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1.
Microbiol Spectr ; 10(3): e0096122, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35604161

RESUMO

In the opportunistic pathogen Pseudomonas aeruginosa, many virulence traits are finely regulated by quorum sensing (QS), an intercellular communication system that allows the cells of a population to coordinate gene expression in response to cell density. The key aspects underlying the functionality of the complex regulatory network governing QS in P. aeruginosa are still poorly understood, including the interplay between the effector protein PqsE and the transcriptional regulator RhlR in controlling the QS regulon. Different studies have focused on the characterization of PqsE- and RhlR-controlled genes in genetic backgrounds in which RhlR activity can be modulated by PqsE and pqsE expression is controlled by RhlR, thus hampering identification of the distinct regulons controlled by PqsE and RhlR. In this study, a P. aeruginosa PAO1 mutant strain with deletion of multiple QS elements and inducible expression of pqsE and/or rhlR was generated and validated. Transcriptomic analyses performed on this genetic background allowed us to unambiguously define the regulons controlled by PqsE and RhlR when produced alone or in combination. Transcriptomic data were validated via reverse transcription-quantitative PCR (RT-qPCR) and transcriptional fusions. Overall, our results showed that PqsE has a negligible effect on the P. aeruginosa transcriptome in the absence of RhlR, and that multiple RhlR subregulons exist with distinct dependency on PqsE. Overall, this study contributes to untangling the regulatory link between the pqs and rhl QS systems mediated by PqsE and RhlR and clarifying the impact of these QS elements on the P. aeruginosa transcriptome. IMPORTANCE The ability of Pseudomonas aeruginosa to cause difficult-to-treat infections relies on its capacity to fine-tune the expression of multiple virulence traits via the las, rhl, and pqs QS systems. Both the pqs effector protein PqsE and the rhl transcriptional regulator RhlR are required for full production of key virulence factors in vitro and pathogenicity in vivo. While it is known that PqsE can stimulate the ability of RhlR to control some virulence factors, no data are available to allow clear discrimination of the PqsE and RhlR regulons. The data produced in this study demonstrate that PqsE mainly impacts the P. aeruginosa transcriptome via an RhlR-dependent pathway and splits the RhlR regulon into PqsE-dependent and PqsE-independent subregulons. Besides contributing to untangling of the complex QS network of P. aeruginosa, our data confirm that both PqsE and RhlR are suitable targets for the development of antivirulence drugs.


Assuntos
Pseudomonas aeruginosa , Percepção de Quorum , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/fisiologia , Regulon , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
2.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33608300

RESUMO

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple-gene expression at the single-cell level has limited the understanding of phenotypic heterogeneity. To investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa, a genetic tool that allows gauging multiple-gene expression at the single-cell level has been generated. This tool, named pRGC, consists of a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP), and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single-cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population without the need for image processing for spectral cross talk correction. In addition, two pRGC variants have been generated for either (i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome that is suitable for long-term experiments in the absence of antibiotic selection or (ii) replication in bacterial genera other than Pseudomonas The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple-gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity.IMPORTANCE Within a bacterial population, single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial subpopulations with distinct phenotypes. The analysis of gene expression at the single-cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation, and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple-gene expression at the single-cell level by fluorescence microscopy without the need for advanced image-processing procedures. A proof of concept has been provided by investigating iron uptake and iron storage gene expression in response to iron availability in P. aeruginosa.


Assuntos
Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Análise de Célula Única/métodos , Genes Reporter , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas
3.
Opt Express ; 27(24): 35245-35256, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31878697

RESUMO

Enzymes are essential to maintain organisms alive. Some of the reactions they catalyze are associated with a change in reagents chirality, hence their activity can be tracked by using optical means. However, illumination affects enzyme activity: the challenge is to operate at low-intensity regime avoiding loss in sensitivity. Here we apply quantum phase estimation to real-time measurement of invertase enzymatic activity. Control of the probe at the quantum level demonstrates the potential for reducing invasiveness with optimized sensitivity at once. This preliminary effort, bringing together methods of quantum physics and biology, constitutes an important step towards full development of quantum sensors for biological systems.


Assuntos
Luz , Teoria Quântica , beta-Frutofuranosidase/metabolismo , Lasers , Fótons , Saccharomyces cerevisiae/enzimologia
4.
Front Microbiol ; 10: 2355, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31649658

RESUMO

The emergence of antibiotic resistant bacterial pathogens is increasing at an unprecedented pace, calling for the development of new therapeutic options. Small molecules interfering with virulence processes rather than growth hold promise as an alternative to conventional antibiotics. Anti-virulence agents are expected to decrease bacterial virulence and to pose reduced selective pressure for the emergence of resistance. In the opportunistic pathogen Pseudomonas aeruginosa the expression of key virulence traits is controlled by quorum sensing (QS), an intercellular communication process that coordinates gene expression at the population level. Hence, QS inhibitors represent promising anti-virulence agents against P. aeruginosa. Virtual screenings allow fast and cost-effective selection of target ligands among vast libraries of molecules, thus accelerating the time and limiting the cost of conventional drug-discovery processes, while the drug-repurposing approach is based on the identification of off-target activity of FDA-approved drugs, likely endowed with low cytotoxicity and favorable pharmacological properties. This study aims at combining the advantages of virtual screening and drug-repurposing approaches to identify new QS inhibitors targeting the pqs QS system of P. aeruginosa. An in silico library of 1,467 FDA-approved drugs has been screened by molecular docking, and 5 hits showing the highest predicted binding affinity for the pqs QS receptor PqsR (also known as MvfR) have been selected. In vitro experiments have been performed by engineering ad hoc biosensor strains, which were used to verify the ability of hit compounds to decrease PqsR activity in P. aeruginosa. Phenotypic analyses confirmed the impact of the most promising hit, the antipsychotic drug pimozide, on the expression of P. aeruginosa PqsR-controlled virulence traits. Overall, this study highlights the potential of virtual screening campaigns of FDA-approved drugs to rapidly select new inhibitors of important bacterial functions.

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