RESUMEN
Acne is one of the most common dermatological conditions, but the details of its pathology are unclear, and current management regimens often have adverse effects. Cutibacterium acnes is known as a major acne-associated bacterium that derives energy from lipase-mediated sebum lipid degradation. C. acnes is commensal, but lipase activity has been observed to differ among C. acnes types. For example, higher populations of the type IA strains are present in acne lesions with higher lipase activity. In the present study, we examined a conserved lipase in types IB and II that was truncated in type IA C. acnes strains. Closed, blocked, and open structures of C. acnes ATCC11828 lipases were elucidated by X-ray crystallography at 1.6-2.4 Å. The closed crystal structure, which is the most common form in aqueous solution, revealed that a hydrophobic lid domain shields the active site. By comparing closed, blocked, and open structures, we found that the lid domain-opening mechanisms of C. acnes lipases (CAlipases) involve the lid-opening residues, Phe-179 and Phe-211. To the best of our knowledge, this is the first structure-function study of CAlipases, which may help to shed light on the mechanisms involved in acne development and may aid in future drug design.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Lipasa/química , Lipasa/metabolismo , Propionibacteriaceae/enzimología , Secuencia de Aminoácidos , Secuencia Conservada , Lisofosfatidilcolinas/metabolismo , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Especificidad de la EspecieRESUMEN
The DJ-1/ThiJ/PfpI superfamily is a group of proteins found in diverse organisms. This superfamily includes versatile proteins, such as proteases, chaperones, heat-shock proteins and human Parkinson's disease protein. Most members of the DJ-1/ThiJ/PfpI superfamily are oligomers and are classified into subfamilies depending on discriminating quaternary structures (DJ-1, YhbO and Hsp types). SAV1875, a conserved protein from Staphylococcus aureus, is a member of the YhbO-type subfamily. However, its structure and function remain unknown. Thus, to understand the function and activity mechanism of this protein, the crystal structure of SAV1875 from S. aureus was determined. The overall fold of SAV1875 is similar to that observed for the DJ-1/ThiJ/PfpI superfamily. The cysteine residue located in the dimeric interface (Cys(105)) forms a catalytic triad with His(106) and Asp(77), and it is spontaneously oxidized to Cys(105)-SO2H in the crystal structure. To study the oxidative propensity of Cys(105) and the corresponding functional differences with changes in cysteine oxidation state, the crystal structures of SAV1875 variants E17N, E17D and C105D, and over-oxidized SAV1875 were determined. We identified SAV1875 as a novel member of the YhbO-type subfamily exhibiting chaperone function. However, if SAV1875 is over-oxidized further with H2O2, its chaperone activity is eliminated. On the basis of our study, we suggest that SAV1875 functions as a chaperone and the redox state of Cys(105) may play an important role.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Staphylococcus aureus/metabolismo , Cristalografía por Rayos X , Oxidación-Reducción , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
This paper proposes a novel method of estimating walking distance based on a precise counting of walking strides using insole sensors. We use an inertial triaxial accelerometer and eight pressure sensors installed in the insole of a shoe to record walkers' movement data. The data is then transmitted to a smartphone to filter out noise and determine stance and swing phases. Based on phase information, we count the number of strides traveled and estimate the movement distance. To evaluate the accuracy of the proposed method, we created two walking databases on seven healthy participants and tested the proposed method. The first database, which is called the short distance database, consists of collected data from all seven healthy subjects walking on a 16 m distance. The second one, named the long distance database, is constructed from walking data of three healthy subjects who have participated in the short database for an 89 m distance. The experimental results show that the proposed method performs walking distance estimation accurately with the mean error rates of 4.8% and 3.1% for the short and long distance databases, respectively. Moreover, the maximum difference of the swing phase determination with respect to time is 0.08 s and 0.06 s for starting and stopping points of swing phases, respectively. Therefore, the stride counting method provides a highly precise result when subjects walk.
RESUMEN
Bacterial chromosomal toxin-antitoxin (TA) systems have been proposed not only to play an important role in the stress response, but also to be associated with antibiotic resistance. Here, we identified the chromosomal HP0892-HP0893 TA proteins in the gastric pathogen, Helicobacter pylori, and structurally characterized their protein-protein interaction. Previously, HP0892 protein was suggested to be a putative TA toxin based on its structural similarity to other RelE family TA toxins. In this study, we demonstrated that HP0892 binds to HP0893 strongly with a stoichiometry of 1:1, and HP0892-HP0893 interaction occurs mainly between the N-terminal secondary structure elements of HP0892 and the C-terminal region of HP0893. HP0892 cleaved mRNA in vitro, preferentially at the 5' end of A or G, and the RNase activity of HP0892 was inhibited by HP0893. In addition, heterologous expression of HP0892 in Escherichia coli cells led to cell growth arrest, and the cell toxicity of HP0892 was neutralized by co-expression with HP0893. From these results and a structural comparison with other TA toxins, it is concluded that HP0892 is a toxin with intrinsic RNase activity and HP0893 is an antitoxin against HP0892 from a TA system of H. pylori. It has been known that hp0893 gene and another TA antitoxin gene, hp0895, of H. pylori, are both genomic open reading frames that correspond to genes that are potentially expressed in response to interactions with the human gastric mucosa. Therefore, it is highly probable that TA systems of H. pylori are involved in virulence of H. pylori.
Asunto(s)
Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica , Helicobacter pylori/metabolismo , Secuencia de Aminoácidos , Antitoxinas/química , Sitios de Unión , Cromatografía en Gel , Clonación Molecular , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Helicobacter pylori/patogenicidad , Humanos , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Ribonucleasas/metabolismo , Homología de Secuencia de Aminoácido , VirulenciaRESUMEN
The toxin-antitoxin (TA) systems widely spread among bacteria and archaea are important for antibiotic resistance and microorganism virulence. The bacterial kingdom uses TA systems to adjust the global level of gene expression and translation through RNA degradation. In Helicobacter pylori, only two TA systems are known thus far. Our previous studies showed that HP0894-HP0895 acts as a TA system and that HP0894 exhibits intrinsic RNase activity. However, the precise molecular basis for interaction with substrate or antitoxin and the mechanism of mRNA cleavage remain unclear. Therefore, in an attempt to shed some light on the mechanism behind the TA system of HP0894-HP0895, here we present the crystal structures of apo- and metal-bound H. pylori 0894 at 1.28Å and 1.89Å, respectively. Through the combined approach of structural analysis and structural homology search, the amino acids involved in mRNase active site were monitored and the reorientations of different residues were discussed in detail. In the mRNase active site of HP0894 toxin, His84 acts as a catalytic residue and reorients itself to exhibit this type of activity, acting as a general acid in an acid-base catalysis reaction, while His47 and His60 stabilize the transition state. Lys52, Glu58, Asp64 and Arg80 have phosphate binding and specific sequence recognition. Glu58 also acts as a general base, and substrate reorientation is caused by Phe88. Based on experimental findings, a model for antitoxin binding could be suggested.
Asunto(s)
Proteínas Bacterianas/química , Cobre/química , Helicobacter pylori/enzimología , Ribonucleasas/química , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Dominio Catalítico , Cobre/metabolismo , Cristalografía por Rayos X , Helicobacter pylori/genética , Unión Proteica , Estabilidad del ARN/fisiología , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismoRESUMEN
HU, one of the major nucleoid-associated proteins, interacts with the minor groove of DNA in a nonspecific manner to induce DNA bending or to stabilize bent DNA. In this study, crystal structures are reported for both free HU from Staphylococcus aureus Mu50 (SHU) and SHU bound to 21-mer dsDNA. The structures, in combination with electrophoretic mobility shift assays (EMSAs), isothermal titration calorimetry (ITC) measurements and molecular-dynamics (MD) simulations, elucidate the overall and residue-specific changes in SHU upon recognizing and binding to DNA. Firstly, structural comparison showed the flexible nature of the ß-sheets of the DNA-binding domain and that the ß-arms bend inwards upon complex formation, whereas the other portions are nearly unaltered. Secondly, it was found that the disruption and formation of salt bridges accompanies DNA binding. Thirdly, residue-specific free-energy analyses using the MM-PBSA method with MD simulation data suggested that the successive basic residues in the ß-arms play a central role in recognizing and binding to DNA, which was confirmed by the EMSA and ITC analyses. Moreover, residue Arg55 resides in the hinge region of the flexible ß-arms, exhibiting a remarkable role in their flexible nature. Fourthly, EMSAs with various DNAs revealed that SHU prefers deformable DNA. Taken together, these data suggest residue-specific roles in local shape and base readouts, which are primarily mediated by the flexible ß-arms consisting of residues 50-80.
Asunto(s)
Proteínas Bacterianas/química , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/química , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Conformación Proteica , Alineación de SecuenciaRESUMEN
VapD-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3D structure of these proteins. Recently, a relationship between the Cas2 family of ribonucleases associated with the CRISPR system of microbial immunity and VapD was suggested. Here, we show for the first time the structure of a member of the VapD family and present a relationship of VapD with Cas2 family and toxin-antitoxin (TA) systems. The crystal structure of HP0315 from Helicobacter pylori was solved at a resolution of 2.8 Å. The structure of HP0315, which has a modified ferredoxin-like fold, is very similar to that of the Cas2 family. Like Cas2 proteins, HP0315 shows endoribonuclease activity. HP0315-cleaved mRNA, mainly before A and G nucleotides preferentially, which means that HP0315 has purine-specific endoribonuclease activity. Mutagenesis studies of HP0315 revealed that D7, L13, S43 and D76 residues are important for RNase activity, in contrast, to the Cas2 family. HP0315 is arranged as an operon with HP0316, which was found to be an antitoxin-related protein. However, HP0315 is not a component of the TA system. Thus, HP0315 may be an evolutionary intermediate which does not belong to either the Cas2 family or TA system.
Asunto(s)
Proteínas Bacterianas/química , Endorribonucleasas/química , Helicobacter pylori/enzimología , Glicoproteínas de Membrana/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Multimerización de Proteína , Estabilidad Proteica , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
In traditional oriental medicine, apricot (Prunus armeniaca L.) seed has been used to treat skin diseases such as furuncle, acne vulgaris and dandruff, as well as coughing, asthma and constipation. This study describes the phytochemical profile and antimicrobial potential of the essential oil obtained from apricot seeds (Armeniacae Semen). The essential oil isolated by hydrodistillation was analysed by gas chromatography-mass spectroscopy. Benzaldehyde (90.6%), mandelonitrile (5.2%) and benzoic acid (4.1%) were identified. Disc diffusion, agar dilution and gaseous contact methods were performed to determine the antimicrobial activity against 16 bacteria and two yeast species. The minimum inhibitory concentrations ranged from 250 to 4000, 500 to 2000 and 250 to 1000 µg/mL for Gram-positive bacteria, Gram-negative bacteria and yeast strains, respectively. The minimum inhibitory doses by gaseous contact ranged from 12.5 to 50, 12.5 to 50 and 3.13 to 12.5 mg/L air for Gram-positive bacteria, Gram-negative bacteria and yeast strains, respectively. The essential oil exhibited a variable degree of antimicrobial activity against a range of bacteria and yeasts tested.
Asunto(s)
Antiinfecciosos/farmacología , Aceites Volátiles/farmacología , Prunus/química , Semillas/química , Cromatografía de Gases y Espectrometría de Masas , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Aceites Volátiles/química , Aceites de Plantas/química , Aceites de Plantas/farmacología , Levaduras/efectos de los fármacosRESUMEN
Endoribonuclease YbeY is specific to the single-stranded RNA of ribosomal RNAs and small RNAs. This enzyme is essential for the maturation and quality control of ribosomal RNA in a wide range of bacteria and for virulence in some pathogenic bacteria. In this study, we determined the crystal structure of YbeY from Staphylococcus aureus at a resolution of 1.9 Å in the presence of zinc chloride. The structure showed a zinc ion at the active site and two molecules of tricarboxylic acid citrate, which were also derived from the crystallization conditions. Our structure showed the zinc ion-bound local environment at the molecular level for the first time. Molecular comparisons were performed between the carboxylic moieties of citrate and the phosphate moiety of the RNA backbone, and a model of YbeY in complex with a single strand of RNA was subsequently constructed. Our findings provide molecular insights into how the YbeY enzyme recognizes single-stranded RNA in bacteria.
Asunto(s)
Endorribonucleasas , Staphylococcus aureus , Endorribonucleasas/genética , Staphylococcus aureus/genética , Virulencia , ARN , ZincRESUMEN
Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Helicobacter pylori/metabolismo , Complejos Multiproteicos/metabolismo , Multimerización de Proteína/fisiología , Ribonucleasas/metabolismo , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Helicobacter pylori/genética , Complejos Multiproteicos/genética , Estructura Terciaria de Proteína , Ribonucleasas/genéticaRESUMEN
BACKGROUND: This study was performed to determine the extended spectrum of quinolone resistance caused by increased mutations within the target enzymes of quinolones. METHODS: The minimum inhibitory concentrations (MICs) for ciprofloxacin, sparfloxacin, trovafloxacin and DW286 were determined against 98 ciprofloxacin-resistant Staphylococcus aureus strains. Also, PCR-amplified grlA, grlB, gyrA and gyrB DNA fragments were sequenced and amino acid changes were analyzed. RESULTS: The MIC(50) values of quinolones decreased with later-generation compounds, i.e. >or=64 microg/ml for ciprofloxacin, 16 microg/ml for sparfloxacin, 2 microg/ml for trovafloxacin and 0.25 microg/ml for DW286. Combinations of amino acid changes within GrlA (Ser-80, Tyr-83 or Glu-84), GrlB (Pro-451, Pro-585 or Asp-432) and GyrA (Ser-84, Ser-85 or Glu-88) were constructed. The combination of Ser-80-->Phe within GrlA and Ser-84-->Leu within GyrA was the fundamental combination in alterations involved in ciprofloxacin resistance, and additional alterations extended quinolone resistance. CONCLUSION: A larger number of alterations within GrlA and GyrA further extended the spectrum of quinolone resistance.
Asunto(s)
Antibacterianos/farmacología , Fluoroquinolonas/farmacología , Staphylococcus aureus/efectos de los fármacos , Secuencia de Aminoácidos , Girasa de ADN/genética , ADN Bacteriano , Farmacorresistencia Bacteriana/genética , Proteínas de Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Mutación , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Transactivadores/genéticaRESUMEN
HslUV is a bacterial heat shock protein complex consisting of the AAA+ ATPase component HslU and the protease component HslV. HslV is a threonine (Thr) protease employing the N-terminal Thr residue in the mature protein as the catalytic residue. To date, HslUV from Gram-negative bacteria has been extensively studied. However, the mechanisms of action and activation of HslUV from Gram-positive bacteria, which have an additional N-terminal sequence before the catalytic Thr residue, remain to be revealed. In this study, we determined the crystal structures of HslV from the Gram-positive bacterium Staphylococcus aureus with and without HslU in the crystallization conditions. The structural comparison suggested that a structural transition to the symmetric form of HslV was triggered by ATP-bound HslU. More importantly, the additional N-terminal sequence was cleaved in the presence of HslU and ATP, exposing the Thr9 residue at the N-terminus and activating the ATP-dependent protease activity. Further biochemical studies demonstrated that the exposed N-terminal Thr residue is critical for catalysis with binding to the symmetric HslU hexamer. Since eukaryotic proteasomes have a similar additional N-terminal sequence, our results will improve our understanding of the common molecular mechanisms for the activation of proteasomes.
Asunto(s)
Proteasas ATP-Dependientes/metabolismo , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Conformación Proteica , Staphylococcus aureusRESUMEN
Staphylococcus aureus is a round-shaped, gram-positive bacterium that can cause numerous infectious diseases ranging from mild infections such as skin infections and food poisoning to life-threatening infections such as sepsis, endocarditis and toxic shock syndrome. Various antibiotic-resistant strains of S. aureus have frequently emerged, threatening human lives significantly. Despite much research on the genetics of S. aureus, many of its genes remain unknown functionally and structurally. To counteract its toxins and to prevent the antibiotic resistance of S. aureus, our understanding of S. aureus should be increased at the proteomic scale. SAV0927 was first sequenced in an antibiotic resistant S. aureus strain. The gene is a conserved hypothetical protein, and its homologues appear to be restricted to Firmicutes. In this study, we determined the crystal structure of SAV0927 at 2.5 Å resolution. The protein was primarily dimeric both in solution and in the crystals. The asymmetric unit contained five dimers that are stacked linearly with ~80° rotation by each dimer, and these interactions further continued in the crystal packing, resulting in a long linear polymer. The crystal structures, together with the network analysis, provide functional implications for the SAV0927-mediated protein network.
Asunto(s)
Proteínas Bacterianas/química , Farmacorresistencia Bacteriana/fisiología , Staphylococcus aureus/metabolismo , Antibacterianos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Cristalografía por Rayos X , Farmacorresistencia Bacteriana/genética , Humanos , Staphylococcus aureus Resistente a Meticilina , Modelos Moleculares , Conformación Proteica en Hélice alfa , Dominios Proteicos , Proteómica , Alineación de Secuencia , Staphylococcus aureus/genéticaRESUMEN
Translational attenuation has been proposed to be the mechanism by which the erm(B) gene is induced. Here, we report genetic and biochemical evidence, obtained by using erythromycin as the inducing antibiotic, that supports this hypothesis. We also show that erythromycin increases the level of the erm(B) transcript by stalling the ribosome on the leader mRNA and thereby facilitating the stabilization and processing of the mRNA. Erythromycin-induced mRNA stabilization and processing were observed with an ochre stop at codons 11 to 13 of the leader but not with an ochre stop at codon 10. This suggests that erythromycin does not stall the ribosome before codon 11 of the leader reaches the aminoacyl site. Secondary structure analyses of the erm(B) transcripts by in vitro and in vivo chemical probing techniques identified conformational changes in the transcripts that result from induction by erythromycin. These findings demonstrate that stalling of erythromycin-bound ribosomes at leader codon 11 causes the refolding of mRNA into a conformation in which the translational initiation site for the structural gene is unmasked and renders erm(B) translationally active.
Asunto(s)
Eritromicina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Codón/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , Ribosomas/metabolismoRESUMEN
OBJECTIVES: We identified Staphylococcus aureus strains having blunt inhibitory zones around the clindamycin or josamycin discs proximal to the erythromycin disc filled with slight bacterial growth. This ambiguous phenotype was termed as 'macrolide-lincosamide-streptogramin B antimicrobial (MLS(B)) resistance having a foggy D-shaped inhibitory zone' (fMLS(B)), and we tried to analyse its molecular mechanisms. METHODS: Forty-one clinically isolated strains of fMLS(B) S. aureus were studied. The regulatory region of the erm(A) gene, which was found to be the only molecular mechanism of fMLS(B), was sequenced. Then, beta-galactosidase assays were performed to observe their expression patterns through the nucleotide sequential alteration. RESULTS: According to the sequencing electropherogram, a grouping was made of a homogeneous nucleotide sequence group (73.2%) and a heterogeneous nucleotide sequence group (26.8%). The former group was composed of a 25 bp tandem duplication type and a 25 bp tandem triplication type. Their beta-galactosidase activity was similar to that of constitutive MLS(B) resistance due to its high basal level. Nevertheless, the predicted mRNA secondary structure of the regulatory region maintains the stem-loops of inducible wild-type erm(A), and thereby its inducible character might be expected in vivo. Strains in the latter group were proven to have two different erm(A) genes, and then dual effect of expression was observed. CONCLUSIONS: The ambiguous phenotype of fMLS(B) is due to its possessing the dual character of inducible and constitutive expression of erm(A). The dual character is due to having one erm(A) gene of dual character or coexistence of two characterized erm(A) genes simultaneously.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Macrólidos/farmacología , Staphylococcus aureus/efectos de los fármacos , Estreptogramina B/farmacología , Combinación de Medicamentos , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Lincosamidas , Pruebas de Sensibilidad Microbiana/métodos , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Frequencies of spontaneous mutation from inducible resistance to constitutive resistance were determined for the four clinical isolates of erythromycin-resistant enterococci, including one isolate with ermB gene and three clinical isolates with ermA gene. The rate of ermB mutation was higher than that of ermA mutation by more than 10 fold. Sequence analysis of the regulatory regions of erm genes revealed that mutation type of ermB was just point mutation, by contraries the mutation type of ermA was either deletion or tandem duplication. These results showed distinct characteristics in mutation patterns of ermB and ermA.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Enterococcus faecalis/genética , Enterococcus faecium/genética , Macrólidos/farmacología , Metiltransferasas/genética , Mutación , Análisis Mutacional de ADN , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/enzimología , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/enzimología , Eritromicina/farmacología , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genotipo , Josamicina/farmacología , Fenotipo , Mutación Puntual , Eliminación de SecuenciaRESUMEN
The characteristic fold of a protein is the decisive factor for its biological function. However, small structural changes to amino acids can also affect their function, for example in the case of post-translational modification (PTM). Many different types of PTMs are known, but for some, including chlorination, studies elucidating their importance are limited. A recent study revealed that the YjgF/YER057c/UK114 family (YjgF family) member RidA from Escherichia coli shows chaperone activity after chlorination. Thus, to identify the functional and structural differences of RidA upon chlorination, we studied an RidA homolog from Staphylococcus aureus: YabJ. The overall structure of S. aureus YabJ was similar to other members of the YjgF family, showing deep pockets on its surface, and the residues composing the pockets were well conserved. S. aureus YabJ was highly stable after chlorination, and the chlorinated state is reversible by treatment with DTT. However, it shows no chaperone activity after chlorination. Instead, YabJ from S. aureus shows chlorination-induced ribonuclease activity, and the activity is diminished after subsequent reduction. Even though the yabJ genes from Staphylococcus and Bacillus are clustered with regulators that are expected to code nucleic acid-interacting proteins, the nucleic acid-related activity of bacterial RidA has not been identified before. From our study, we revealed the structure and function of S. aureus YabJ as a novel chlorination-activated ribonuclease. The present study will contribute to an in-depth understanding of chlorination as a PTM.
Asunto(s)
Proteínas Bacterianas/química , Ribonucleasas/química , Infecciones Estafilocócicas/enzimología , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos/genética , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Escherichia coli/genética , Halogenación/genética , Humanos , Unión Proteica , Conformación Proteica/efectos de los fármacos , Pliegue de Proteína , Procesamiento Proteico-Postraduccional/genética , Ribonucleasas/genética , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidadRESUMEN
The structure and dynamics of a 37-residue antimicrobial peptide gaegurin 4 (GGN4) isolated from the skin of the native Korean frog, Rana rugosa, was determined in SDS micelles by NMR spectroscopy. The solution structure of the peptide in SDS micelles was determined from 352 NOE-derived distance constraints and 22 backbone torsion angle constraints. Dynamic properties for the amide backbone were characterized by (1)H-(15)N heteronuclear NOE experiments. The structural study revealed two amphipathic helices spanning residues 2-10 and 16-32 and that the helices were connected by a flexible loop. An intraresidue disulfide bridge was formed between residues Cys31 and Cys37 near the C-terminus. The loop region (11-15) connecting the two helices are were slightly more flexible than these helices themselves. From the fact that since there is no contact NOEs between two helices, it is implied that the GGN4 peptide shows an independent motion of both helices which has an angle of about 60 degrees -120 degrees from each other.
Asunto(s)
Anfibios/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Canales Iónicos/química , Canales Iónicos/metabolismo , Micelas , Dodecil Sulfato de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Concentración de Iones de Hidrógeno , Canales Iónicos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , TemperaturaRESUMEN
The antibacterial activities of clindamycin, synercid, telithromycin, linezolid and mupirocin were evaluated against erythromycin-resistant Gram-positive coccal clinical isolates collected in Korean hospitals. In Staphylococcus aureus, synercid, linezolid and mupirocin were the most active agents. Against coagulase-negative staphylococci (CNS), synercid, linezolid and mupirocin were also active. Telithromycin and synercid resistance was common against enterococci, only linezolid and mupirocin were active. The reason of low activity of telithromycin against staphylococci and enterococci is because most of the isolates were constitutively resistant to erythromycin. Synercid, telithromycin, linezolid and mupirocin were active against streptococci.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Eritromicina/farmacología , Cocos Grampositivos/efectos de los fármacos , Acetamidas/farmacología , Clindamicina/farmacología , Infecciones por Bacterias Grampositivas/microbiología , Cocos Grampositivos/aislamiento & purificación , Humanos , Cetólidos/farmacología , Corea (Geográfico) , Linezolid , Pruebas de Sensibilidad Microbiana , Mupirocina/farmacología , Oxazolidinonas/farmacología , Virginiamicina/farmacologíaRESUMEN
The DJ-1/ThiJ/PfpI superfamily of proteins is highly conserved across all biological kingdoms showing divergent multifunctions, such as chaperone, catalase, protease, and kinase. The common theme of these functions is responding to and managing various cellular stresses. DJ-1/ThiJ/PfpI superfamily members are classified into three subfamilies according to their quaternary structure (DJ-1-, YhbO-, and Hsp-types). The Hsp-type subfamily includes Hsp31, a chaperone and glyoxalase III. SAV0551, an Hsp-type subfamily member from Staphylococcus aureus, is a hypothetical protein that is predicted as Hsp31. Thus, to reveal the function and reaction mechanism of SAV0551, the crystal structure of SAV0551 was determined. The overall folds in SAV0551 are similar to other members of the Hsp-type subfamily. We have shown that SAV0551 functions as a chaperone and that the surface structure is crucial for holding unfolded substrates. As many DJ-1/ThiJ/PfpI superfamily proteins have been characterized as glyoxalase III, our study also demonstrates SAV0551 as a glyoxalase III that is independent of any cofactors. The reaction mechanism was evaluated via a glyoxylate-bound structure that mimics the hemithioacetal reaction intermediate. We have confirmed that the components required for reaction are present in the structure, including a catalytic triad for a catalytic action, His78 as a base, and a water molecule for hydrolysis. Our functional studies based on the crystal structures of native and glyoxylate-bound SAV0551 will provide a better understanding of the reaction mechanism of a chaperone and glyoxalase III.