RESUMO
Acanthopanax (A.) henryi (Oliv.) Harms contain many bioactive compounds commonly used in traditional Chinese medicine. The objective of the present study was to investigate the antibacterial activity of the single constituent, Eleutheroside K (ETSK) isolated from the leaves of A. henryi (Oliv.) Harms, against methicillin-resistant Staphylococcus (S.) aureus (MRSA). Broth microdilution assay was used to measure the minimal inhibitory concentration (MIC) and the MIC values of ETSK against eight clinical S. aureus strains were all 50 µg ml-1 . At sub-inhibitory concentrations, a synergistic effect between oxacillin (OXA) and ETSK was confirmed using checkerboard dilution assay and time-kill curve analysis. The bacteriostatic effect became more pronounced when ETSK was used in combination with detergent (Triton X-100) or ATPase inhibitor (N, N'-dicyclohexylcarbodiimide). According to western blot analysis, the down-regulated expression of Penicillin-binding protein 2a (PBP2a) further validated that the bacterial activity was inhibited when treated with ETSK in a dose-dependent manner. Results based on our study verified that ETSK significantly suppressed MRSA infections and emphasized the potential application of ETSK as a novel anti-MRSA natural drug.
Assuntos
Antibacterianos/farmacologia , Eleutherococcus/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxacilina/farmacologia , Extratos Vegetais/farmacologia , Dicicloexilcarbodi-Imida/farmacologia , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Eleutherococcus/química , Resistência a Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Octoxinol/farmacologia , Proteínas de Ligação às Penicilinas/biossíntese , Folhas de Planta/químicaRESUMO
Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.
Assuntos
Parede Celular/genética , Farmacorresistência Bacteriana/genética , Endopeptidases/biossíntese , Peptidoglicano/biossíntese , Andinocilina/farmacologia , Parede Celular/metabolismo , Endopeptidases/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/biossíntese , Proteínas de Ligação às Penicilinas/genéticaRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) decontamination regimens predominantly use chlorhexidine bathing in combination with mupirocin nasal ointment. However, resistances in Staphylococcus aureus strains are increasingly common and there is a need of alternative, safe and feasible protocols. This interventional cohort study performed at the Albert Schweitzer Hospital in Graz, Austria, aimed to (1) determine MRSA prevalence at different body sites and (2) assess the efficacy of the decontamination using octenidine-based leave-on products added to existing robust infection control measures. All inpatients of this tertiary care hospital being treated in geriatric medical wards (GWs) and apallic care units (ACUs) were screened for MRSA and decontamination rates were determined after one, two or three decontamination cycles, respectively. At baseline, MRSA was detected in 25 of the 126 patients screened (19.8%). We found MRSA in 13/126 (10.3%) swabs from nasal vestibules, in 12/126 (9.5%) skin swabs, in 11/51 (21.6%) swabs from PEG-stomata or suprapubic catheters and in 8/13 (61.5%) tracheostomata swabs. A maximum of three 5-day decontamination cycles reduced the number of MRSA positive patients by 68.0%. Excluding non-compliant and deceased patients, decontamination reduced MRSA carriage by 93.3% (n = 15). No adverse events related to the applied decontamination regimen occurred. Exclusive screening of the nose might underreport MRSA prevalence rates. In this study, decontamination with octenidine-based leave-on products was safe and effective in a critical patient population.
Assuntos
Antibacterianos/uso terapêutico , Desinfecção/métodos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Piridinas/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia , Idoso , Idoso de 80 Anos ou mais , Áustria/epidemiologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Portador Sadio/microbiologia , Estudos de Coortes , Feminino , Humanos , Iminas , Masculino , Pessoa de Meia-Idade , Cavidade Nasal/microbiologia , Proteínas de Ligação às Penicilinas/biossíntese , Proteínas de Ligação às Penicilinas/genética , Pele/microbiologia , Infecções Estafilocócicas/diagnóstico , Centros de Atenção Terciária , Traqueia/microbiologia , Cateteres Urinários/microbiologiaRESUMO
Studies with methicillin-resistant Staphylococcus aureus (MRSA) strain COL have shown that the optimal resistance phenotype requires not only mecA but also a large number of "auxiliary genes" identified by Tn551 mutagenesis. The majority of auxiliary mutants showed greatly increased levels of oxacillin resistance when grown in the presence of sub-MICs of mupirocin, suggesting that the mechanism of reduced resistance in the auxiliary mutants involved the interruption of a stringent stress response, causing reduced production of penicillin-binding protein 2A (PBP 2A).
Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Staphylococcus aureus Resistente à Meticilina/genética , Meticilina/farmacologia , Mupirocina/farmacologia , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/genética , Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Genoma Bacteriano/genética , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/biossínteseRESUMO
Penicillin binding protein 4 (PBP4) can provide high-level ß-lactam resistance in Staphylococcus aureus A series of missense and promoter mutations associated with pbp4 were detected in strains that displayed high-level resistance. We show here that the missense mutations facilitate the ß-lactam resistance mediated by PBP4 and the promoter mutations lead to overexpression of pbp4 Our results also suggest a cooperative interplay among PBPs for ß-lactam resistance.
Assuntos
Proteínas de Ligação às Penicilinas/genética , Regiões Promotoras Genéticas/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Genoma Bacteriano/genética , Mutação de Sentido Incorreto/genética , Proteínas de Ligação às Penicilinas/biossíntese , Penicilinas/metabolismo , Penicilinas/farmacologiaRESUMO
OBJECTIVES: Recent surveillance of MRSA colonizing patients and healthcare workers in two African countries (Angola and São Tomé and Príncipe) reported the frequent recovery of oxacillin-susceptible MRSA (OS-MRSA): Staphylococcus aureus strains that gave positive results with the mecA DNA probe, but had low oxacillin MIC values characteristic of susceptible S. aureus. This apparent dissociation of the drug-resistant phenotype from mecA-the primary genetic determinant of resistance-prompted us to perform a more detailed analysis on nine of the African OS-MRSA strains. METHODS: Oxacillin MIC values were determined by Etest and population analysis profiles with and without induction of the stringent stress response by mupirocin. Biochemical profiling using SDS-PAGE followed by western blotting was used for the detection of PBP2A protein produced. RESULTS: Cultures of the African MRSA strains (ST88-IVa and ST8-V) showed heterogeneous oxacillin resistance in which the majority of cells exhibited low oxacillin MICs (≤0.75 mg/L), but highly resistant subpopulations were also present with oxacillin MIC values up to several hundred mg/L and with frequencies of 10(-4) to 10(-6). The same strains after induction of the stringent stress response by mupirocin 'converted' the heterogeneous phenotypes into a more homogeneous and higher level resistance. After induction by oxacillin and mupirocin, each of the nine African OS-MRSA strains produced PBP2A-the protein product of mecA. CONCLUSIONS: The resistant phenotype of OS-MRSA resembles the phenotypes of historically early MRSA clones. The nature of genetic determinants responsible for the heterogeneous phenotypes of OS-MRSA remains to be determined.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/biossíntese , África/epidemiologia , Angola/epidemiologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Eletroforese em Gel de Poliacrilamida , Monitoramento Epidemiológico , Humanos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Fenótipo , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Resistência beta-Lactâmica/genéticaRESUMO
BACKGROUND: Staphylococcus aureus is the major cause of global and nosocomial infections with a significant impact in hospitals worldwide. Our objective was to investigate clinical and molecular characteristics of S. aureus isolates causing infections in patients admitted to hospitals from Recife city, Brazil, and investigate the prevalence of oxacillin-susceptible mecA-positive S. aureus (OS-MRSA) in the region, as well as genetically characterize the isolates and compare with epidemic clones. RESULTS: We characterized 89 isolates in total, 31 clinical methicillin-resistant S. aureus (MRSA) and 58 methicillin-sensitive (MSSA) isolates by PFGE, MLST, spa typing and SCCmec genotyping. Isolates belonging to international MRSA clones were present: Brazilian epidemic clone (BEC) (61 % of MRSA isolates), Paediatric (36 %), New York/Japan (3 %). Some MSSA isolates were related to MRSA clones: USA400-related (10 % of MSSA isolates), Berlin clone (2 %), Paediatric (14 %), New York/Japan (2 %) and Southwest Pacific clone (17 %). MLST revealed new sequence types (ST's): ST2381, ST2382, and ST2383 and new spa types: 10548 and 10550. Among isolates phenotypically identified as MSSA by antimicrobial susceptibility assays, we verified 30 oxacillin-susceptible isolates, which exhibited the mecA gene, without mec complex amplification and were thus classified as OS-MRSA. We observed clonal spread of MRSA and MSSA, including OS-MRSA, within several areas of the main hospital investigated and closely related isolates between hospitals analyzed. CONCLUSIONS: The results of this study suggest a possible spread of the strains in hospital environment that could be responsible for nosocomial infections. We documented the presence of several MRSA clones, as well as new MLST and spa types, that were responsible for severe infections in hospitalized patients. The finding of OS-MRSA isolates could have implications for therapy, because testing for mecA and PBP2a is not a routine procedure performed by clinical microbiology laboratories in Brazil and, as consequence, these isolates could be misclassified as MSSA. Our data alert to the necessity to develop more effective strategies for epidemiological control of S. aureus in order to avoid an increase of hospital infections provoked by this pathogen. We reinforce the use of genetic methods, in addition to phenotypic tests, for a precise identification of MRSA.
Assuntos
Proteínas de Bactérias/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/genética , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Sequência de Bases , Brasil/epidemiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Proteínas de Ligação às Penicilinas/biossíntese , PrevalênciaRESUMO
UNLABELLED: SIPI-8294, as an erythromycin derivative, has only weak antibacterial effects on MRSA and MSSA. Interestingly, synergistic effect of SIPI-8294 with oxacillin was observed both in vitro and in vivo. Western blot and RT-PCR results demonstrate that mecA expressions were suppressed by SIPI-8294 in MRSA. Furthermore, the knock out of mecA in ATCC 43300 led to the loss of synergy of the combinations while mecA complemented strain showed almost the same synergistic capability compared to the wild type strain. However, the knock out of mecR1 and mecI in MRSA displayed no impact on the synergy of the combinations and the ability of SIPI-8294 to suppress mecA expression. In summary, our study has demonstrated that SIPI-8294 could dramatically reverse MRSA resistance to ß-lactams both in vitro and in vivo owing to inhibiting mecA expression. However, mecR1 and mecI, as the pivotal regulatory genes of mecA, do not participate in SIPI-8294-mecA pathway. The research indicates that it may be a promising strategy for combating MRSA infections with the combinations of SIPI-8294 and ß-lactam antibiotics. The research of the mechanism is important for structure modification and new drug development. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the mechanism of synergy between SIPI-8294 and ß-lactams against MRSA on the molecular level. In this study, SIPI-8294 showed strong synergistic effects on ß-lactam antibiotics both in vitro and in vivo owing to inhibiting mecA expression. As pivotal regulatory genes of mecA, mecR1 and mecI do not participate in SIPI-8294-mecA pathway and are not involved in the synergism of SIPI-8294 and ß-lactams. The research indicates that it may be a promising strategy for combating MRSA infections with the combinations of SIPI-8294 and ß-lactams. The research is important for structure modification and new drug development.
Assuntos
Proteínas de Bactérias/biossíntese , Eritromicina/análogos & derivados , Eritromicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/biossíntese , beta-Lactamas/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Sinergismo Farmacológico , Meticilina/farmacologia , Resistência a Meticilina , Testes de Sensibilidade Microbiana , Proteínas Repressoras/genéticaRESUMO
UNLABELLED: Membrane proteins need to be properly inserted and folded in the membrane in order to perform a range of activities that are essential for the survival of bacteria. The Sec translocon and the YidC insertase are responsible for the insertion of the majority of proteins into the cytoplasmic membrane. YidC can act in combination with the Sec translocon in the insertion and folding of membrane proteins. However, YidC also functions as an insertase independently of the Sec translocon for so-called YidC-only substrates. In addition, YidC can act as a foldase and promote the proper assembly of membrane protein complexes. Here, we investigate the effect of Escherichia coli YidC depletion on the assembly of penicillin binding proteins (PBPs), which are involved in cell wall synthesis. YidC depletion does not affect the total amount of the specific cell division PBP3 (FtsI) in the membrane, but the amount of active PBP3, as assessed by substrate binding, is reduced 2-fold. A similar reduction in the amount of active PBP2 was observed, while the levels of active PBP1A/1B and PBP5 were essentially similar. PBP1B and PBP3 disappeared from higher-Mw bands upon YidC depletion, indicating that YidC might play a role in PBP complex formation. Taken together, our results suggest that the foldase activity of YidC can extend to the periplasmic domains of membrane proteins. IMPORTANCE: This study addresses the role of the membrane protein insertase YidC in the biogenesis of penicillin binding proteins (PBPs). PBPs are proteins containing one transmembrane segment and a large periplasmic or extracellular domain, which are involved in peptidoglycan synthesis. We observe that in the absence of YidC, two critical PBPs are not correctly folded even though the total amount of protein in the membrane is not affected. Our findings extend the function of YidC as a foldase for membrane protein (complexes) to periplasmic domains of membrane proteins.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Ligação às Penicilinas/biossíntese , Membrana Celular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Proteínas de Ligação às Penicilinas/genética , Plasmídeos , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , VirulênciaRESUMO
Expression of the methicillin-resistant S. aureus (MRSA) phenotype results from the expression of the extra penicillin-binding protein 2A (PBP2A), which is encoded by mecA and acquired horizontally on part of the SCCmec cassette. PBP2A can catalyze dd-transpeptidation of peptidoglycan (PG) because of its low affinity for ß-lactam antibiotics and can functionally cooperate with the PBP2 transglycosylase in the biosynthesis of PG. Here, we focus upon the role of the membrane-bound PrsA foldase protein as a regulator of ß-lactam resistance expression. Deletion of prsA altered oxacillin resistance in three different SCCmec backgrounds and, more importantly, caused a decrease in PBP2A membrane amounts without affecting mecA mRNA levels. The N- and C-terminal domains of PrsA were found to be critical features for PBP2A protein membrane levels and oxacillin resistance. We propose that PrsA has a role in posttranscriptional maturation of PBP2A, possibly in the export and/or folding of newly synthesized PBP2A. This additional level of control in the expression of the mecA-dependent MRSA phenotype constitutes an opportunity to expand the strategies to design anti-infective agents.
Assuntos
Proteínas de Bactérias/genética , Lipoproteínas/genética , Proteínas de Membrana/genética , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/genética , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/biossíntese , Peptidoglicano/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Dobramento de Proteína , RNA Mensageiro/genéticaRESUMO
Many bacteria are able to assume a transient cell wall-deficient (or L-form) state under favourable osmotic conditions. Cell wall stress such as exposure to ß-lactam antibiotics can enforce the transition to and maintenance of this state. L-forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted Escherichia coli as a model system for the controlled transition to and reversion from the L-form state, and have studied these dynamics with genetics, cell biology and 'omics' technologies. As such, a transposon mutagenesis screen underscored the requirement for the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L-forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E. coliâ L-forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labelled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD-carboxypeptidases PBP5 and PBP6 are threefold and fourfold upregulated in L-forms, indicating a specific role for regulation of crosslinking during L-form proliferation.
Assuntos
Parede Celular/metabolismo , Escherichia coli/metabolismo , Lipídeos de Membrana/metabolismo , Peptidoglicano/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Biblioteca Gênica , Modelos Biológicos , Proteínas de Ligação às Penicilinas/biossíntese , Proteínas de Ligação às Penicilinas/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/biossíntese , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Resistência beta-Lactâmica/genética , beta-Lactamas/farmacologiaRESUMO
In this study we combined experimentation with mathematical modeling to unravel the in vivo kinetic properties of the enzymes and transporters of the penicillin biosynthesis pathway in a high yielding Penicillium chrysogenum strain. The experiment consisted of a step response experiment with the side chain precursor phenyl acetic acid (PAA) in a glucose-limited chemostat. The metabolite data showed that in the absence of PAA all penicillin pathway enzymes were expressed, leading to the production of a significant amount of 6-aminopenicillanic acid (6APA) as end product. After the stepwise perturbation with PAA, the pathway produced PenG within seconds. From the extra- and intracellular metabolite measurements, hypotheses for the secretion mechanisms of penicillin pathway metabolites were derived. A dynamic model of the penicillin biosynthesis pathway was then constructed that included the formation and transport over the cytoplasmic membrane of pathway intermediates, PAA and the product penicillin-G (PenG). The model parameters and changes in the enzyme levels of the penicillin biosynthesis pathway under in vivo conditions were simultaneously estimated using experimental data obtained at three different timescales (seconds, minutes, hours). The model was applied to determine changes in the penicillin pathway enzymes in time, calculate fluxes and analyze the flux control of the pathway. This led to a reassessment of the in vivo behavior of the pathway enzymes and in particular Acyl-CoA:Isopenicillin N Acyltransferase (AT).
Assuntos
Penicilinas/biossíntese , Fenilacetatos/metabolismo , Aciltransferases/biossíntese , Aciltransferases/genética , Algoritmos , Carbono/metabolismo , Meios de Cultura , Filtração , Glucose/metabolismo , Cinética , Redes e Vias Metabólicas , Modelos Biológicos , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/metabolismo , Proteínas de Ligação às Penicilinas/biossíntese , Proteínas de Ligação às Penicilinas/genética , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismoRESUMO
Penicillin-binding protein 3 (PBP3) of Pseudomonas aeruginosa is the primary target of ß-lactams used to treat pseudomonas infections. Meanwhile, structure change and overproduction of PBP3 play important roles in the drug resistance of P. aeruginosa. Therefore, studies on the gene and structure of PBP3 are urgently needed. P. aeruginosa CMCC 10104 is a type culture strain common used in China. However, there is no report on its genomic and proteomic profiles. In this study, based on ftsI of P. aeruginosa PAO1, the gene encoding PBP3 was cloned from CMCC 10104. A truncated version of the ftsI gene, omitting the bases encoding the hydrophobic leader peptide (amino acids 1-34), was amplified by PCR. The cloned DNA shared 99.76% identity with ftsI from PAO1. Only four bases were different (66 C-A, 1020 T-C, 1233 T-C, and 1527 T-C). However, there were no differences between their deduced amino acid sequences. The recombinant PBP3 (rPBP3), containing a 6-histidine tag, was expressed in Escherichia coli BL21 (DE3). Immobilized metal affinity chromatography (IMAC) with Ni(2+)-NTA agarose was used for its purification. The purified rPBP3 was identified by SDS-PAGE and western blot analysis, and showed a single band at about 60kDa with purity higher than 95%. The penicillin-binding assay indicated that the obtained rPBP3 was functional and not hindered by the presence of the C-terminal His-tag. The protocol described in this study offers a method for obtaining purified recombinant PBP3 from P. aeruginosa CMCC 10104.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Proteínas de Ligação às Penicilinas/isolamento & purificação , Pseudomonas aeruginosa/química , Deleção de Sequência , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Dados de Sequência Molecular , Proteínas de Ligação às Penicilinas/biossíntese , Proteínas de Ligação às Penicilinas/genética , Penicilinas/química , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Sinais Direcionadores de Proteínas/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Resistência beta-Lactâmica/genéticaRESUMO
The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Inibidores do Crescimento/administração & dosagem , Inibidores do Crescimento/química , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Ligação às Penicilinas/biossíntese , Proteínas de Ligação às Penicilinas/química , Especificidade por Substrato , Tioureia/administração & dosagem , Tioureia/análogos & derivadosRESUMO
Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Parede Celular/química , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Staphylococcus aureus Resistente à Meticilina/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Muramilpentapeptídeo Carboxipeptidase/biossíntese , Muramilpentapeptídeo Carboxipeptidase/química , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Proteínas de Ligação às Penicilinas/química , Peptidoglicano/biossíntese , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/química , Vancomicina/farmacologia , Parede Celular/metabolismo , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Proteínas de Ligação às Penicilinas/biossíntese , Peptidoglicano/química , Staphylococcus aureus/metabolismoRESUMO
OBJECTIVES: Vancomycin use for neonatal coagulase-negative staphylococci (CoNS) sepsis is based on a high CoNS carriage rate of mecA, encoding penicillin-binding protein (PBP)-2a, with low affinity for, and associated with resistance to, ß-lactam antibiotics. The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the ß-lactam antibiotics oxacillin, cefazolin and amoxicillin/clavulanate was determined for 85 CoNS blood isolates randomly obtained from our collection of isolates from neonates with CoNS sepsis. METHODS: The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the ß-lactam antibiotics oxacillin, cefazolin and amoxicillin/clavulanate was determined for randomly obtained CoNS blood isolates from our collection of isolates from neonates with CoNS sepsis. The mecA gene was detected using multiplex PCR, and PBP-2a expression was determined using a latex agglutination (LA) test (Oxoid). ß-Lactam susceptibility was determined using the Phoenix automated system and, in addition, by Etest with interpretation of MIC values according to the reference MIC breakpoints adopted from the CLSI guidelines M100-S20, Infobase™. RESULTS: Among 85 CoNS blood isolates, 73 (86%) were mecA positive and 12 (14%) were mecA negative. None of the mecA-negative isolates expressed PBP-2a and all were ß-lactam susceptible. All mecA-positive CoNS isolates were oxacillin resistant, although most oxacillin MICs were not very high, ranging from 2 to 8 mg/L for the majority of isolates. Only 8/73 (11%) mecA-positive CoNS isolates had oxacillin MICs ≥32 mg/L (range 32 to >256 mg/L). mecA-positive CoNS blood isolates, although fully resistant to oxacillin, were almost universally susceptible to cefazolin and amoxicillin/clavulanate, which was associated with a low expression rate of PBP-2a. CONCLUSIONS: ß-Lactam antibiotics are useful for the treatment of neonatal CoNS sepsis, reserving vancomycin for selected cases.
Assuntos
Antibacterianos/uso terapêutico , Expressão Gênica , Proteínas de Ligação às Penicilinas/biossíntese , Sepse/microbiologia , Staphylococcus/enzimologia , Staphylococcus/genética , beta-Lactamas/uso terapêutico , Coagulase/metabolismo , DNA Bacteriano/genética , Humanos , Recém-Nascido , Resistência a Meticilina , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Reação em Cadeia da Polimerase , Staphylococcus/efeitos dos fármacos , Staphylococcus/isolamento & purificação , Vancomicina/uso terapêuticoRESUMO
Intense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.
Assuntos
Aciltransferases/biossíntese , Aciltransferases/genética , Dosagem de Genes , Engenharia Metabólica , Proteínas de Ligação às Penicilinas/biossíntese , Proteínas de Ligação às Penicilinas/genética , Penicilinas/biossíntese , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Expressão Gênica , Família MultigênicaRESUMO
In its natural habitats, Bacillus subtilis is exposed to changing osmolarity, necessitating adaptive stress responses. Transcriptomic and proteomic approaches can provide a picture of the dynamic changes occurring in salt-stressed B. subtilis cultures because these studies provide an unbiased view of cells coping with high salinity. We applied whole-genome microarray technology and metabolic labeling, combined with state-of-the-art proteomic techniques, to provide a global and time-resolved picture of the physiological response of B. subtilis cells exposed to a severe and sudden osmotic upshift. This combined experimental approach provided quantitative data for 3,961 mRNA transcription profiles, 590 expression profiles of proteins detected in the cytosol, and 383 expression profiles of proteins detected in the membrane fraction. Our study uncovered a well-coordinated induction of gene expression subsequent to an osmotic upshift that involves large parts of the SigB, SigW, SigM, and SigX regulons. Additionally osmotic upregulation of a large number of genes that do not belong to these regulons was observed. In total, osmotic upregulation of about 500 B. subtilis genes was detected. Our data provide an unprecedented rich basis for further in-depth investigation of the physiological and genetic responses of B. subtilis to hyperosmotic stress.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteômica/métodos , Sais/farmacologia , Fator sigma/metabolismo , Estresse Fisiológico/fisiologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação Bacteriana da Expressão Gênica/genética , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Ligação às Penicilinas/biossíntese , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/biossíntese , Peptidoglicano/genética , Regulon/genética , Fator sigma/genética , Espectrometria de Massas em Tandem , Ácidos Teicoicos/biossíntese , Ácidos Teicoicos/genéticaRESUMO
Bacterial shape is physically determined by the peptidoglycan cell wall. The cell-wall-synthesis machinery responsible for rod shape in Escherichia coli is the processive 'Rod complex'. Previously, cytoplasmic MreB filaments were thought to govern formation and localization of Rod complexes based on local cell-envelope curvature. Using single-particle tracking of the transpeptidase and Rod-complex component PBP2, we found that PBP2 binds to a substrate different from MreB. Depletion and localization experiments of other putative Rod-complex components provide evidence that none of those provide the sole rate-limiting substrate for PBP2 binding. Consistently, we found only weak correlations between MreB and envelope curvature in the cylindrical part of cells. Residual correlations do not require curvature-based Rod-complex initiation but can be attributed to persistent rotational motion. We therefore speculate that the local cell-wall architecture provides the cue for Rod-complex initiation, either through direct binding by PBP2 or through an unknown intermediate.
Assuntos
Parede Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Frações Subcelulares/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Ligação às Penicilinas/biossínteseRESUMO
Rhodococcus exhibits strong adaptability to environmental stressors and plays a crucial role in environmental bioremediation. However, seasonal changes in ambient temperature, especially rapid temperature drops exert an adverse effect on in situ bioremediation. In this paper, we studied the cell morphology and fatty acid composition of an aniline-degrading strain Rhodococcus sp. CNS16 at temperatures of 30⯰C, 20⯰C, and 10⯰C. At suboptimal temperatures, cell morphology of CNS16 changed from short rod-shaped to long rod or irregular shaped, and the proportion of unsaturated fatty acids was upregulated. Transcriptomic technologies were then utilized to gain detailed insights into the adaptive mechanisms of CNS16 subjected to suboptimal temperatures. The results showed that the number of gene responses was significantly higher at 10⯰C than that at 20⯰C. The inhibition of peptidoglycan synthase expression and up-regulation of Filamentous Temperature Sensitive as well as unsaturated fatty acid synthesis genes at suboptimal temperatures might be closely related to corresponding changes in cell morphology and fatty acids composition. Strain CNS16 showed loss of catalase and superoxide dismutase activity, and utilized thioredoxin-dependent thiol peroxidase to resist oxidative stress. The up-regulation of carotenoid and Vitamin B2 synthesis at 10⯰C might also be involved in the resistance to oxidative stress. Amino acid metabolism, coenzyme and vitamin metabolism, ABC transport, and energy metabolism are essential for peptidoglycan synthesis and regulation of cellular metabolism; therefore, synergistically resisting environmental stress. This study provides a mechanistic basis for the regulation of aniline degradation in Rhodococcus sp. CNS16 at low temperatures.