ABSTRACT
BACKGROUND: Carriage rates of Staphylococcus aureus on affected skin in atopic dermatitis (AD) are approximately 70%. Increasing disease severity during flares and overall disease severity correlate with increased burden of S. aureus. Treatment in AD therefore often targets S. aureus with topical and systemic antimicrobials. OBJECTIVES: To determine whether antimicrobial sensitivities and genetic determinants of resistance differed in S. aureus isolates from the skin of children with AD and healthy child nasal carriers. METHODS: In this case-control study, we compared S. aureus isolates from children with AD (n = 50) attending a hospital dermatology department against nasal carriage isolates from children without skin disease (n = 49) attending a hospital emergency department for noninfective conditions. Using whole genome sequencing we generated a phylogenetic framework for the isolates based on variation in the core genome, then compared antimicrobial resistance phenotypes and genotypes between disease groups. RESULTS: Staphylococcus aureus from cases and controls had on average similar numbers of phenotypic resistances per isolate. Case isolates differed in their resistance patterns, with fusidic acid resistance (FusR ) being significantly more frequent in AD (P = 0·009). The genetic basis of FusR also differentiated the populations, with chromosomal mutations in fusA predominating in AD (P = 0·049). Analysis revealed that FusR evolved multiple times and via multiple mechanism in the population. Carriage of plasmid-derived qac genes, which have been associated with reduced susceptibility to antiseptics, was eight times more frequent in AD (P = 0·016). CONCLUSIONS: The results suggest that strong selective pressure drives the emergence and maintenance of specific resistances in AD.
Subject(s)
Anti-Infective Agents, Local/adverse effects , Dermatitis, Atopic/microbiology , Drug Resistance, Bacterial/drug effects , Staphylococcal Skin Infections/microbiology , Staphylococcus aureus/physiology , Administration, Cutaneous , Anti-Infective Agents, Local/administration & dosage , Carrier State/diagnosis , Carrier State/drug therapy , Carrier State/microbiology , Case-Control Studies , Child , Child, Preschool , Dermatitis, Atopic/diagnosis , Dermatitis, Atopic/drug therapy , Drug Resistance, Bacterial/genetics , Female , Healthy Volunteers , Humans , Infant , Infant, Newborn , Male , Microbial Sensitivity Tests , Mutation , Nasal Mucosa/microbiology , Peptide Elongation Factor G/genetics , Peptide Elongation Factor G/isolation & purification , Severity of Illness Index , Skin/microbiology , Staphylococcal Skin Infections/diagnosis , Staphylococcal Skin Infections/drug therapy , Staphylococcus aureus/isolation & purificationABSTRACT
The fusA gene encoding a thermophilic protein EF-G with multiple rare condons was cloned from Thermoanaerobacter tengcongensis (TteEF-G) and overexpressed in Escherichia coli by cotransfering a RIG plasmid to overcome the potential codon-bias problem originated from Arg, Ile and Gly. The recombinant protein was identified by MALDI-TOF-MS for molecular mass with approximation of 76 kDa and by trypsin digestion coupled LC-MS/MS for peptide sequence coverage of 61.3%. The in vivo complementary assay indicates that TteEF-G could significantly rescue the E. coli LJ14 (frr(ts)) at the non-permission temperature of 42 degrees C in the bi-transformant of TteRRF and TteEF-G. This study indicated that coexpression of rare codons' cognate tRNA is a useful method for protein overexpression in E. coli.
Subject(s)
Peptide Elongation Factor G/biosynthesis , Thermoanaerobacter/chemistry , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Molecular Sequence Data , Peptide Elongation Factor G/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
Elongation factor G (EF-G) catalyzes the translocation step of protein biosynthesis. Genomic analysis suggests that two isoforms of this protein occur in mitochondria. The region of the cDNA coding for the mature sequence of isoform 1 of human mitochondrial EF-G (EF-G1(mt)) has been cloned and expressed in Escherichia coli. The recombinant protein has been purified to near homogeneity by chromatography on Ni-NTA resins and cation exchange high performance liquid chromatography. EF-G1(mt) is active on both bacterial and mitochondrial ribosomes. Human EF-G1(mt) is considerably more resistant to fusidic acid than many bacterial translocases. A molecular model for EF-G1(mt) has been created and analyzed in the context of its relationship to the translocases from other systems.
Subject(s)
Peptide Elongation Factor G/biosynthesis , Peptide Elongation Factor G/chemistry , Amino Acid Sequence , Chromatography , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Cloning, Molecular , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Expressed Sequence Tags , Fusidic Acid/chemistry , Glutamates/chemistry , Humans , Mitochondria/metabolism , Models, Molecular , Molecular Sequence Data , Nickel , Peptide Elongation Factor G/isolation & purification , Potassium Chloride/chemistry , Protein Isoforms , Protein Transport , Ribosomes/metabolism , Sequence Homology, Amino Acid , Time FactorsABSTRACT
Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome. Their functional cycles depend on GTP binding and its hydrolysis. The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome. In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 10(3)-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation. By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G.GDP complex to the ribosome are not influenced. These findings indicate an essential contribution of domain III to activation of GTP hydrolysis. These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.
Subject(s)
Guanosine Triphosphate/metabolism , Peptide Elongation Factor G/chemistry , Peptide Elongation Factor G/metabolism , Ribosomes/metabolism , Thermus thermophilus/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Fusidic Acid/metabolism , Guanosine Diphosphate/metabolism , Hydrolysis , Kinetics , Models, Molecular , Peptide Elongation Factor G/genetics , Peptide Elongation Factor G/isolation & purification , Protein Biosynthesis , Protein Structure, Tertiary , Puromycin/metabolism , Sequence Deletion/geneticsABSTRACT
The fus gene of the translation factor G (EF-G) from the hyperthermophilic bacterium Aquifex aeolicus was cloned under control of a phage promoter and overexpressed in Escherichia coli with the T7 RNA polymerase system. A heat denaturation step at 95 degrees C was used to purify the protein from the cell extract. This approach simplified the chromatographic procedures and decreased the protein loss since most of Escherichia coli proteins were denatured and precipitated. Ten milligrams of the highly purified protein was isolated from 4 liters of induced culture. The overproduced EF-G was active in ribosome-dependent GTP hydrolysis and a poly(U)-directed polyphenylalanine translation system with E. coli 70S ribosomes. The method presented here might facilitate functional and structural studies of important components of the protein biosynthesis system.
