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1.
Int J Nanomedicine ; 15: 5147-5163, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764942

RESUMO

Background: In the last decades, nosocomial infections caused by drug-resistant Pseudomonas aeruginosa became a common problem in healthcare facilities. Antibiotics are becoming less effective as new resistant strains appear. Therefore, the development of novel enhanced activity antibacterial agents becomes very significant. A combination of nanomaterials with different physical and chemical properties enables us to generate novel multi-functional derivatives. In this study, graphene oxide and polyvinylpyrrolidone-stabilized silver nanoparticles hybrid nanocomposite (GO-Ag HN) were synthesized. The relation between antibiotic resistance and GO-Ag HN potential toxicity to clinical P. aeruginosa strains, their antibiotic resistance, and molecular mechanisms were assessed. Methods: Chemical state, particle size distribution, and morphology of synthesized GO-Ag NH were investigated using spectroscopy and microscopy techniques (UV-Vis, FTIR, XPS, TEM, SEM, AFM). Broad-spectrum antibiotic resistance of P. aeruginosa strains was determined using E-test. Antibiotic resistance genes were identified using polymerase chain reaction (PCR). Results: In this study, the toxicity of the GO-Ag NH to the isolated clinical P. aeruginosa strains has been investigated. A high antibiotic resistance level (92%) was found among P. aeruginosa strains. The most prevalent antibiotic resistance gene among tested strains was the AMPC beta-lactamase gene (65.6%). UV-vis, FTIR, and XPS studies confirmed the formation of the silver nanoparticles on the GO nanosheets. The functionalization process occurred through the interaction between Ag nanoparticles, GO, and polyvinylpyrrolidone used for nanoparticle stabilization. SEM analysis revealed that GO nanosheets undergo partial fragmentation during hybrid nanocomposite preparation, which remarkably increases the number of sharp edges and their mediated cutting effect. TEM analysis showed that GO-Ag HN spherical Ag nanoparticles mainly 9-12 nm in size were irregularly precipitated on the GO nanosheet surface. A higher density of Ag NPs was observed in the sheets' wrinkles, corrugations, and sharp edges. This hybrid nanocomposite poses enhanced antibacterial activity against carbapenem-resistant P. aeruginosa strains through a possible synergy between toxicity mechanisms of GO nanosheets and Ag nanoparticles. With incubation time increasing up to 10 minutes, the survival of P. aeruginosa decreased significantly. Conclusion: A graphene oxide and silver nanoparticles hybrid composite has been shown to be a promising material to control nosocomial infections caused by bacteria strains resistant to most antibiotics.


Assuntos
Farmacorresistência Bacteriana/genética , Grafite/química , Grafite/farmacologia , Nanopartículas Metálicas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Prata/química , Antibacterianos/química , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos
2.
PLoS Biol ; 18(8): e3000805, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810152

RESUMO

Antibiotics are losing efficacy due to the rapid evolution and spread of resistance. Treatments targeting bacterial virulence factors have been considered as alternatives because they target virulence instead of pathogen viability, and should therefore exert weaker selection for resistance than conventional antibiotics. However, antivirulence treatments rarely clear infections, which compromises their clinical applications. Here, we explore the potential of combining antivirulence drugs with antibiotics against the opportunistic human pathogen Pseudomonas aeruginosa. We combined two antivirulence compounds (gallium, a siderophore quencher, and furanone C-30, a quorum sensing [QS] inhibitor) together with four clinically relevant antibiotics (ciprofloxacin, colistin, meropenem, tobramycin) in 9×9 drug concentration matrices. We found that drug-interaction patterns were concentration dependent, with promising levels of synergies occurring at intermediate drug concentrations for certain drug pairs. We then tested whether antivirulence compounds are potent adjuvants, especially when treating antibiotic resistant (AtbR) clones. We found that the addition of antivirulence compounds to antibiotics could restore growth inhibition for most AtbR clones, and even abrogate or reverse selection for resistance in five drug combination cases. Molecular analyses suggest that selection against resistant clones occurs when resistance mechanisms involve restoration of protein synthesis, but not when efflux pumps are up-regulated. Altogether, our work provides a first systematic analysis of antivirulence-antibiotic combinatorial treatments and suggests that such combinations have the potential to be both effective in treating infections and in limiting the spread of antibiotic resistance.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Colistina/farmacologia , Furanos/farmacologia , Gálio/farmacologia , Meropeném/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Tobramicina/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Biossíntese de Proteínas/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/efeitos dos fármacos , Virulência
3.
Proc Natl Acad Sci U S A ; 117(32): 19455-19464, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32703812

RESUMO

A better understanding of how antibiotic exposure impacts the evolution of resistance in bacterial populations is crucial for designing more sustainable treatment strategies. The conventional approach to this question is to measure the range of concentrations over which resistant strain(s) are selectively favored over a sensitive strain. Here, we instead investigate how antibiotic concentration impacts the initial establishment of resistance from single cells, mimicking the clonal expansion of a resistant lineage following mutation or horizontal gene transfer. Using two Pseudomonas aeruginosa strains carrying resistance plasmids, we show that single resistant cells have <5% probability of detectable outgrowth at antibiotic concentrations as low as one-eighth of the resistant strain's minimum inhibitory concentration (MIC). This low probability of establishment is due to detrimental effects of antibiotics on resistant cells, coupled with the inherently stochastic nature of cell division and death on the single-cell level, which leads to loss of many nascent resistant lineages. Our findings suggest that moderate doses of antibiotics, well below the MIC of resistant strains, may effectively restrict de novo emergence of resistance even though they cannot clear already-large resistant populations.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Teóricos , Plasmídeos/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Análise de Célula Única , Processos Estocásticos
4.
PLoS Genet ; 16(6): e1008848, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32530919

RESUMO

Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Flagelos/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Mutação , Polissacarídeos Bacterianos/biossíntese , Pseudomonas aeruginosa/patogenicidade , Seleção Genética
5.
Gene ; 755: 144909, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32569720

RESUMO

In the microbial world, bacteria are the most effective agents in petroleum hydrocarbons (PHs) degradation, utilization/mineralization and they serve as essential degraders of crude oil contaminated environment. Some genes and traits are involved in the hydrocarbon utilization process for which transcriptome analyses are important to identify differentially expressed genes (DEGs) among different conditions, leading to a new understanding of genes or pathways associated with crude oil degradation. In this work, three crude oil utilizing Pseudomonas aeruginosa strains designated as N002, TP16 and J001 subjected to transcriptome analyses revealed a total of 81, 269 and 137 significant DEGs. Among them are 80 up-regulated genes and one downregulated gene of N002, 121 up- regulated and 148 down-regulated genes of TP16, 97 up-regulated and 40 down-regulated genes of J001 which are involved in various metabolic pathways. TP16 strain has shown more number of DEGs upon crude oil treatment in comparison to the other two strains. Through quantitative real time polymerase chain reaction (qRT-PCR), the selected DEGs of each strain from transcriptome data were substantiated. The results have shown that the up- regulated and down-regulated genes observed by qRT-PCR were consistent with transcriptome data. Taken together, our transcriptome results have revealed that TP16 is a potential P. aeruginosa strain for functional analysis of identified potential DEGs involved in crude oil degradation.


Assuntos
Biodegradação Ambiental , Petróleo/microbiologia , Pseudomonas aeruginosa/genética , Bactérias/genética , Regulação para Baixo , Poluentes Ambientais/efeitos adversos , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/genética , Hidrocarbonetos/metabolismo , Transcriptoma/genética , Regulação para Cima
6.
Nat Commun ; 11(1): 3136, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561716

RESUMO

Class 2 CRISPR-Cas proteins have been widely developed as genome editing and transcriptional regulating tools. Class 1 type I CRISPR-Cas constitutes ~60% of all the CRISPR-Cas systems. However, only type I-B and I-E systems have been used to control mammalian gene expression and for genome editing. Here we demonstrate the feasibility of using type I-F system to regulate human gene expression. By fusing transcription activation domain to Pseudomonas aeruginosa type I-F Cas proteins, we activate gene transcription in human cells. In most cases, type I-F system is more efficient than other CRISPR-based systems. Transcription activation is enhanced by elongating the crRNA. In addition, we achieve multiplexed gene activation with a crRNA array. Furthermore, type I-F system activates target genes specifically without off-target transcription activation. These data demonstrate the robustness and programmability of type I-F CRISPR-Cas in human cells.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/isolamento & purificação , Células HEK293 , Humanos , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ativação Transcricional , Transfecção
7.
Proc Natl Acad Sci U S A ; 117(24): 13689-13698, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467157

RESUMO

Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call "serial enrichment for efficient recombineering" (SEER). By performing SEER in Escherichia coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Pseudomonas aeruginosa/genética , Recombinação Genética , Engenharia Genética , Genoma Bacteriano , Mutação
8.
Nucleic Acids Res ; 48(11): 5967-5985, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32406921

RESUMO

During infection of a host, Pseudomonas aeruginosa orchestrates global gene expression to adapt to the host environment and counter the immune attacks. P. aeruginosa harbours hundreds of regulatory genes that play essential roles in controlling gene expression. However, their contributions to the bacterial pathogenesis remain largely unknown. In this study, we analysed the transcriptomic profile of P. aeruginosa cells isolated from lungs of infected mice and examined the roles of upregulated regulatory genes in bacterial virulence. Mutation of a novel regulatory gene pvrA (PA2957) attenuated the bacterial virulence in an acute pneumonia model. Chromatin immunoprecipitation (ChIP)-Seq and genetic analyses revealed that PvrA directly regulates genes involved in phosphatidylcholine utilization and fatty acid catabolism. Mutation of the pvrA resulted in defective bacterial growth when phosphatidylcholine or palmitic acid was used as the sole carbon source. We further demonstrated that palmitoyl coenzyme A is a ligand for the PvrA, enhancing the binding affinity of PvrA to its target promoters. An arginine residue at position 136 was found to be essential for PvrA to bind palmitoyl coenzyme A. Overall, our results revealed a novel regulatory pathway that controls genes involved in phosphatidylcholine and fatty acid utilization and contributes to the bacterial virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Genes Bacterianos/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Animais , Arginina/metabolismo , Sequência de Bases , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ligantes , Camundongos , Modelos Moleculares , Mutação , Ácido Palmítico/metabolismo , Palmitoil Coenzima A/metabolismo , Fosfatidilcolinas/metabolismo , Pneumonia Bacteriana/microbiologia , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Transcriptoma , Virulência/genética
9.
BMC Bioinformatics ; 21(1): 159, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32349677

RESUMO

BACKGROUND: Genomic islands are associated with microbial adaptations, carrying genomic signatures different from the host. Some methods perform an overall test to identify genomic islands based on their local features. However, regions of different scales will display different genomic features. RESULTS: We proposed here a novel method "2SigFinder ", the first combined use of small-scale and large-scale statistical testing for genomic island detection. The proposed method was tested by genomic island boundary detection and identification of genomic islands or functional features of real biological data. We also compared the proposed method with the comparative genomics and composition-based approaches. The results indicate that the proposed 2SigFinder is more efficient in identifying genomic islands. CONCLUSIONS: From real biological data, 2SigFinder identified genomic islands from a single genome and reported robust results across different experiments, without annotated information of genomes or prior knowledge from other datasets. 2SigHunter identified 25 Pathogenicity, 1 tRNA, 2 Virulence and 2 Repeats from 27 Pathogenicity, 1 tRNA, 2 Virulence and 2 Repeats, and detected 101 Phage and 28 HEG out of 130 Phage and 36 HEGs in S. enterica Typhi CT18, which shows that it is more efficient in detecting functional features associated with GIs.


Assuntos
Algoritmos , Genoma Bacteriano , Ilhas Genômicas/genética , Genômica/métodos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Virulência
10.
Chemosphere ; 252: 126349, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32443257

RESUMO

Biosurfactants have potential applications in the remediation of petroleum-contaminated sites. Several strategies can be used to reduce the production costs of these surfactants and make the process more environmentally friendly. In this study, we combined some of these strategies to produce the rhamnolipid-type biosurfactant, including the use of the genetically modified strain Pseudomonas aeruginosa-estA, an industrial coproduct as a carbon source, a simple and low-cost medium, and a simple downstream process. The process resulted in a high yield (17.6 g L-1), even using crude glycerin as the carbon source, with substrate in product conversion factor (YRML/s) of 0.444. The cell-free supernatant (CFS) was not toxic to Artemia salina and selected mammalian cell lineages, suggesting that it can be used directly in the environment without further purification steps. Qualitative analysis showed that CFS has excellent dispersion in the oil-displacement test, emulsifying (IE24 = 65.5%), and tensoactive properties. When salinity, temperature and pressure were set to seawater conditions, the values for interfacial tension between crude oil and water were below 1.0 mN m-1. Taken together, these results demonstrate that it is possible to obtain a nontoxic crude rhamnolipid product, with high productivity, to replace petroleum-based surfactants in oil spill cleanups and other environmental applications.


Assuntos
Biodegradação Ambiental , Glicolipídeos/metabolismo , Petróleo/metabolismo , Animais , Artemia , Carbono , Emulsões , Petróleo/análise , Poluição por Petróleo/análise , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Tensão Superficial , Tensoativos/química , Temperatura
11.
J Med Microbiol ; 69(4): 492-504, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32427563

RESUMO

Pseudomonas aeruginosa is one of the most important opportunistic pathogens, whose clinical relevance is not only due to the high morbidity/mortality of the infections caused, but also to its striking capacity for antibiotic resistance development. In the current scenario of a shortage of effective antipseudomonal drugs, it is essential to have thorough knowledge of the pathogen's biology from all sides, so as to find weak points for drug development. Obviously, one of these points could be the peptidoglycan, given its essential role for cell viability. Meanwhile, immune weapons targeting this structure could constitute an excellent model to be taken advantage of in order to design new therapeutic strategies. In this context, this review gathers all the information regarding the activity of mammalian peptidoglycan-targeting innate immunity (namely lysozyme and peptidoglycan recognition proteins), specifically against P. aeruginosa. All the published studies were considered, from both in vitro and in vivo fields, including works that envisage these weapons as options not only to potentiate their innate effects within the host or for use as exogenously administered treatments, but also harnessing their inflammatory and immune regulatory capacity to finally reduce damage in the patient. Altogether, this review has the objective of anticipating and discussing whether these innate immune resources, in combination or not with other drugs attacking certain P. aeruginosa targets leading to its increased sensitization, could be valid therapeutic antipseudomonal allies.


Assuntos
Peptidoglicano/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Antibacterianos/farmacologia , Humanos , Imunidade Inata , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética
12.
Nat Commun ; 11(1): 2600, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451409

RESUMO

Light-dependent or light-stimulated catalysis provides a multitude of perspectives for implementation in technological or biomedical applications. Despite substantial progress made in the field of photobiocatalysis, the number of usable light-responsive enzymes is still very limited. Flavoproteins have exceptional potential for photocatalytic applications because the name-giving cofactor intrinsically features light-dependent reactivity, undergoing photoreduction with a variety of organic electron donors. However, in the vast majority of these enzymes, photoreactivity of the enzyme-bound flavin is limited or even suppressed. Here, we present a flavoprotein monooxygenase in which catalytic activity is controllable by blue light illumination. The reaction depends on the presence of nicotinamide nucleotide-type electron donors, which do not support the reaction in the absence of light. Employing various experimental approaches, we demonstrate that catalysis depends on a protein-mediated photoreduction of the flavin cofactor, which proceeds via a radical mechanism and a transient semiquinone intermediate.


Assuntos
Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/metabolismo , Oxigenases de Função Mista/metabolismo , NAD/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Cristalografia por Raios X , Flavoproteínas Transferidoras de Elétrons/química , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Luz , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Moleculares , NADP/metabolismo , Oxirredução , Processos Fotoquímicos , Pseudomonas aeruginosa/genética
13.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 375-384, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254062

RESUMO

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Š(spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC.


Assuntos
Proteínas de Transporte de Ânions/química , Proteínas de Bactérias/química , Heme/análogos & derivados , Pseudomonas aeruginosa/enzimologia , Proteínas de Transporte de Ânions/genética , Proteínas de Bactérias/genética , Cristalografia por Raios X , Heme/química , Modelos Moleculares , Óperon , Multimerização Proteica , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/genética
14.
Proc Natl Acad Sci U S A ; 117(19): 10520-10529, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332166

RESUMO

In the opportunistic pathogen Pseudomonas aeruginosa, RsmA is an RNA-binding protein that plays critical roles in the control of virulence, interbacterial interactions, and biofilm formation. Although RsmA is thought to exert its regulatory effects by binding full-length transcripts, the extent to which RsmA binds nascent transcripts has not been addressed. Moreover, which transcripts are direct targets of this key posttranscriptional regulator is largely unknown. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, with cells grown in the presence and absence of the RNA polymerase inhibitor rifampicin, we identify hundreds of nascent transcripts that RsmA associates with in P. aeruginosa We also find that the RNA chaperone Hfq targets a subset of those nascent transcripts that RsmA associates with and that the two RNA-binding proteins can exert regulatory effects on common targets. Our findings establish that RsmA associates with many transcripts as they are being synthesized in P. aeruginosa, identify the transcripts targeted by RsmA, and suggest that RsmA and Hfq may act in a combinatorial fashion on certain transcripts. The binding of posttranscriptional regulators to nascent transcripts may be commonplace in bacteria where distinct regulators can function alone or in concert to achieve control over the translation of transcripts as soon as they emerge from RNA polymerase.


Assuntos
Pseudomonas aeruginosa/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Pseudomonas aeruginosa/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Virulência
15.
Nat Commun ; 11(1): 1865, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313027

RESUMO

Bacterial Rhs proteins containing toxic domains are often secreted by type VI secretion systems (T6SSs) through unclear mechanisms. Here, we show that the T6SS Rhs-family effector TseI of Aeromonas dhakensis is subject to self-cleavage at both the N- and the C-terminus, releasing the middle Rhs core and two VgrG-interacting domains (which we name VIRN and VIRC). VIRC is an endonuclease, and the immunity protein TsiI protects against VIRC toxicity through direct interaction. Proteolytic release of VIRC and VIRN is mediated, respectively, by an internal aspartic protease activity and by two conserved glutamic residues in the Rhs core. Mutations abolishing self-cleavage do not block secretion, but reduce TseI toxicity. Deletion of VIRN or the Rhs core abolishes secretion. TseI homologs from Pseudomonas syringae, P. aeruginosa, and Vibrio parahaemolyticus are also self-cleaved. VIRN and VIRC interact with protein VgrG1, while the Rhs core interacts with protein TecI. We propose that VIRN and the Rhs core act as T6SS intramolecular chaperones to facilitate toxin secretion and function.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Aeromonas/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Genes Bacterianos , Mutação , Óperon , Peptídeo Hidrolases , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo
16.
J Med Microbiol ; 69(6): 895-905, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32242794

RESUMO

Introduction. Pseudomonas aeruginosa grows in extracellular DNA (eDNA)-enriched biofilms and infection sites. eDNA is generally considered to be a structural biofilm polymer required for aggregation and biofilm maturation. In addition, eDNA can sequester divalent metal cations, acidify growth media and serve as a nutrient source.Aim. We wanted to determine the genome-wide influence on the transcriptome of planktonic P. aeruginosa PAO1 grown in the presence of eDNA.Methodology. RNA-seq analysis was performed to determine the genome-wide effects on gene expression of PAO1 grown with eDNA. Transcriptional lux fusions were used to confirm eDNA regulation and to validate phenotypes associated with growth in eDNA.Results. The transcriptome of eDNA-regulated genes included 89 induced and 76 repressed genes (FDR<0.05). A large number of eDNA-induced genes appear to be involved in utilizing DNA as a nutrient. Several eDNA-induced genes are also induced by acidic pH 5.5, and eDNA/acidic pH promoted an acid tolerance response in P. aeruginosa. The cyoABCDE terminal oxidase is induced by both eDNA and pH 5.5, and contributed to the acid tolerance phenotype. Quantitative metal analysis confirmed that DNA binds to diverse metals, which helps explain why many genes involved in a general uptake of metals were controlled by eDNA. Growth in the presence of eDNA also promoted intracellular bacterial survival and influenced virulence in the acute infection model of fruit flies.Conclusion. The diverse functions of the eDNA-regulated genes underscore the important role of this extracellular polymer in promoting antibiotic resistance, virulence, acid tolerance and nutrient utilization; phenotypes that contribute to long-term survival.


Assuntos
DNA Bacteriano/fisiologia , Regulação Bacteriana da Expressão Gênica , Homeostase , Metais/metabolismo , Nutrientes/metabolismo , Pseudomonas aeruginosa/genética , Animais , Drosophila/microbiologia , Concentração de Íons de Hidrogênio , Camundongos , Células RAW 264.7 , Análise de Sequência de RNA , Transcriptoma , Sistemas de Secreção Tipo III/fisiologia , Sistemas de Secreção Tipo VI/fisiologia , Virulência
17.
PLoS Comput Biol ; 16(3): e1007608, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32119670

RESUMO

The evolution of antimicrobial resistance (AMR) poses a persistent threat to global public health. Sequencing efforts have already yielded genome sequences for thousands of resistant microbial isolates and require robust computational tools to systematically elucidate the genetic basis for AMR. Here, we present a generalizable machine learning workflow for identifying genetic features driving AMR based on constructing reference strain-agnostic pan-genomes and training random subspace ensembles (RSEs). This workflow was applied to the resistance profiles of 14 antimicrobials across three urgent threat pathogens encompassing 288 Staphylococcus aureus, 456 Pseudomonas aeruginosa, and 1588 Escherichia coli genomes. We find that feature selection by RSE detects known AMR associations more reliably than common statistical tests and previous ensemble approaches, identifying a total of 45 known AMR-conferring genes and alleles across the three organisms, as well as 25 candidate associations backed by domain-level annotations. Furthermore, we find that results from the RSE approach are consistent with existing understanding of fluoroquinolone (FQ) resistance due to mutations in the main drug targets, gyrA and parC, in all three organisms, and suggest the mutational landscape of those genes with respect to FQ resistance is simple. As larger datasets become available, we expect this approach to more reliably predict AMR determinants for a wider range of microbial pathogens.


Assuntos
Biologia Computacional/métodos , Farmacorresistência Bacteriana/genética , Genoma Bacteriano/genética , Antibacterianos/farmacologia , Anti-Infecciosos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/genética , Fluoroquinolonas/farmacologia , Humanos , Aprendizado de Máquina , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Staphylococcus aureus/genética , Sequenciamento Completo do Genoma/métodos
18.
Am J Epidemiol ; 189(8): 841-849, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32128575

RESUMO

In 2013-2014, an outbreak involving 14 patients infected by an extensively drug-resistant strain of Pseudomonas aeruginosa was detected in a hospital in Madrid, Spain. Our objective was to evaluate an alternative strategy for investigating the outbreak in depth by means of molecular and genomic approaches. Pulsed-field gel electrophoresis (PFGE) was applied as a first-line approach, followed by a more refined whole genome sequencing analysis. Single nucleotide polymorphisms identified by whole genome sequencing were used to design a specific polymerase chain reaction (PCR) for screening unsuspected cases infected by the outbreak strain. Whole genome sequencing alerted us to the existence of greater genetic diversity than was initially assumed, splitting the PFGE-associated outbreak isolates into 4 groups, 2 of which represented coincidental transmission unrelated to the outbreak. A multiplex allele-specific PCR targeting outbreak-specific single nucleotide polymorphisms was applied to 290 isolates, which allowed us to identify 25 additional cases related to the outbreak during 2011-2017. Whole genome sequencing coupled with an outbreak-strain-specific PCR enabled us to markedly redefine the initial picture of the outbreak by 1) ruling out initially suspected cases, 2) defining likely independent coincidental transmission events, 3) predating the starting point of the outbreak, 4) capturing new unsuspected cases, and 5) revealing that the outbreak was still active.


Assuntos
Infecção Hospitalar/epidemiologia , Surtos de Doenças , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/genética , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla , Humanos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/transmissão , Sequenciamento Completo do Genoma
20.
J Vis Exp ; (156)2020 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-32150164

RESUMO

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.


Assuntos
Alginatos/análise , Técnicas Bacteriológicas/métodos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Alginatos/metabolismo , Meios de Cultura/metabolismo , Fibrose Cística/microbiologia , Humanos , Mutação , Fenótipo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pirimidinas/metabolismo
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