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1.
Front Microbiol ; 15: 1357708, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38435690

RESUMO

Pseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains.

2.
Trends Microbiol ; 28(4): 315-326, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31932141

RESUMO

Cystic fibrosis (CF) patients are at particular risk of infection by microorganisms that are resistant to several antibiotics. About 3% of CF patients are colonized by Burkholderia cenocepacia, and this represents a major threat because of its intrinsic high level of drug resistance and the lack of a safe and effective treatment protocol. The development of anti-Burkholderia vaccines is a valuable and complementary approach, but only a few studies have been reported to date. In this review we discuss recent advances in the vaccine field and how new technologies, including structural reverse vaccinology, could drive the design of an effective vaccine against B. cenocepacia for use in preventive and therapeutic applications.


Assuntos
Antibacterianos/farmacologia , Infecções por Burkholderia/tratamento farmacológico , Infecções por Burkholderia/prevenção & controle , Burkholderia cenocepacia/efeitos dos fármacos , Vacinas/farmacologia , Animais , Burkholderia cenocepacia/genética , Fibrose Cística/microbiologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Pulmão/microbiologia , Microbiota , Fatores de Virulência
3.
Artigo em Inglês | MEDLINE | ID: mdl-30297366

RESUMO

To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Burkholderia cenocepacia/genética , Proteínas do Citoesqueleto/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Burkholderia/tratamento farmacológico , Infecções por Burkholderia/microbiologia , Burkholderia cenocepacia/efeitos dos fármacos , Burkholderia cenocepacia/isolamento & purificação , Caenorhabditis elegans/microbiologia , Fibrose Cística/microbiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Técnicas de Silenciamento de Genes , Genes Essenciais , Proteínas de Fluorescência Verde/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Mutação
4.
Front Pharmacol ; 9: 836, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30108505

RESUMO

Quorum sensing (QS) is a bacterial intercellular communication process which controls the production of major virulence factors, such as proteases, siderophores, and toxins, as well as biofilm formation. Since the inhibition of this pathway reduces bacterial virulence, QS is considered a valuable candidate drug target, particularly for the treatment of opportunistic infections, such as those caused by Burkholderia cenocepacia in cystic fibrosis patients. Diketopiperazine inhibitors of the acyl homoserine lactone synthase CepI have been recently described. These compounds are able to impair the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm, being also active in a Caenorhabditis elegans infection model. However, the precise mechanism of action of the compounds, as well as their effect on the cell metabolism, fundamental for candidate drug optimization, are still not completely defined. Here, we performed a proteomic analysis of B. cenocepacia cells treated with one of these inhibitors, and compared it with a cepI deleted strain. Our results demonstrate that the effects of the compound are similar to the deletion of cepI, clearly confirming that these molecules function as inhibitors of the acyl homoserine lactone synthase. Moreover, to deepen our knowledge about the binding mechanisms of the compound to CepI, we exploited previously published in silico structural insights about this enzyme structure and validated different candidate binding pockets on the enzyme surface using site-directed mutagenesis and biochemical analyses. Our experiments identified a region near the predicted S-adenosylmethionine binding site critically involved in interactions with the inhibitor. These results could be useful for future structure-based optimization of these CepI inhibitors.

5.
Front Microbiol ; 8: 1592, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28878751

RESUMO

Burkholderia cenocepacia is an opportunistic pathogen particularly dangerous for cystic fibrosis (CF) patients. It can cause a severe decline in CF lung function possibly developing into a life-threatening systemic infection known as cepacia syndrome. Antibiotic resistance and presence of numerous virulence determinants in the genome make B. cenocepacia extremely difficult to treat. Better understanding of its resistance profiles and mechanisms is crucial to improve management of these infections. Here, we present the clinical distribution of B. cenocepacia described in the last 6 years and methods for identification and classification of epidemic strains. We also detail new antibiotics, clinical trials, and alternative approaches reported in the literature in the last 5 years to tackle B. cenocepacia resistance issue. All together these findings point out the urgent need of new and alternative therapies to improve CF patients' life expectancy.

6.
Sci Rep ; 6: 32487, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27580679

RESUMO

Burkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Burkholderia cenocepacia/efeitos dos fármacos , Dicetopiperazinas/farmacologia , Inibidores Enzimáticos/farmacologia , Ligases/antagonistas & inibidores , Percepção de Quorum/efeitos dos fármacos , 4-Butirolactona/análogos & derivados , 4-Butirolactona/antagonistas & inibidores , 4-Butirolactona/biossíntese , 4-Butirolactona/genética , Animais , Antibacterianos/síntese química , Biofilmes/crescimento & desenvolvimento , Burkholderia cenocepacia/enzimologia , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/patogenicidade , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Dicetopiperazinas/síntese química , Inibidores Enzimáticos/síntese química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Humanos , Ligases/genética , Ligases/metabolismo , Percepção de Quorum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Virulência
7.
Biochemistry ; 55(23): 3241-50, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27198181

RESUMO

Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes that are important for bacterial virulence, such as biofilm formation and protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule in the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Burkholderia cenocepacia/metabolismo , Ácidos Graxos/metabolismo , Percepção de Quorum , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Cromatografia Gasosa-Espectrometria de Massas , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato
8.
Front Microbiol ; 6: 815, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26300878

RESUMO

Burkholderia cenocepacia is a major concern for people suffering from cystic fibrosis as it contributes to serious respiratory tract infections. The lack of drugs effective against this opportunistic pathogen, along with the high level of resistance to multiple antibiotics, render the treatment of these infections particularly difficult. Here a new compound, belonging to the 2,1,3-benzothiadiazol-5-yl family (10126109), with a bactericidal effect and a minimal inhibitory concentration (MIC) of 8 µg/ml against B. cenocepacia, is described. The compound is not cytotoxic and effective against B. cenocepacia clinical isolates and members of all the known B. cepacia complex species. Spontaneous mutants resistant to 10126109 were isolated and mutations in the MerR transcriptional regulator BCAM1948 were identified. In this way, a mechanism of resistance to this new molecule was described, which relies on the overexpression of the RND-9 efflux pump. Indeed, rnd-9 overexpression was confirmed by quantitative reverse transcription PCR, and RND-9 was identified in the membrane fractions of the mutant strains. Moreover, the increase in the MIC values of different drugs in the mutant strains, together with complementation experiments, suggested the involvement of RND-9 in the efflux of 10126109, thus indicating again the central role of efflux transporters in B. cenocepacia drug resistance.

9.
Antimicrob Agents Chemother ; 58(12): 7424-9, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25267676

RESUMO

Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested.


Assuntos
Sequência de Bases , Biofilmes/efeitos dos fármacos , Burkholderia cenocepacia/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Genes MDR , Plâncton/efeitos dos fármacos , Deleção de Sequência , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes/crescimento & desenvolvimento , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/crescimento & desenvolvimento , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Óperon , Plâncton/genética , Plâncton/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tobramicina/farmacologia
10.
Antimicrob Agents Chemother ; 58(4): 2415-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24395233

RESUMO

The discovery of new compounds that are able to inhibit the growth of Burkholderia cenocepacia is of primary importance for cystic fibrosis patients. Here, the mechanism of resistance to a new pyridine derivative already shown to be effective against Mycobacterium tuberculosis and to have good activity toward B. cenocepacia was investigated. Increased expression of a resistance-nodulation-cell division (RND) efflux system was detected in the resistant mutants, thus confirming their important roles in B. cenocepacia antibiotic resistance.


Assuntos
Antibacterianos/farmacologia , Antituberculosos/farmacologia , Burkholderia cenocepacia/efeitos dos fármacos , Piridinas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Farmacorresistência Bacteriana/genética
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