the outer membrane was deleted (FhuA Δ1-159 Δsignal), resulting in protein accumulation in easy to isolate inclusion bodies. Urea was used to solubilise the unfolded protein and dialysis against phosphate-buffer containing the commercially available diblock copolymer PE-PEG[Polyethylene-Poly(ethyleneglycol)] lead to protein refolding. Circular dichroism spectroscopy revealed a high ß-sheet percentage within the refolded protein secondary structure indicating the successful reconstitution of FhuA Δ1-159 Δsignal native state. Furthermore the channel functionality of FhuA Δ1-159 Δsignal was verified by measuring the in and out-flux through the protein when inserted into liposome membrane, using the HRP/TMB (HRP=Horse Radish Peroxidase, TMB=3,3',5,5'-tetramethylbenzidine) assay system.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Cuerpos de Inclusión/metabolismo , Polietilenglicoles/química , Polietileno/química , Proteínas de la Membrana Bacteriana Externa/genética , Dicroismo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Eliminación de Gen , Replegamiento Proteico , Estructura Secundaria de ProteínaRESUMEN
A protein involved in the transport of glutathione has been identified, cloned and characterized from the fission yeast Schizosaccharomyces pombe. Database searches revealed the Sz. pombe ORF SPAC29B12.10c as a close homologue to several members of the OPT family, including the Saccharomyces cerevisiae high-affinity glutathione transporter Hgt1p. The gene product of SPAC29B12.10c has been identified as a protein, named SpOPT1, localized within the plasma membrane, transporting the tripeptide glutathione. Disruption of SPAC29B12.10c led to strains inable to grow on media containing glutathione as a sole source of sulphur, due to the inability to internalize the tripeptide. Disruptants contained significantly less glutathione than wild-type cells. Furthermore, DeltaSpopt1 strains were non-viable in a glutathione biosynthesis-defective (Deltagsh2) background. However, it was possible to complement the disruption of Spopt1 by overexpressing the intact ORF in the disrupted strain.