Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.033
Filtrar
1.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809732

RESUMO

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.


Assuntos
Paenibacillus polymyxa/metabolismo , Riboswitch/genética , Serina/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Códon/genética , Sequência Conservada , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Nitrogenase/metabolismo , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Paenibacillus polymyxa/efeitos dos fármacos , Paenibacillus polymyxa/genética , Paenibacillus polymyxa/crescimento & desenvolvimento , RNA Bacteriano/química , RNA Bacteriano/genética , Serina/farmacologia
2.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809178

RESUMO

The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell.


Assuntos
Epóxido Hidrolases/antagonistas & inibidores , Tioureia/farmacologia , Tuberculose/tratamento farmacológico , Ureia/farmacologia , Adamantano/análogos & derivados , Adamantano/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Compostos de Fenilureia/farmacologia , Tioacetazona/farmacologia , Tioureia/análogos & derivados , Tuberculose/enzimologia , Tuberculose/microbiologia , Ureia/química
3.
J Med Microbiol ; 70(4)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33830911

RESUMO

Introduction. Antipathogenic or antivirulence strategy is to target a virulence pathway that is dispensable for growth, in the hope to mitigate the selection for drug resistance.Hypothesis/Gap Statment. Peroxide stress responses are one of the conserved virulence pathways in bacterial pathogens and thus good targets for antipathogenic strategy.Aim. This study aims to identify a new chemical compound that targets OxyR, the peroxide sensor required for the full virulence of the opportunistic human pathogen, Pseudomonas aeruginosa.Methodology. Computer-based virtual screening under consideration of the 'eNTRy' rules and molecular docking were conducted on the reduced form of the OxyR regulatory domain (RD). Selected hits were validated by their ability to phenocopy the oxyR null mutant and modulate the redox cycle of OxyR.Results. We first isolated three robust chemical hits that inhibit OxyR without affecting prototrophic growth or viability. One (compound 1) of those affected the redox cycle of OxyR in response to H2O2 treatment, in a way to impair its function. Compound 1 displayed selective antibacterial efficacy against P. aeruginosa in Drosophila infection model, without antibacterial activity against Staphylococcus aureus.Conclusion. These results suggest that compound 1 could be an antipathogenic hit inhibiting the P. aeruginosa OxyR. More importantly, our study provides an insight into the computer-based discovery of new-paradigm selective antibacterials to treat Gram-negative bacterial infections presumably with few concerns of drug resistance.


Assuntos
Antibacterianos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Transativadores/antagonistas & inibidores , Animais , Drosophila , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Simulação de Acoplamento Molecular , Mutação , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/mortalidade , Pseudomonas aeruginosa/genética , Taxa de Sobrevida , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Virulência/efeitos dos fármacos , Virulência/genética
4.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802668

RESUMO

Avibactam belongs to the new class of diazabicyclooctane ß-lactamase inhibitors. Its inhibitory spectrum includes class A, C and D enzymes, including P. aeruginosa AmpC. Nonetheless, recent reports have revealed strain-dependent avibactam AmpC induction. In the present work, we wanted to assess the mechanistic basis underlying AmpC induction and determine if derepressed PDC-X mutated enzymes from ceftazidime/avibactam-resistant clinical isolates were further inducible. We determined avibactam concentrations that half-maximally inhibited (IC50) bocillin FL binding. Inducer ß-lactams were also studied as comparators. Live cells' time-course penicillin-binding proteins (PBPs) occupancy of avibactam was studied. To assess the ampC induction capacity of avibactam and comparators, qRT-PCR was performed in wild-type PAO1, PBP4, triple PBP4, 5/6 and 7 knockout derivatives and two ceftazidime/avibactam-susceptible/resistant XDR clinical isolates belonging to the epidemic high-risk clone ST175. PBP4 inhibition was observed for avibactam and ß-lactam comparators. Induction capacity was consistently correlated with PBP4 binding affinity. Outer membrane permeability-limited PBP4 binding was observed in the live cells' assay. As expected, imipenem and cefoxitin showed strong induction in PAO1, especially for carbapenem; avibactam induction was conversely weaker. Overall, the inducer effect was less remarkable in ampC-derepressed mutants and nonetheless absent upon avibactam exposure in the clinical isolates harboring mutated AmpC variants and their parental strains.


Assuntos
Compostos Azabicíclicos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Mutação/genética , Proteínas de Ligação às Penicilinas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/metabolismo , Proteínas de Bactérias/metabolismo , Cefoxitina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Imipenem/farmacologia , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos
5.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803167

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide and has acquired multiple resistance to a wide range of antibiotics. Hence, there is a pressing need to explore novel strategies to overcome the increase in antimicrobial resistance. The present study aims to investigate the efficacy and mechanism of plant-derived antimicrobials, trans-cinnamaldehyde (TCA) in decreasing MRSA's resistance to eight conventional antibiotics. A checkerboard dilution test and time-kill curve assay are used to determine the synergistic effects of TCA combined with the antibiotics. The results indicated that TCA increased the antibacterial activity of the antibiotics by 2-16-fold. To study the mechanism of the synergism, we analyzed the mecA transcription gene and the penicillin-binding protein 2a level of MRSA treated with TCA by quantitative RT-PCR or Western blot assay. The gene transcription and the protein level were significantly inhibited. Additionally, it was verified that TCA can significantly inhibit the biofilm, which is highly resistant to antibiotics. The expression of the biofilm regulatory gene hld of MRSA after TCA treatment was also significantly downregulated. These findings suggest that TCA maybe is an exceptionally potent modulator of antibiotics.


Assuntos
Acroleína/análogos & derivados , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Acroleína/agonistas , Acroleína/farmacologia , Biofilmes/crescimento & desenvolvimento , Sinergismo Farmacológico
6.
Int J Nanomedicine ; 16: 1849-1867, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33707943

RESUMO

Background: With the development of bacterial resistance, the range of effective antibiotics is increasingly becoming more limited. The effective use of nanoscale antimicrobial peptides (AP) in therapeutic and diagnostic methods is a strategy for new antibiotics. Methods: Combining both AP and cadmium selenide (CdSe) into a composite material may result in a reagent with novel properties, such as enhanced antibacterial activity, fluorescence and favorable stability in aqueous solution. Results: AP-loaded CdSe NPs (AP-CdSe NPs) showed strong antibacterial activity against multidrug-resistant (MDR) Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in vitro and in vivo. Colony-forming unit (CFU) and minimum inhibitory concentration (MIC) assays showed that AP-CdSe NPs have highly effective antibacterial activity. The quantitative analysis of apoptosis by flow cytometry analysis further confirmed that MDR E. coli and S. aureus treated with AP-CdSe NPs had death rates of 98.76% and 99.13%, respectively. Also, AP-CdSe NPs was found to inhibit bacterial activity in an in vivo bacteremia model in mice infected with S. aureus. In addition, the antibacterial mechanism of AP-CdSe NPs was determined by RNA sequencing analysis. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed the molecular mechanism of the antibacterial effect of AP-CdSe NPs. Importantly, histopathology analysis, and hematological toxicity analysis indicated that AP-CdSe NPs had few side effects. Conclusion: These results demonstrate that AP loaded on CdSe NPs had a higher water solubility, bioavailability and antibacterial effect compared with raw AP. This study reports findings that are helpful for the design and development of antibacterial treatment strategies based on AP.


Assuntos
Antibacterianos/farmacologia , Compostos de Cádmio/química , Luminescência , Nanopartículas/química , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Pontos Quânticos/química , Compostos de Selênio/química , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Bacteriemia/microbiologia , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Endocitose/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/ultraestrutura , Feminino , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Camundongos Nus , Testes de Sensibilidade Microbiana , Microscopia de Fluorescência , Nanopartículas/ultraestrutura , Proteínas Citotóxicas Formadoras de Poros/efeitos adversos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/ultraestrutura
7.
Int J Mol Sci ; 22(4)2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672733

RESUMO

Sigma factor C (SigC) contributes to Mycobacterium tuberculosis virulence in various animal models, but the stress response coordinated by this transcription factor was undefined. The results presented here indicate that SigC prevents copper starvation. Whole genome expression studies demonstrate short-term (4-h) induction of sigC, controlled from a tetracycline-inducible promoter, upregulates ctpB and genes in the nonribosomal peptide synthase (nrp) operon. These genes are expressed at higher levels after 48-h sigC induction, but also elevated are genes encoding copper-responsive regulator RicR and RicR-regulated copper toxicity response operon genes rv0846-rv0850, suggesting prolonged sigC induction results in excessive copper uptake. No growth and global transcriptional differences are observed between a sigC null mutant relative to its parent strain in 7H9 medium. In a copper-deficient medium, however, growth of the sigC deletion strain lags the parent, and 40 genes (including those in the nrp operon) are differentially expressed. Copper supplementation reverses the growth defect and silences most transcriptional differences. Together, these data support SigC as a transcriptional regulator of copper acquisition when the metal is scarce. Attenuation of sigC mutants in severe combined immunodeficient mice is consistent with an inability to overcome innate host defenses that sequester copper ions to deprive invading microbes of this essential micronutrient.


Assuntos
Cobre/farmacologia , Imunidade/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Fator sigma/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Sulfato de Cobre/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Camundongos SCID , Viabilidade Microbiana/efeitos dos fármacos , Mutação/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fenótipo , Transcrição Genética/efeitos dos fármacos , Virulência/efeitos dos fármacos , Virulência/genética
8.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557119

RESUMO

Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdD, hcdE, hcdF, and hcdG were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for P. mandelii 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.


Assuntos
Álcool Desidrogenase/metabolismo , Biodegradação Ambiental , Pseudomonas/metabolismo , Umbeliferonas/metabolismo , Álcool Desidrogenase/genética , Catálise , Cumarínicos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano , Estrutura Molecular , Família Multigênica , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Filogenia , Pseudomonas/classificação , Pseudomonas/enzimologia , Pseudomonas/genética , Reação em Cadeia da Polimerase em Tempo Real , Umbeliferonas/química
9.
Appl Environ Microbiol ; 87(9)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33608290

RESUMO

Listeria monocytogenes is a deadly intracellular pathogen mostly associated with consumption of ready-to-eat foods. This study investigated the effectiveness of total beef fat (BF-T) from flaxseed-fed cattle and its fractions enriched with monounsaturated fatty acids (BF-MUFA) and polyunsaturated fatty acids (BF-PUFA), along with commercially available long-chain fatty acids (LC-FA), as natural antimicrobials against L. monocytogenes BF-T was ineffective at concentrations up to 6 mg/ml, while L. monocytogenes was susceptible to BF-MUFA and BF-PUFA, with MICs at pH 7 of 0.33 ± 0.21 mg/ml and 0.06 ± 0.03 mg/ml, respectively. The MIC of C14:0 was significantly lower than those of C16:0 and C18:0 (P < 0.05). Fatty acids c9-C16:1, C18:2n-6, and C18:3n-3 showed stronger inhibitory activity than c9-C18:1 and conjugated C18:2, with MICs of <1 mg/ml. Furthermore, global transcriptional analysis by transcriptome sequencing (RNA-seq) was performed to characterize the response of L. monocytogenes to selected fatty acids. Functional analysis indicated that antimicrobial LC-UFA repressed the expression of genes associated with nutrient transmembrane transport, energy generation, and oxidative stress resistance. On the other hand, upregulation of ribosome assembly and translation process is possibly associated with adaptive and repair mechanisms activated in response to LC-UFA. Virulence genes and genes involved in bile, acid, and osmotic stresses were largely downregulated, and more so for c9-C16:1, C18:2n-6, and C18:3n-3, likely through interaction with the master virulence regulator PrfA and the alternative sigma factor σB IMPORTANCE Listeria monocytogenes is a bacterial pathogen known for its ability to survive and thrive under adverse environments and, as such, its control poses a significant challenge, especially with the trend of minimally processed and ready-to-eat foods. This work investigated the effectiveness of fatty acids from various sources as natural antimicrobials against L. monocytogenes and evaluated their potential role in L. monocytogenes pathogenicity modulation, using the strain ATCC 19111. The findings show that long-chain unsaturated fatty acids (LC-UFA), including unsaturated beef fat fractions from flaxseed-fed cattle, could have the potential to be used as effective antimicrobials for L. monocytogenes through controlling growth as well as virulence attenuation. This not only advances our understanding of the mode of action of LC-UFA against L. monocytogenes but also suggests the potential for use of beef fat or its fractions as natural antimicrobials for controlling foodborne pathogens.


Assuntos
Gorduras/farmacologia , Ácidos Graxos/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Carne Vermelha , Animais , Antibacterianos/farmacologia , Bovinos , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/crescimento & desenvolvimento
10.
Biochem Biophys Res Commun ; 537: 50-56, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33385805

RESUMO

INTRODUCTION: Although therapeutic agents for methicillin-resistant Staphylococcus aureus (MRSA) are clinically available, MRSA infection is still a life-threatening disease. Bacterial attachment and biofilm formation contribute significantly to the initiation of MRSA infection. Controlling MRSA's attachment and biofilm formation might reduce the frequency of MRSA infection. According to recent data, some amino acids can reduce MRSA's attachment on plates; however, their precise inhibitory mechanisms remain unclear. Therefore, we explored the effect of the amino acids on bacterial adhesion and biofilm formation in vitro and in vivo MRSA infection models. METHODS: We tested the inhibitory effect of amino acids on MRSA and Escherichia coli (E. coli) in the attachment assay. Moreover, we evaluated the therapeutic potential of amino acids on the in vivo catheter infection model. RESULTS: Among the amino acids, D-Serine (D-Ser) was found to reduce MRSA's ability to attach on plate assay. The proliferation of MRSA was not affected by the addition of D-Ser; thus, D-Ser likely only played a role in preventing attachment and biofilm formation. Then, we analyzed the expression of genes related to attachment and biofilm formation. D-Ser was found to reduce the expressions of AgrA, SarS, IcaA, DltD, and SdrD. Moreover, the polyvinyl chloride catheters treated with D-Ser had fewer MRSA colonies. D-Ser treatment also reduced the severity of infection in the catheter-induced peritonitis model. Moreover, D-Ser reduced the attachment ability of E. coli. CONCLUSION: D-Ser inhibits the attachment and biofilm formation of MRSA by reducing the expression of the related genes. Also, the administration of D-Ser reduces the severity of catheter infection in the mouse model. Therefore, D-Ser may be a promising therapeutic option for MRSA as well as E. coli infection.


Assuntos
Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Serina/farmacologia , Animais , Cateteres/microbiologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Camundongos Endogâmicos BALB C , Peritonite/microbiologia , Peritonite/patologia , Cloreto de Polivinila
11.
Carbohydr Polym ; 255: 117484, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436244

RESUMO

Wound dressing composed of chitosan, based crosslinked gelatin/ polyvinyl pyrrolidone, embedded silver nanoparticles were fabricated using solution casting method. The membrane was characterized by FTIR, SEM and TGA. Glutaraldehyde (0.5 %) was used for the crosslinking of membrane components and associated with 7-folds boosted mechanical performance, 28 % more hydrolytic stability, 3-folds thickness reduction and morphological roughness. Silver nanoparticles were characterized by UV-vis, XRD and TEM for an average size of 9.9 nm. The membrane with higher concentration of silver nanoparticles showed maximum antibacterial activity against human pathogenic bacteria; and the measured inhibition zones ranged from 1.5 to 3 cm. The activity of the particles ranged from severe to complete reduction in Penicillin, Erythromycin and Macrolide family's resistance genes expression such as ß-Lactamase, mecA and erm. This developed membrane can serve as promising and cost-effective system against severe diabetic and burn wound infections.


Assuntos
Antibacterianos/farmacologia , Bandagens , Quitosana/química , Citrullus colocynthis/química , Gelatina/química , Povidona/química , Prata/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Macrolídeos/farmacologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Metiltransferases/genética , Metiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Penicilinas/farmacologia , Cultura Primária de Células , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Salmonella typhi/efeitos dos fármacos , Salmonella typhi/crescimento & desenvolvimento , Prata/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , beta-Lactamases/genética , beta-Lactamases/metabolismo
12.
Molecules ; 26(1)2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466475

RESUMO

The demand for reduced chemical preservative usage is currently growing, and natural preservatives are being developed to protect seafood. With its excellent antibacterial properties, linalool has been utilized widely in industries. However, its antibacterial mechanisms remain poorly studied. Here, untargeted metabolomics was applied to explore the mechanism of Shewanella putrefaciens cells treated with linalool. Results showed that linalool exhibited remarkable antibacterial activity against S. putrefaciens, with 1.5 µL/mL minimum inhibitory concentration (MIC). The growth of S. putrefaciens was suppressed completely at 1/2 MIC and 1 MIC levels. Linalool treatment reduced the membrane potential (MP); caused the leakage of alkaline phosphatase (AKP); and released the DNA, RNA, and proteins of S. putrefaciens, thus destroying the cell structure and expelling the cytoplasmic content. A total of 170 differential metabolites (DMs) were screened using metabolomics analysis, among which 81 species were upregulated and 89 species were downregulated after linalool treatment. These DMs are closely related to the tricarboxylic acid (TCA) cycle, glycolysis, amino acid metabolism, pantothenate and CoA biosynthesis, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism. In addition, linalool substantially affected the activity of key enzymes, such as succinate dehydrogenase (SDH), pyruvate kinase (PK), ATPase, and respiratory chain dehydrogenase. The results provided some insights into the antibacterial mechanism of linalool against S. putrefaciens and are important for the development and application of linalool in seafood preservation.


Assuntos
Monoterpenos Acíclicos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Metaboloma/efeitos dos fármacos , Shewanella putrefaciens/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , Inseticidas/farmacologia , Shewanella putrefaciens/crescimento & desenvolvimento , Shewanella putrefaciens/metabolismo
13.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430070

RESUMO

The nosocomial opportunistic Gram-negative bacterial pathogen Acinetobacter baumannii is resistant to multiple antimicrobial agents and an emerging global health problem. The polymyxin antibiotic colistin, targeting the negatively charged lipid A component of the lipopolysaccharide on the bacterial cell surface, is often considered as the last-resort treatment, but resistance to colistin is unfortunately increasing worldwide. Notably, colistin-susceptible A. baumannii can also develop a colistin dependence after exposure to this drug in vitro. Colistin dependence might represent a stepping stone to resistance also in vivo. However, the mechanisms are far from clear. To address this issue, we combined proteogenomics, high-resolution microscopy, and lipid profiling to characterize and compare A. baumannii colistin-susceptible clinical isolate (Ab-S) of to its colistin-dependent subpopulation (Ab-D) obtained after subsequent passages in moderate colistin concentrations. Incidentally, in the colistin-dependent subpopulation the lpxA gene was disrupted by insertion of ISAjo2, the lipid A biosynthesis terminated, and Ab-D cells displayed a lipooligosaccharide (LOS)-deficient phenotype. Moreover, both mlaD and pldA genes were perturbed by insertions of ISAjo2 and ISAba13, and LOS-deficient bacteria displayed a capsule with decreased thickness as well as other surface imperfections. The major changes in relative protein abundance levels were detected in type 6 secretion system (T6SS) components, the resistance-nodulation-division (RND)-type efflux pumps, and in proteins involved in maintenance of outer membrane asymmetry. These findings suggest that colistin dependence in A. baumannii involves an ensemble of mechanisms seen in resistance development and accompanied by complex cellular events related to insertional sequences (ISs)-triggered LOS-deficiency. To our knowledge, this is the first study demonstrating the involvement of ISAjo2 and ISAba13 IS elements in the modulation of the lipid A biosynthesis and associated development of dependence on colistin.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Acinetobacter/genética , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/patogenicidade , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Mutagênese Insercional/genética
14.
mBio ; 12(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402533

RESUMO

Despite dogma suggesting that lipopolysaccharide/lipooligosaccharide (LOS) was essential for viability of Gram-negative bacteria, several Acinetobacter baumannii clinical isolates produced LOS- colonies after colistin selection. Inactivation of the conserved class A penicillin-binding protein, PBP1A, was a compensatory mutation that supported isolation of LOS- A. baumannii, but the impact of PBP1A mutation was not characterized. Here, we show that the absence of PBP1A causes septation defects and that these, together with ld-transpeptidase activity, support isolation of LOS- A. baumannii PBP1A contributes to proper cell division in A. baumannii, and its absence induced cell chaining. Only isolates producing three or more septa supported selection of colistin-resistant LOS- A. baumannii PBP1A was enriched at the midcell, where the divisome complex facilitates daughter cell formation, and its localization was dependent on glycosyltransferase activity. Transposon mutagenesis showed that genes encoding two putative ld-transpeptidases (LdtJ and LdtK) became essential in the PBP1A mutant. Both LdtJ and LdtK were required for selection of LOS- A. baumannii, but each had distinct enzymatic activities in the cell. Together, these findings demonstrate that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity remodel the cell envelope to support selection of colistin-resistant LOS- A. baumannii IMPORTANCE The increasing prevalence of antibiotic treatment failure associated with Gram-negative bacterial infections highlights an urgent need to develop new alternative therapeutic strategies. The last-line antimicrobial colistin (polymyxin E) targets the ubiquitous outer membrane lipopolysaccharide (LPS)/LOS membrane anchor, lipid A, which is essential for viability of most diderms. However, several LOS- Acinetobacter baumannii clinical isolates were recovered after colistin selection, suggesting a conserved resistance mechanism. Here, we characterized a role for penicillin-binding protein 1A in A. baumannii septation and intrinsic ß-lactam susceptibility. We also showed that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity support isolation of colistin-resistant LOS- A. baumannii.


Assuntos
Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Lipopolissacarídeos/deficiência , Proteínas de Ligação às Penicilinas/metabolismo , Peptidil Transferases/metabolismo , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Lipídeo A/metabolismo , Lipopolissacarídeos/genética , Testes de Sensibilidade Microbiana , Peptidoglicano Glicosiltransferase
15.
Gene ; 764: 145055, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32882332

RESUMO

Cyanobacteria are model photosynthetic prokaryotic organisms often used in biotechnology to produce biofuels including alcohols. The effect of alcohols on cyanobacterial cell physiology and specifically on membrane fluidity is poorly understood. Previous research on various primary aliphatic alcohols found that alcohols with a short hydrocarbon chain (C1-C3) do not affect expression of genes related to membrane physical state. In addition, less water-soluble alcohols with a hydrocarbon chain longer than C8 are found to have a reduced ability to reach cellular membranes hence do not drastically change membrane physical state or induce expression of stress-responsive genes. Therefore, hexan-1-ol (C6) is suggested to have the most profound effect on cyanobacterial membrane physical state. Here, we studied the effects of hexan-1-ol on the cyanobacterium Synechocystis sp. PCC 6803 transcriptome. The transcriptome data obtained is compared to the previously reported analysis of gene expression induced by benzyl alcohol and butan-1-ol. The set of genes whose expression is induced after exposure to all three studied alcohols is identified. The expression under alcohol stress for several general stress response operons is analyzed, and examples of antisense interactions of RNA are investigated.


Assuntos
Membrana Celular/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Hexanóis/toxicidade , Estresse Fisiológico/genética , Synechocystis/genética , 1-Butanol/toxicidade , Álcool Benzílico/toxicidade , Óperon/efeitos dos fármacos , Óperon/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA-Seq , Estresse Fisiológico/efeitos dos fármacos , Synechocystis/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
16.
Ecotoxicol Environ Saf ; 207: 111273, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32916524

RESUMO

Toxic pollutant (TP) detection in situ using analytical instruments or whole-cell biosensors is inconvenient. Designing and developing genetically coded biosensors in vitro for real-world TP detection is a promising alternative. However, because the bioactivity and stability of some key biomolecules are weakened in vitro, the response and regulation of reporter protein become difficult. Here, we established a genetically encoded biosensor in vitro with an arsenical resistance operon repressor (ArsR) and GFP reporter gene. Given that the wildtype ArsR did not respond to arsenic and activate GFP expression in vitro, we found, after screening, an evolved ArsR mutant ep3 could respond to arsenic and exhibited an approximately 3.4-fold fluorescence increase. Arsenic induced expression of both wildtype ArsR and ep3 mutant in vitro, however, only ep3 mutant regulated the expression of reporter gene. Furthermore, the effects of cell extracts, temperature, pH, incubation, and equilibrium time were investigated, and the equilibration of reaction mixtures for 30 min at 37 °C was found to be essential for in vitro arsenic detection prior to treatment with arsenic. Based on our data, we established a standard procedure for arsenic detection in vitro. Our results will facilitate the practical application of genetically encoded biosensors in TP monitoring.


Assuntos
Arsênico/análise , Técnicas Biossensoriais/métodos , Poluentes Ambientais/análise , Arsênico/metabolismo , Arsenicais/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Óperon/efeitos dos fármacos
17.
Sci Rep ; 10(1): 21975, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33319862

RESUMO

Acinetobacter baumannii (AB) is rising as a human pathogen of critical priority worldwide as it is the leading cause of chronic opportunistic infections in healthcare settings and the condition is ineradicable with antibiotic therapy. AB possesses the ability to form biofilm on abiotic as well as biotic surfaces which plays a major role in its pathogenesis and resistance in clinical settings. Hence, the demand for an alternative therapy to combat the biofilm-associated infections is increasing. The present study explored the antibiofilm potential of myrtenol, a bicyclic monoterpene present in various plants against reference and clinical strains of AB. Myrtenol (200 µg/mL) exhibited a strong antibiofilm activity without exerting any harmful effect on growth and metabolic viability of AB strains. Microscopic analyses confirmed the reduction in the biofilm thickness and surface coverage upon myrtenol treatment. Especially, myrtenol was found to be effective in disrupting the mature biofilms of tested AB strains. Furthermore, myrtenol inhibited the biofilm-associated virulence factors of AB strains such as extracellular polysaccharide, cell surface hydrophobicity, oxidant resistance, swarming and twitching motility. Transcriptional analysis unveiled the suppression of the biofilm-associated genes such as bfmR, csuA/B, bap, ompA, pgaA, pgaC, and katE by myrtenol. Notably, myrtenol improved the susceptibility of AB strains towards conventional antibiotics such as amikacin, ciprofloxacin, gentamicin and trimethoprim. Thus, the present study demonstrates the therapeutic potential of myrtenol against biofilm-associated infections of AB.


Assuntos
Acinetobacter baumannii/fisiologia , Acinetobacter baumannii/patogenicidade , Antibacterianos/farmacologia , Monoterpenos Bicíclicos/farmacologia , Biofilmes/efeitos dos fármacos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/crescimento & desenvolvimento , Aderência Bacteriana/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Modelos Biológicos , Movimento , Oxidantes/toxicidade , Polissacarídeos/farmacologia , Virulência/efeitos dos fármacos
18.
Nat Commun ; 11(1): 6430, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33353937

RESUMO

The trp operon of Chlamydia trachomatis is organized differently from other model bacteria. It contains trpR, an intergenic region (IGR), and the biosynthetic trpB and trpA open-reading frames. TrpR is a tryptophan-dependent repressor that regulates the major promoter (PtrpR), while the IGR harbors an alternative promoter (PtrpBA) and an operator sequence for the iron-dependent repressor YtgR to regulate trpBA expression. Here, we report that YtgR repression at PtrpBA is also dependent on tryptophan by regulating YtgR levels through a rare triple-tryptophan motif (WWW) in the YtgCR precursor. Inhibiting translation during tryptophan limitation at the WWW motif subsequently promotes Rho-independent transcription termination of ytgR, thereby de-repressing PtrpBA. Thus, YtgR represents an alternative strategy to attenuate trpBA expression, expanding the repertoire for trp operon attenuation beyond TrpL- and TRAP-mediated mechanisms described in other bacteria. Furthermore, repurposing the iron-dependent repressor YtgR underscores the fundamental importance of maintaining tryptophan-dependent attenuation of the trpRBA operon.


Assuntos
Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Ferro/metabolismo , Óperon/genética , Triptofano/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Chlamydia trachomatis/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Indóis/farmacologia , Modelos Biológicos , Regiões Promotoras Genéticas , Biossíntese de Proteínas/efeitos dos fármacos , Domínios Proteicos , RNA de Transferência de Triptofano/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(52): 33530-33539, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318202

RESUMO

Two-component systems (TCSs) in bacteria are molecular circuits that allow the perception of and response to diverse stimuli. These signaling circuits rely on phosphoryl-group transfers between transmitter and receiver domains of sensor kinase and response regulator proteins, and regulate several cellular processes in response to internal or external cues. Phosphorylation, and thereby activation, of response regulators has been demonstrated to occur by their cognate histidine kinases but also by low molecular weight phosphodonors such as acetyl phosphate and carbamoyl phosphate. Here, we present data indicating that the intermediates of the de novo syntheses of purines and histidine, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) and/or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-triphosphate (ZTP), activate the response regulator UvrY, by promoting its autophosphorylation at the conserved aspartate at position 54. Moreover, these Z nucleotides are shown to also activate the nonrelated response regulators ArcA, CpxR, RcsB, and PhoQ. We propose that ZMP and/or ZTP act as alarmones for a wide range of response regulators in vivo, providing a novel mechanism by which they could impact gene expression in response to metabolic cues.


Assuntos
Escherichia coli/metabolismo , Nucleotídeos/farmacologia , Transdução de Sinais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Mutação/genética , Fosfatos/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
20.
Proc Natl Acad Sci U S A ; 117(52): 33519-33529, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318204

RESUMO

Pseudomonas aeruginosa causes severe multidrug-resistant infections that often lead to bacteremia and sepsis. Physiologically relevant conditions can increase the susceptibility of pathogens to antibiotics, such as azithromycin (AZM). When compared to minimal-inhibitory concentrations (MICs) in laboratory media, AZM had a 16-fold lower MIC in tissue culture medium with 5% Mueller Hinton broth (MHB) and a 64-fold lower MIC in this tissue culture medium with 20% human serum. AZM also demonstrated increased synergy in combination with synthetic host-defense peptides DJK-5 and IDR-1018 under host-like conditions and in a murine abscess model. To mechanistically study the altered effects of AZM under physiologically relevant conditions, global transcriptional analysis was performed on P. aeruginosa with and without effective concentrations of AZM. This revealed that the arn operon, mediating arabinosaminylation of lipopolysaccharides and related regulatory systems, was down-regulated in host-like media when compared to MHB. Inactivation of genes within the arn operon led to increased susceptibility of P. aeruginosa to AZM and great increases in synergy between AZM and other antimicrobial agents, indicating that dysregulation of the arn operon might explain increased AZM uptake and synergy in host-like media. Furthermore, genes involved in central and energy metabolism and ribosome biogenesis were dysregulated more in physiologically relevant conditions treated with AZM, likely due to general changes in cell physiology as a result of the increased effectiveness of AZM in these conditions. These data suggest that, in addition to the arn operon, there are multiple factors in host-like environments that are responsible for observed changes in susceptibility.


Assuntos
Azitromicina/farmacologia , Meios de Cultura/farmacologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sinergismo Farmacológico , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Óperon/genética , Peptídeos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Soro
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...