RESUMO
Gram-negative bacteria expressing class A ß-lactamases pose a serious health threat due to their ability to inactivate all ß-lactam antibiotics. The acyl-enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A ß-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved ß-lactamase inhibitor avibactam to trap the acyl-enzyme complex of class A ß-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKa) compared with acyl-enzyme complexes with ß-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKa to be â¼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl-enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.
Assuntos
Compostos Azabicíclicos/química , Compostos Azabicíclicos/farmacologia , Prótons , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , beta-Lactamases/efeitos dos fármacos , Acilação , Compostos Azabicíclicos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos dos fármacos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Inibidores de beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
In Staphylococcus aureus, the two-component system SaeRS is responsible for regulating various virulence factors essential for the success of this pathogen. SaeRS can be stimulated by neutrophil-derived products but has also recently been shown to be inactivated by the presence of free fatty acids. A mechanism for how fatty acids negatively impacts SaeRS has not been described. We found that unsaturated fatty acids, as well as fatty acids not commonly found in Staphylococcal membranes, prevent the activation of SaeRS at a lower concentration than their saturated counterparts. These fatty acids can negatively impact SaeRS without altering the respiratory capacity of the bacterium. To uncover a potential mechanism for how fatty acids impact SaeRS function/activity, we utilized a naturally occurring point mutation found in S. aureus as well as chimeric SaeS proteins. Using these tools, we identified that the native transmembrane domains of SaeS dictate the transcriptional response to fatty acids in S. aureus. Our data support a model where free fatty acids alter the activity of the two-component system SaeRS directly through the sensor kinase SaeS and is dependent on the transmembrane domains of the protein.
Assuntos
Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Ácidos Graxos/farmacologia , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Respiração , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , VirulênciaRESUMO
Pathogenic Leptospira spp. are the causative agents of the waterborne zoonotic disease leptospirosis. Leptospira are challenged by numerous adverse conditions, including deadly reactive oxygen species (ROS), when infecting their hosts. Withstanding ROS produced by the host innate immunity is an important strategy evolved by pathogenic Leptospira for persisting in and colonizing hosts. In L. interrogans, genes encoding defenses against ROS are repressed by the peroxide stress regulator, PerR. In this study, RNA sequencing was performed to characterize both the L. interrogans response to low and high concentrations of hydrogen peroxide and the PerR regulon. We showed that Leptospira solicit three main peroxidase machineries (catalase, cytochrome C peroxidase and peroxiredoxin) and heme to detoxify oxidants produced during peroxide stress. In addition, canonical molecular chaperones of the heat shock response and DNA repair proteins from the SOS response were required for Leptospira recovering from oxidative damage. Identification of the PerR regulon upon exposure to H2O2 allowed to define the contribution of this regulator in the oxidative stress response. This study has revealed a PerR-independent regulatory network involving other transcriptional regulators, two-component systems and sigma factors as well as non-coding RNAs that putatively orchestrate, in concert with PerR, the oxidative stress response. We have shown that PerR-regulated genes encoding a TonB-dependent transporter and a two-component system (VicKR) are involved in Leptospira tolerance to superoxide. This could represent the first defense mechanism against superoxide in L. interrogans, a bacterium lacking canonical superoxide dismutase. Our findings provide an insight into the mechanisms required by pathogenic Leptospira to overcome oxidative damage during infection-related conditions. This will participate in framing future hypothesis-driven studies to identify and decipher novel virulence mechanisms in this life-threatening pathogen.
Assuntos
Peróxido de Hidrogênio/farmacologia , Leptospira/patogenicidade , Estresse Oxidativo/efeitos dos fármacos , Peróxidos/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Ferro/metabolismo , Leptospira/efeitos dos fármacos , Leptospira interrogans/efeitos dos fármacos , Leptospira interrogans/genética , Leptospirose/genética , Chaperonas Moleculares/metabolismo , Estresse Oxidativo/fisiologia , Virulência/efeitos dos fármacos , Virulência/fisiologiaAssuntos
Anticorpos/uso terapêutico , Vacinas contra COVID-19 , Biologia Celular , Biologia do Desenvolvimento , Nariz Eletrônico , Espectrometria de Massas/instrumentação , Neurociências , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/imunologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Bioimpressão/tendências , COVID-19/epidemiologia , COVID-19/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/provisão & distribuição , Biologia Celular/instrumentação , Biologia Celular/tendências , Biologia do Desenvolvimento/métodos , Biologia do Desenvolvimento/tendências , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Holografia/tendências , Humanos , Imunoglobulina E/química , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina E/uso terapêutico , Canais Iônicos/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/efeitos da radiação , Camundongos , Microscopia/instrumentação , Microscopia/tendências , Sondas Moleculares/análise , Neoplasias/tratamento farmacológico , Neurociências/métodos , Neurociências/tendências , Optogenética/tendências , Análise de Célula Única , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Rationale: Bedaquiline and delamanid offer the possibility of more effective and less toxic treatment for multidrug-resistant (MDR) tuberculosis (TB). With this treatment, however, some patients remain at high risk for an unfavorable treatment outcome. The endTB Observational Study is the largest multicountry cohort of patients with rifampin-resistant TB or MDR-TB treated in routine care with delamanid- and/or bedaquiline-containing regimens according to World Health Organization guidance.Objectives: We report the frequency of sputum culture conversion within 6 months of treatment initiation and the risk factors for nonconversion.Methods: We included patients with a positive baseline culture who initiated a first endTB regimen before April 2018. Two consecutive negative cultures collected 15 days or more apart constituted culture conversion. We used generalized mixed models to derive marginal predictions for the probability of culture conversion in key subgroups.Measurements and Main Results: A total of 1,109 patients initiated a multidrug treatment containing bedaquiline (63%), delamanid (27%), or both (10%). Of these, 939 (85%) experienced culture conversion within 6 months. In adjusted analyses, patients with HIV had a lower probability of conversion (0.73; 95% confidence interval [CI], 0.62-0.84) than patients without HIV (0.84; 95% CI, 0.79-0.90; P = 0.03). Patients with both cavitary disease and highly positive sputum smear had a lower probability of conversion (0.68; 95% CI, 0.57-0.79) relative to patients without either (0.89; 95% CI, 0.84-0.95; P = 0.0004). Hepatitis C infection, diabetes mellitus or glucose intolerance, and baseline resistance were not associated with conversion.Conclusions: Frequent sputum conversion in patients with rifampin-resistant TB or MDR-TB who were treated with bedaquiline and/or delamanid underscores the need for urgent expanded access to these drugs. There is a need to optimize treatment for patients with HIV and extensive disease.
Assuntos
Antituberculosos/uso terapêutico , Proteínas de Bactérias/efeitos dos fármacos , Diarilquinolinas/uso terapêutico , Nitroimidazóis/uso terapêutico , Oxazóis/uso terapêutico , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or "binding boxes" within the PAP sequence. In the important human pathogen Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a predictive model of pump-contacts that could be exploited in the future for the development of targeted therapeutics and efflux pump inhibitors.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Infecções Bacterianas/tratamento farmacológico , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Feminino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos BALB C , Periplasma/efeitos dos fármacos , Periplasma/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/metabolismoRESUMO
Despite sharing conserved substrate-binding residues, members of 3-hydroxyisobutyrate dehydrogenase (HIBADH) superfamily show remarkable differences in substrate preference. Cysteine residues were identified within a radius of 6 Å surrounding both the active site and the substrate entry site of HIBADH enzyme from Mycobacterium tuberculosis (MtHIBADH). Chemical modification with thiol-modifying reagents, pCMB and DTNB, abrogated the dehydrogenase activity of the enzyme. The loss in activity followed pseudo-first-order kinetics as a function of the concentration of pCMB. S-HIBA (substrate) binding provided partial protection, while NAD (cofactor) binding provided ~70% protection from thiol-modifying reagent. Site-directed mutagenesis of cysteine residues present in the MtHIBADH enzyme identified the indispensable role of Cys-210 residue, located at C-terminal domain, for its dehydrogenase activity. Cys-210 mutation to serine reduced the dehydrogenase activity by ~2-fold while mutation to alanine strikingly reduced the activity by ~140-fold. C210A mutation did not perturb the state of oligomerization of the enzyme but perturbed the secondary structure content. Structural analysis revealed the involvement of Cys-210 residue in inter-chain interaction with Gln-178, which acts as hydrogen bond donor and coordinates with Cys-210 and Gly-208 of the adjacent subunit. The data demonstrate a critical role of Cys-210 residue in maintaining the conformation and rigidity of loop composed of substrate-interacting residues involved in the entry of S-HIBA substrate in MtHIBADH.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína/química , Alanina/química , Oxirredutases do Álcool/química , Oxirredutases do Álcool/efeitos dos fármacos , Oxirredutases do Álcool/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Catálise , Dinitrobenzenos/farmacologia , Ligação de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , NAD/metabolismo , Conformação Proteica , Proteínas Recombinantes/metabolismo , Serina/química , Relação Estrutura-Atividade , Especificidade por Substrato , Ácido p-Cloromercurobenzoico/farmacologiaRESUMO
Urease as a potential target of antimicrobial drugs has received considerable attention given its versatile roles in microbial infection. Development of effective urease inhibitors, however, is a significant challenge due to the deeply buried active site and highly specific substrate of a bacterial urease. Conventionally, urease inhibitors are designed by either targeting the active site or mimicking substrate of urease, which is not efficient. Up to now, only one effective inhibitor-acetohydroxamic acid (AHA)-is clinically available, but it has adverse side effects. Herein, we demonstrate that a clinically used drug, colloidal bismuth subcitrate, utilizes an unusual way to inhibit urease activity, i.e., disruption of urease maturation process via functional perturbation of a metallochaperone, UreG. Similar phenomena were also observed in various pathogenic bacteria, suggesting that UreG may serve as a general target for design of new types of urease inhibitors. Using Helicobacter pylori UreG as a showcase, by virtual screening combined with experimental validation, we show that two compounds targeting UreG also efficiently inhibited urease activity with inhibitory concentration (IC)50 values of micromolar level, resulting in attenuated virulence of the pathogen. We further demonstrate the efficacy of the compounds in a mammalian cell infection model. This study opens up a new opportunity for the design of more effective urease inhibitors and clearly indicates that metallochaperones involved in the maturation of important microbial metalloenzymes serve as new targets for devising a new type of antimicrobial drugs.
Assuntos
Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Urease/antagonistas & inibidores , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/fisiologia , Proteínas de Transporte/fisiologia , Domínio Catalítico , Helicobacter pylori/metabolismo , Metalochaperonas/farmacologia , Proteínas de Ligação a Fosfato , Urease/fisiologia , VirulênciaRESUMO
Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism. Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5' exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5' exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0-8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0-6.5. The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.
Assuntos
Proteínas de Bactérias/efeitos da radiação , DNA Polimerase I/efeitos da radiação , Deinococcus/genética , Proteínas Recombinantes/efeitos da radiação , Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Sequência de Bases , DNA Polimerase I/química , DNA Polimerase I/genética , Reparo do DNA , DNA Bacteriano/genética , Deinococcus/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-AtividadeRESUMO
Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.
Assuntos
Peptídeo Hidrolases/metabolismo , Periodontite/microbiologia , Inibidores de Proteases/farmacologia , Tannerella forsythia/enzimologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Descoberta de Drogas , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Acoplamento Molecular , Estrutura Molecular , Peptídeo Hidrolases/efeitos dos fármacos , Inibidores de Proteases/química , Tannerella forsythia/isolamento & purificação , TemperaturaRESUMO
The unique architecture of the mycobacterial cell envelope plays an important role in Mycobacterium tuberculosis (Mtb) pathogenesis. A critical protein in cell envelope biogenesis in mycobacteria, required for transport of precursors, trehalose monomycolates (TMMs), is the Mycobacterial membrane protein large 3 (MmpL3). Due to its central role in TMM transport, MmpL3 has been an attractive therapeutic target and a key target for several preclinical agents. In 2019, the first crystal structures of the MmpL3 transporter and its complexes with lipids and inhibitors were reported. These structures revealed several unique structural features of MmpL3 and provided invaluable information on the mechanism of TMM transport. This review aims to highlight the recent advances made in the function of MmpL3 and summarises structural findings. The overall goal is to provide a mechanistic perspective of MmpL3-mediated lipid transport and inhibition, and to highlight the prospects for potential antituberculosis therapies.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Desenvolvimento de Medicamentos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico , Lipídeos/química , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Ácidos Micólicos/metabolismo , Conformação ProteicaRESUMO
Rapid and accurate diagnosis of bacterial infections resistant to multiple antibiotics requires development of new bio-sensors for differentiated detection of multiple targets. This work demonstrates 7x multiplexed detection for antibiotic-resistance bacterial screening on an optofluidic platform. We utilize spectrally multiplexed multi-spot excitation for simultaneous detection of nucleic acid strands corresponding to bacterial targets and resistance genes. This is enabled by multi-mode interference (MMI) waveguides integrated in an optofluidic device. We employ a combinatorial three-color labeling scheme for the nucleic acid assays to scale up their multiplexing capability to seven different nucleic acids, representing three species and four resistance genes.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Enterobacter aerogenes/genética , Escherichia coli/genética , Klebsiella pneumoniae/genética , Técnicas de Amplificação de Ácido Nucleico , Antibacterianos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Enterobacter aerogenes/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Testes de Sensibilidade Microbiana , Dispositivos ÓpticosRESUMO
Staphylococcus aureus expresses many Microbial Surface Recognizing Adhesive Matrix Molecules (MSCRAMM's) to recognize host extracellular matrix (ECM) molecules to initiate colonization. The MSCRAMM, fibronectin binding protein A (FnBPA), is an important adhesin for S. aureus infection. FnBPA also binds with fibrinogen (Fg) by using a unique ligand binding mechanism called dock, lock and latch. Nanoparticles, especially nanosilver particles have been widely used in a variety of biomedical applications which includes disease diagnosis and treatment, drug delivery and implanted medical device coating. In a biological system, when protein molecules encounter nanoparticle, they can be absorbed onto its surface which results in the formation of protein corona. In the present study, we have analysed the fibrinogen binding ability of rFnBPA(189-512) in the presence of silver nanoparticles by employing techniques like gel shift assay, Western blot, size exclusion chromatography, enzyme-linked immunosorbent assay, bio-layer interferometry and circular dichroism spectroscopy. The results indicate that rFnBPA(189-512) is unable to bind to Fg in the presence of a nanoparticle. This could be due to the inaccessibility of the Fg binding site and conformational change in rFnBPA(189-512). With nanoparticles, rFnBPA(189-512) undergoes significant structural changes as the ß-sheet content has drastically reduced to 10% from the initial 60% at higher concentration of the nanoparticle. Pathogenic bacteria interact with its surrounding environment through their surface molecules which includes MSCRAMMs. Therefore MSCRAMMs play an important role when bacteria encounter nanoparticles. The results of the present study suggest that the orientation of the protein during the absorption on the surface of a nanoparticle as well as the concentration of the nanoparticle, will dictate the function of the absorbed protein and in this case the Fg binding property of rFnBPA(189-512).
Assuntos
Adesinas Bacterianas , Aderência Bacteriana/efeitos dos fármacos , Nanopartículas Metálicas , Staphylococcus aureus/metabolismo , Adesinas Bacterianas/biossíntese , Adesinas Bacterianas/efeitos dos fármacos , Adesinas Bacterianas/isolamento & purificação , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Fibrinogênio/efeitos dos fármacos , Fibrinogênio/metabolismo , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Ligação Proteica , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
Arrabidaea brachypoda is a native shrub of the Brazilian Cerrado widely used in the folk medicine for treatment of renal diseases and articular pains. This study aimed to, first, evaluate the antimicrobial activity of both extracts and isolated molecules Brachydins BR-A and BR-B obtained from the flowers of A. brachypoda against Staphylococcus aureus, Escherchia coli and Candida albicans species. A second objective was to investigate if these natural products were able to potentiate the Norfloxacin activity against the strain Staphylococcus aureus SA1199-B that overexpress the norA gene encoding the NorA efflux pump. Extracts and isolated compounds were analyzed by HPLC-PDA and LC-ESI-MS respectively. Minimal inhibitory concentrations of Norfloxacin or Ethidium Bromide (EtBr) were determined in the presence or absence of ethanolic extract, dichloromethane fraction, as well as BR-A or BR-B by microdilution method. Only BR-B showed activity against Candida albicans. Addition of ethanolic extract, dichloromethane fraction or BR-B to the growth media at sub-inhibitory concentrations enhanced the activity of both Norfloxacin and EtBr against S. aureus SA1199-B, indicating that these natural products and its isolated compound BR-B were able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. Moreover, BR-B inhibited the EtBr efflux in the SA1199-B strain confirming that it is a NorA inhibitor. Isolated BR-B was able to inhibit an important mechanism of multidrug-resistance very prevalent in S. aureus strains, thus its use in combination with Norfloxacin could be considered as an alternative for the treatment of infections caused by S. aureus strains overexpressing norA.
Assuntos
Proteínas de Bactérias/efeitos dos fármacos , Bignoniaceae/metabolismo , Flavonoides/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Candida albicans/efeitos dos fármacos , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Etídio/farmacologia , Flavonoides/isolamento & purificação , Fluoroquinolonas/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Extratos Vegetais/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismoRESUMO
Pneumolysin (PLY), a pore-forming cytotoxin and a major virulence determinant, is a member of the cholesterol-dependent cytolysin (CDC) family and essential for promoting Streptococcus pneumoniae (S.pneumoniae) infection. Due to the action characteristics of hemolysin itself, the pneumolysin released after killing bacteria with conventional antibiotics still has the ability to damage host cells; therefore, drug treatments directly inhibiting hemolysin activity are the most effective. Hemolysis assays were used to confirm that quercetin can inhibit the activity of PLY, protecting cells in vitro, and an oligomerization assay was used to determine the mechanism of quercetin to suppress PLY activity. Live/Dead testing, lactate dehydrogenase (LDH) release analysis and a murine model of endonasal pulmonary infection were used to explore the capability of quercetin to protect cells and mice from S. pneumoniae-mediated damage in vivo and in vitro. The results indicated that quercetin significantly reduced PLY-induced hemolytic activity and cytotoxicity via repressing the formation of oligomers. In addition, treatment with quercetin can reduce PLY-mediated cell injury, improve the survival rate of mice infected with a lethal dose of S. pneumoniae, alleviate the pathological damage of lung tissue and inhibit the release of cytokines (IL-1ß and TNF-α) in bronchoalveolar lavage fluid. Considering the importance of these events in antimicrobial resistant S. pneumoniae pathogenesis, our results indicated that quercetin may be a novel potential drug candidate for the treatment of clinical pneumococcal infections.
Assuntos
Infecções Pneumocócicas/tratamento farmacológico , Quercetina/farmacologia , Animais , Proteínas de Bactérias/efeitos dos fármacos , Linhagem Celular , Hemólise/efeitos dos fármacos , Interleucina-1beta/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/metabolismo , Estreptolisinas , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Virulência/efeitos dos fármacosRESUMO
The use of traditional foods and beverages or their bioactive compounds as anti-virulence agents is a new alternative method to overcome the increased global emergence of antimicrobial resistance in enteric pathogens. In the present study, we investigated the anti-virulence activity of a polyphenolic fraction previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against Vibrio cholerae O1. The isolated fraction was mainly composed of the polyphenols catechin and isorhamnetin. The fraction, the individual polyphenols and the combination of the individual polyphenols significantly inhibited bacterial swarming motility and expression of flagellar regulatory genes motY and flaC, even at sub-inhibitory concentrations. The polyphenolic compounds also decreased bacterial protease secretion and mucin penetration in vitro. In vivo study revealed that the polyphenolic fraction significantly inhibited V. cholerae induced fluid accumulation in the rabbit ileal loop model and intestinal colonization in suckling mice model. Therefore, the anti-virulence activity of the Kombucha polyphenolic fraction involved inhibition of motility and protease secretion of V. cholerae, thus preventing bacterial penetration through the mucin layer as well as fluid accumulation and bacterial colonization in the intestinal epithelial cells. The overall results implied that Kombucha might be considered as a potential alternative source of anti-virulence polyphenols against V. cholerae. To the best of our knowledge, this is the first report on the anti-virulence activity of Kombucha, mostly attributed to its polyphenolic content.
Assuntos
Chá de Kombucha , Polifenóis/farmacologia , Vibrio cholerae/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Catequina/farmacologia , Movimento Celular/efeitos dos fármacos , Cólera/tratamento farmacológico , Expressão Gênica/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/microbiologia , Camundongos , Peptídeo Hidrolases/efeitos dos fármacos , Extratos Vegetais/farmacologia , Quercetina/análogos & derivados , Quercetina/farmacologia , Coelhos , Vibrio cholerae/patogenicidade , Virulência/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Helicobacter pylori (H. pylori) infection is highly prevalent, and has developed antimicrobial resistance to virtually all existing antibiotics. Currently, treatment of H. pylori infection (involving proton pump inhibitors and broad-spectrum antibiotics) is suboptimal, with high failure rates. Thus, there is a pressing need to develop new anti-H. pylori therapies. Cbf-K16, a cathelicidin-like antimicrobial peptide, presented broad antimicrobial activity during our previous research. This study further evaluated the therapeutic potential and the mode of action underlying Cbf-K16 against clarithromycin- and amoxicillin-resistant H. pylori SS1. The MIC and MBC of Cbf-K16 against the tested H. pylori were 16 and 32⯵g/ml, respectively, and its killing kinetics was time-dependent, reflecting the thorough elimination of drug-resistant bacteria within 24â¯h. This peptide also protected H. pylori-infected gastric epithelial cells (GES-1) from death by reducing the cell supernatant and intracellular bacterial counts by 1.9 and 2.9-log10 units, respectively. These data indicated the powerful antimicrobial effects of Cbf-K16in vitro. Meanwhile, notable antimicrobial activity in the mouse gastritis model was observed, with decreasing bacterial counts by 3.9-log10 units in stomach tissues and Cbf-K16 could effectively suppress the secretion of inflammatory cytokine IL-8. For its mode of action, Cbf-K16 not only neutralized the negative potential and increased the membrane uptake of NPN and PI by 78.5% and 85.1%, respectively, but also bound to genomic DNA, which in turn downregulated the expression of adhesion genes (alpA and alpB) and virulence gene (cagA), indicating its effective activities on membrane disruption, DNA-binding and gene expression. The data above demonstrated that Cbf-K16 possessed effective antimicrobial and anti-inflammatory activities and downregulated the expression of adhesion- and cytotoxin-associated genes of drug-resistant H. pylori SS1, making it a potential candidate for anti-infective therapy.
Assuntos
Adesinas Bacterianas/efeitos dos fármacos , Catelicidinas/farmacologia , Infecções por Helicobacter , Helicobacter pylori/efeitos dos fármacos , Interleucina-8/efeitos dos fármacos , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Antígenos de Bactérias/efeitos dos fármacos , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Farmacorresistência Bacteriana , Genes Bacterianos , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Humanos , Interleucina-8/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Virulência/efeitos dos fármacos , Virulência/genéticaRESUMO
Antibiotic resistance is a growing global challenge to public health. Polymyxin is considered to be the last-resort antibiotic against most gram-negative bacteria. Recently, discoveries of a plasmid-mediated, transferable mobilized polymyxin resistance gene (mcr-1) in many countries have heralded the increased threat of the imminent emergence of pan-drug-resistant super bacteria. MCR-1 is an inner membrane protein that enables bacteria to develop resistance to polymyxin by transferring phosphoethanolamine to lipid A. However, the mechanism associated with polymyxin resistance has yet to be elucidated, and few drugs exist to address this issue. Here, we review our current understanding regarding MCR-1 and small molecule inhibitors to provide a detailed enzymatic mechanism of MCR-1 and the associated implications for drug design.
Assuntos
Proteínas de Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/efeitos dos fármacos , Polimixinas/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Colistina/química , Colistina/farmacologia , Desenho de Fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos , Plasmídeos/efeitos dos fármacos , Polimixinas/químicaRESUMO
Considering the emergence of multidrug resistance (MDR) in prevalent human pathogen, Mycobacterium tuberculosis (MTB), there is parallel spurt in development of novel strategies aimed to disrupt MDR. The cell envelope of MTB comprises a wealth of lipid moieties contributing towards long-term survival of pathogen that could be exploited as efficient antitubercular target owing to advancements made in mass spectrometry-based lipidomics technology. This study aimed to utilize the lipidomics approach to unveil several lipid associated changes in response to natural antimycobacterial compound vanillin (Van) in Mycobacterium smegmatis, a surrogate for MTB. Lipidomic analyses revealed that that Van alters the composition of fatty acid (FA), glycerolipid (GL), glycerophospholipid (GP), and saccharolipids (SL). Furthermore, Van leads to potentiation of ampicillin and displayed additive effect. The differential expressions of various lipid biosynthetic pathway genes by RT-PCR corroborated with the lipidomics data. Lastly, we demonstrated enhanced survival of Mycobacterium-infected Caenorhabditis elegans model in presence of Van. Thus, lipidomics approach provided detailed insight into mechanisms of membrane disruption by Van in Mycobacterium smegmatis. Our work offers the basis of further understanding the regulation of lipid homeostasis in MTB so that better therapeutic targets could be identified to combat MDR.
Assuntos
Benzaldeídos/farmacologia , Membrana Celular/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Mycobacterium smegmatis/efeitos dos fármacos , Antituberculosos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Parede Celular/química , Parede Celular/efeitos dos fármacos , Ácidos Graxos/metabolismo , Glicerofosfolipídeos/metabolismo , Glicolipídeos/metabolismo , Humanos , Lipidômica/métodos , Mycobacterium tuberculosis/efeitos dos fármacosRESUMO
The high frequency and incidence of foodborne outbreaks related to fresh vegetables consumption is a major public health concern and an economic burden worldwide. This study evaluated the effect of individual and combined application of ultrasound (40 kHz, 100 W) and ozone on the inactivation of foodborne Escherichia coli and Salmonella, as well as their impact on cabbage color and vitamin C content. Plate count, scanning electron microscopy (SEM), and flow cytometry (FCM) following single or double staining with carboxyfluorescein diacetate and (or) propidium iodide were used to determine bacterial inactivation parameters, such as cell culturability, membrane integrity, intracellular enzyme activity, and injured and dead cells. The results of FCM and SEM showed that ultrasound treatment affected bacteria mainly by acting on the cell membrane and inactivating intracellular esterase, which resulted in bacterial death. Furthermore, when combined with ozone at 1.5 mg/L, the maximum reduction of bacterial populations was observed at 8 min with no damage on the surface of treated leaves. Therefore, fresh products sanitization using a combination of ultrasound and ozone has the potential to be an alternative for maintaining the color and vitamin C content of green cabbage.