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1.
Sci Rep ; 12(1): 10404, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35729352

RESUMO

In Gram-negative pathogens, the stringent response regulator DksA controls the expression of hundreds of genes, including virulence-related genes. Interestingly, Pseudomonas aeruginosa has two functional DksA paralogs: DksA1 is constitutively expressed and has a zinc-finger motif, while DksA2 is expressed only under zinc starvation conditions and does not contain zinc. DksA1 stimulates the production of virulence factors in vitro and is required for full pathogenicity in vivo. DksA2 can replace these DksA1 functions. Here, the role of dksA paralogs in P. aeruginosa tolerance to H2O2-induced oxidative stress has been investigated. The P. aeruginosa dksA1 dksA2 mutant showed impaired H2O2 tolerance in planktonic and biofilm-growing cultures and increased susceptibility to macrophages-mediated killing compared to the wild type. Complementation with either dksA1 or dksA2 genes restored the wild type phenotypes. The DksA-dependent tolerance to oxidative stress involves, at least in part, the positive transcriptional control of both katA and katE catalase-encoding genes. These data support the hypothesis that DksA1 and DksA2 are eco-paralogs with indistinguishable function but optimal activity under different environmental conditions, and highlight their mutual contribution to P. aeruginosa virulence.


Assuntos
Peróxido de Hidrogênio , Pseudomonas aeruginosa , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Macrófagos/metabolismo , Pseudomonas aeruginosa/fisiologia , Zinco/metabolismo
2.
Front Microbiol ; 13: 845231, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35547141

RESUMO

The chronic lung infection caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Antivirulence drugs targeting P. aeruginosa quorum sensing (QS) systems are intensively studied as antibiotics substitutes or adjuvants. Previous studies, carried out in non-CF P. aeruginosa reference strains, showed that the old drugs niclosamide and clofoctol could be successfully repurposed as antivirulence drugs targeting the las and pqs QS systems, respectively. However, frequent emergence of QS-defective mutants in the CF lung undermines the use of QS inhibitors in CF therapy. Here, QS signal production and susceptibility to niclosamide and clofoctol have been investigated in 100 P. aeruginosa CF isolates, with the aim of broadening current knowledge on the potential of anti-QS compounds in CF therapy. Results showed that 85, 78, and 69% of the CF isolates from our collection were proficient for the pqs, rhl, and las QS systems, respectively. The ability of both niclosamide and clofoctol to inhibit QS and virulence in vitro was highly variable and strain-dependent. Niclosamide showed an overall low range of activity and its negative effect on las signal production did not correlate with a decreased production of virulence factors. On the other hand, clofoctol displayed a broader QS inhibitory effect in CF isolates, with consequent reduction of the pqs-controlled virulence factor pyocyanin. Overall, this study highlights the importance of testing new antivirulence drugs against large panels of P. aeruginosa CF clinical isolates before proceeding to further pre-clinical studies and corroborates previous evidence that strains naturally resistant to QS inhibitors occur among CF isolates. However, it is also shown that resistance to pqs inhibitors is less frequent than resistance to las inhibitors, thus supporting the development of pqs inhibitors for antivirulence therapy in CF.

3.
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
4.
Antibiotics (Basel) ; 11(3)2022 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-35326787

RESUMO

To cope with stressful conditions, including antibiotic exposure, bacteria activate the SOS response, a pathway that induces error-prone DNA repair and mutagenesis mechanisms. In most bacteria, the SOS response relies on the transcriptional repressor LexA and the co-protease RecA, the latter being also involved in homologous recombination. The role of the SOS response in stress- and antibiotic-induced mutagenesis has been characterized in detail in the model organism Escherichia coli. However, its effect on antibiotic resistance in the human pathogen Pseudomonas aeruginosa is less clear. Here, we analyzed a recA deletion mutant and confirmed, by conjugation and gene expression assays, that RecA is required for homologous recombination and SOS response induction in P. aeruginosa. MIC assays demonstrated that RecA affects P. aeruginosa resistance only towards fluoroquinolones and genotoxic agents. The comparison of antibiotic-resistant mutant frequency between treated and untreated cultures revealed that, among the antibiotics tested, only fluoroquinolones induced mutagenesis in P. aeruginosa. Notably, both RecA and error-prone DNA polymerases were found to be dispensable for this process. These data demonstrate that the SOS response is not required for antibiotic-induced mutagenesis in P. aeruginosa, suggesting that RecA inhibition is not a suitable strategy to target antibiotic-induced emergence of resistance in this pathogen.

5.
Microorganisms ; 10(2)2022 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-35208877

RESUMO

DNA polymerase III (Pol III) is the replicative enzyme in bacteria. It consists of three subcomplexes, the catalytic core, the ß clamp, and the clamp loader. While this complex has been thoroughly characterized in the model organism Escherichia coli, much less is known about its functioning and/or its specific properties in other bacteria. Biochemical studies highlighted specific features in the clamp loader subunit ψ of Pseudomonas aeruginosa as compared to its E. coli counterpart, and transposon mutagenesis projects identified the ψ-encoding gene holD among the strictly essential core genes of P. aeruginosa. By generating a P. aeruginosa holD conditional mutant, here we demonstrate that, as previously observed for E. coli holD mutants, HolD-depleted P. aeruginosa cells show strongly decreased growth, induction of the SOS response, and emergence of suppressor mutants at high frequency. However, differently from what was observed in E. coli, the growth of P. aeruginosa cells lacking HolD cannot be rescued by the deletion of genes for specialized DNA polymerases. We also observed that the residual growth of HolD-depleted cells is strictly dependent on homologous recombination functions, suggesting that recombination-mediated rescue of stalled replication forks is crucial to support replication by a ψ-deficient Pol III enzyme in P. aeruginosa.

6.
ACS Infect Dis ; 8(1): 78-85, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-34965085

RESUMO

The decreasing efficacy of existing antibiotics against pulmonary pathogens that affect cystic fibrosis (CF) patients calls for the development of novel antimicrobials. Iron uptake and metabolism are vital processes for bacteria, hence potential therapeutic targets. Gallium [Ga(III)] is a ferric iron-mimetic that inhibits bacterial growth by disrupting iron uptake and metabolism. In this work we evaluate the efficacy of three Ga(III) compounds, namely, Ga(NO3)3, (GaN), Ga(III)-maltolate (GaM), and Ga(III)-protoporphyrin IX (GaPPIX), against a collection of CF pathogens using both reference media and media mimicking biological fluids. All CF pathogens, except Streptococcus pneumoniae, were susceptible to at least one Ga(III) compound. Notably, Mycobacterium abscessus and Stenotrophomonas maltophilia were susceptible to all Ga(III) compounds. Achromobacter xylosoxidans, Burkholderia cepacia complex, and Pseudomonas aeruginosa were more susceptible to GaN and GaM, whereas Staphylococcus aureus and Haemophilus influenzae were more sensitive to GaPPIX. The results of this study support the development of Ga(III)-based therapy as a broad-spectrum strategy to treat CF lung infections.


Assuntos
Fibrose Cística , Gálio , Stenotrophomonas maltophilia , Humanos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa
7.
Environ Microbiol ; 23(9): 5487-5504, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34327807

RESUMO

The stringent response regulator DksA plays a key role in Gram negative bacteria adaptation to challenging environments. Intriguingly, the plant and human pathogen Pseudomonas aeruginosa is unique as it expresses two functional DksA paralogs: DksA1 and DksA2. However, the role of DksA2 in P. aeruginosa adaptive strategies has been poorly investigated so far. Here, RNA-Seq analysis and phenotypic assays showed that P. aeruginosa DksA1 and DksA2 proteins are largely interchangeable. Relative to wild type P. aeruginosa, transcription of 1779 genes was altered in a dksA1 dksA2 double mutant, and the wild type expression level of ≥90% of these genes was restored by in trans complementation with either dksA1 or dksA2. Interestingly, the expression of a small sub-set of genes seems to be preferentially or exclusively complemented by either dksA1 or dksA2. In addition, evidence has been provided that the DksA-dependent regulation of virulence genes expression is independent and hierarchically dominant over two major P. aeruginosa regulatory circuits, i.e., quorum sensing and cyclic-di-GMP signalling systems. Our findings support the prominent role of both DksA paralogs in P. aeruginosa environmental adaptation.


Assuntos
Pseudomonas aeruginosa , Transcriptoma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transcriptoma/genética , Virulência/genética
8.
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
9.
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
10.
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.

11.
Appl Environ Microbiol ; 85(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31324623

RESUMO

The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model.IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.


Assuntos
Acinetobacter/genética , Farmacorresistência Bacteriana Múltipla/genética , Perfilação da Expressão Gênica/métodos , Vetores Genéticos/farmacologia , Acinetobacter/efeitos dos fármacos , Antibacterianos/farmacologia , Escherichia coli/genética
12.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718286

RESUMO

Acinetobacter baumannii is an important nosocomial pathogen. Mechanisms that allow A. baumannii to cause human infection are still poorly understood. Iron is an essential nutrient for bacterial growth in vivo, and the multiplicity of iron uptake systems in A. baumannii suggests that iron acquisition contributes to the ability of A. baumannii to cause infection. In Gram-negative bacteria, active transport of ferrisiderophores and heme relies on the conserved TonB-ExbB-ExbD energy-transducing complex, while active uptake of ferrous iron is mediated by the Feo system. The A. baumannii genome invariably contains three tonB genes (tonB1, tonB2, and tonB3), whose role in iron uptake is poorly understood. Here, we generated A. baumannii mutants with knockout mutations in the feo and/or tonB gene. We report that tonB3 is essential for A. baumannii growth under iron-limiting conditions, whereas tonB1, tonB2, and feoB appear to be dispensable for ferric iron uptake. tonB3 deletion resulted in reduced intracellular iron content despite siderophore overproduction, supporting a key role of TonB3 in iron uptake. In contrast to the case for tonB1 and tonB2, the promoters of tonB3 and feo contain functional Fur boxes and are upregulated in iron-poor media. Both TonB3 and Feo systems are required for growth in complement-free human serum and contribute to resistance to the bactericidal activity of normal human serum, but only TonB3 appears to be essential for virulence in insect and mouse models of infection. Our findings highlight a central role of the TonB3 system for A. baumannii pathogenicity. Hence, TonB3 represents a promising target for novel antibacterial therapies and for the generation of attenuated vaccine strains.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidade , Proteínas de Transporte de Cátions/metabolismo , Ferro/metabolismo , Acinetobacter baumannii/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico Ativo , Proteínas de Transporte de Cátions/genética , Feminino , Heme/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Sideróforos/metabolismo , Virulência
13.
Artigo em Inglês | MEDLINE | ID: mdl-30705882

RESUMO

The bottom-up branch of synthetic biology includes-among others-innovative studies that combine cell-free protein synthesis with liposome technology to generate cell-like systems of minimal complexity, often referred to as synthetic cells. The functions of this type of synthetic cell derive from gene expression, hence they can be programmed in a modular, progressive and customizable manner by means of ad hoc designed genetic circuits. This experimental scenario is rapidly expanding and synthetic cell research already counts numerous successes. Here, we present a review focused on the exchange of chemical signals between liposome-based synthetic cells (operating by gene expression) and biological cells, as well as between two populations of synthetic cells. The review includes a short presentation of the "molecular communication technologies," briefly discussing their promises and challenges.

14.
IEEE Trans Nanobioscience ; 18(1): 43-50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30475724

RESUMO

The recent progresses in bottom-up synthetic biology allow the construction of cell-like systems (also called "synthetic cells") based on the encapsulation of chemicals and biological macromolecules inside lipid vesicles. Synthetic cells are far from being alive, but can be designed in order to imitate biological cells with respect to specific functions. The exchange of chemical signals is one of the most fascinating ones. Experimental papers have shown that synthetic cells can be designed to send and receive molecular signals, inaugurating a new research avenue that can be highly relevant not only for nano-medicine and nano-biotechnology but also for basic understanding of minimal cognitive systems. Here, we shortly present the synthetic cell technology and illustrate how to implement the concept of molecular communication in this field.


Assuntos
Comunicação Celular/fisiologia , Computadores Moleculares , Modelos Biológicos , Biologia Sintética , Células Artificiais/citologia , Células Artificiais/metabolismo , Bactérias/citologia , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Biotecnologia
15.
Sci Rep ; 8(1): 16912, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30442901

RESUMO

Pseudomonas spp. are endowed with a complex pathway for glucose uptake that relies on multiple transporters. In this work we report the construction and characterization of Pseudomonas aeruginosa single and multiple mutants with unmarked deletions of genes encoding outer membrane (OM) and inner membrane (IM) proteins involved in glucose uptake. We found that a triple ΔgltKGF ΔgntP ΔkguT mutant lacking all known IM transporters (named GUN for Glucose Uptake Null) is unable to grow on glucose as unique carbon source. More than 500 genes controlling both metabolic functions and virulence traits show differential expression in GUN relative to the parental strain. Consistent with transcriptomic data, the GUN mutant displays a pleiotropic phenotype. Notably, the genome-wide transcriptional profile and most phenotypic traits differ between the GUN mutant and the wild type strain irrespective of the presence of glucose, suggesting that the investigated genes may have additional roles besides glucose transport. Finally, mutants carrying single or multiple deletions in the glucose uptake genes showed attenuated virulence relative to the wild type strain in Galleria mellonella, but not in Caenorhabditis elegans infection model, supporting the notion that metabolic functions may deeply impact P. aeruginosa adaptation to specific environments found inside the host.


Assuntos
Pleiotropia Genética , Glucose/metabolismo , Modelos Biológicos , Mutação/genética , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Biofilmes/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Carbono/farmacologia , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Mariposas/microbiologia , Oligopeptídeos/metabolismo , Oxirredução , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Piocianina/metabolismo , Percepção de Quorum/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Transcriptoma/genética , Virulência
16.
Artigo em Inglês | MEDLINE | ID: mdl-30201815

RESUMO

The long-term use of antibiotics has led to the emergence of multidrug-resistant bacteria. A promising strategy to combat bacterial infections aims at hampering their adaptability to the host environment without affecting growth. In this context, the intercellular communication system quorum sensing (QS), which controls virulence factor production and biofilm formation in diverse human pathogens, is considered an ideal target. Here, we describe the identification of new inhibitors of the pqs QS system of the human pathogen Pseudomonas aeruginosa by screening a library of 1,600 U.S. Food and Drug Administration-approved drugs. Phenotypic characterization of ad hoc engineered strains and in silico molecular docking demonstrated that the antifungal drugs clotrimazole and miconazole, as well as an antibacterial compound active against Gram-positive pathogens, clofoctol, inhibit the pqs system, probably by targeting the transcriptional regulator PqsR. The most active inhibitor, clofoctol, specifically inhibited the expression of pqs-controlled virulence traits in P. aeruginosa, such as pyocyanin production, swarming motility, biofilm formation, and expression of genes involved in siderophore production. Moreover, clofoctol protected Galleria mellonella larvae from P. aeruginosa infection and inhibited the pqs QS system in P. aeruginosa isolates from cystic fibrosis patients. Notably, clofoctol is already approved for clinical treatment of pulmonary infections caused by Gram-positive bacterial pathogens; hence, this drug has considerable clinical potential as an antivirulence agent for the treatment of P. aeruginosa lung infections.


Assuntos
Antibacterianos/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Fatores de Virulência/antagonistas & inibidores , Virulência/efeitos dos fármacos , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Estados Unidos , United States Food and Drug Administration
17.
FEBS J ; 285(20): 3815-3834, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30106221

RESUMO

Bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c-di-GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c-di-GMP-dependent response. Both the c-di-GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c-di-GMP-related processes. Here, we report a structure-function relationship study on the catalytic portion of the PA0575 protein from Pseudomonas aeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF-EAL portion is a GTP-dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide-dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Guanosina Trifosfato/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pseudomonas aeruginosa/metabolismo , Regulação Alostérica , Cristalografia por Raios X , GMP Cíclico/metabolismo , Hidrólise , Diester Fosfórico Hidrolases/química , Conformação Proteica , Multimerização Proteica
18.
Artigo em Inglês | MEDLINE | ID: mdl-30083519

RESUMO

The Pseudomonas aeruginosa quorum sensing (QS) network plays a key role in the adaptation to environmental changes and the control of virulence factor production in this opportunistic human pathogen. Three interlinked QS systems, namely las, rhl, and pqs, are central to the production of pyocyanin, a phenazine virulence factor which is typically used as phenotypic marker for analysing QS. Pyocyanin production in P. aeruginosa is a complex process involving two almost identical operons termed phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), which drive the production of phenazine-1-carboxylic acid (PCA) which is further converted to pyocyanin by two modifying enzymes PhzM and PhzS. Due to the high sequence conservation between the phz1 and phz2 operons (nucleotide identity > 98%), analysis of their individual expression by RNA hybridization, qRT-PCR or transcriptomics is challenging. To overcome this difficulty, we utilized luminescence based promoter fusions of each phenazine operon to measure in planktonic cultures their transcriptional activity in P. aeruginosa PAO1-N genetic backgrounds impaired in different components of the las, rhl, and pqs QS systems, in the presence or absence of different QS signal molecules. Using this approach, we found that all three QS systems play a role in differentially regulating the phz1 and phz2 phenazine operons, thus uncovering a higher level of complexity to the QS regulation of PCA biosynthesis in P. aeruginosa than previously appreciated. Importance: The way the P. aeruginosa QS regulatory networks are intertwined creates a challenge when analysing the mechanisms governing specific QS-regulated traits. Multiple QS regulators and signals have been associated with the production of phenazine virulence factors. In this work we designed experiments where we dissected the contribution of specific QS switches using individual mutations and complementation strategies to gain further understanding of the specific roles of these QS elements in controlling expression of the two P. aeruginosa phenazine operons. Using this approach we have teased out which QS regulators have either indirect or direct effects on the regulation of the two phenazine biosynthetic operons. The data obtained highlight the sophistication of the QS cascade in P. aeruginosa and the challenges in analysing the control of phenazine secondary metabolites.


Assuntos
Vias Biossintéticas/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Piocianina/biossíntese , Percepção de Quorum , Fusão Gênica Artificial , Perfilação da Expressão Gênica , Genes Reporter , Proteínas Luminescentes/análise , Hibridização de Ácido Nucleico , Fenazinas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
19.
Artigo em Inglês | MEDLINE | ID: mdl-29339383

RESUMO

Understanding bacterial pathogenesis requires adequate genetic tools to assess the role of individual virulence determinants by mutagenesis and complementation assays, as well as for homologous and heterologous expression of cloned genes. Our knowledge of Acinetobacter baumannii pathogenesis has so far been limited by the scarcity of genetic tools to manipulate multidrug-resistant (MDR) epidemic strains, which are responsible for most infections. Here, we report on the construction of new multipurpose shuttle plasmids, namely, pVRL1 and pVRL2, which can efficiently replicate in Acinetobacter spp. and in Escherichia coli The pVRL1 plasmid has been constructed by combining (i) the cryptic plasmid pWH1277 from Acinetobacter calcoaceticus, which provides an origin of replication for Acinetobacter spp.; (ii) a ColE1-like origin of replication; (iii) the gentamicin or zeocin resistance cassette for antibiotic selection; and (iv) a multilinker containing several unique restriction sites. Modification of pVRL1 led to the generation of the pVRL2 plasmid, which allows arabinose-inducible gene transcription with an undetectable basal expression level of cloned genes under uninduced conditions and a high dynamic range of responsiveness to the inducer. Both pVRL1 and pVRL2 can easily be selected in MDR A. baumannii, have a narrow host range and a high copy number, are stably maintained in Acinetobacter spp., and appear to be compatible with indigenous plasmids carried by epidemic strains. Plasmid maintenance is guaranteed by the presence of a toxin-antitoxin system, providing more insights into the mechanism of plasmid stability in Acinetobacter spp.


Assuntos
Acinetobacter/genética , Vetores Genéticos/genética , Acinetobacter/efeitos dos fármacos , Antibacterianos/farmacologia , Bleomicina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Gentamicinas/farmacologia , Plasmídeos/genética
20.
Chem Commun (Camb) ; 54(17): 2090-2093, 2018 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-29334092

RESUMO

Recent developments in bottom-up synthetic biology (e.g., lipid vesicle technology integrated with cell-free protein expression systems) allow the generation of semi-synthetic minimal cells (in short, synthetic cells, SCs) endowed with some distinctive capacities of natural cells. In particular, such approaches provide technological tools and conceptual frameworks for the design and engineering of programmable SCs capable of communicating with natural cells by exchanging chemical signals. Here we describe the generation of giant vesicle-based SCs which, via gene expression, synthesize in their aqueous lumen an enzyme that in turn produces a chemical signal. The latter is a small molecule, which is passively released in the medium and then perceived by the bacterium Pseudomonas aeruginosa, demonstrating that SCs and bacteria can communicate chemically. The results pave the way to a novel basic and applied research area where synthetic cells can communicate with natural cells, for example for exploring minimal cognition, developing chemical information technologies, and producing smart and programmable drug-producing/drug-delivery systems.


Assuntos
4-Butirolactona/análogos & derivados , Células Artificiais/metabolismo , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum , 4-Butirolactona/metabolismo , 4-Butirolactona/farmacologia , Células Artificiais/enzimologia , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Biologia Sintética/métodos
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