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1.
Front Cell Infect Microbiol ; 11: 734296, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34746024

RESUMO

Pseudomonas aeruginosa and Aspergillus fumigatus infections frequently co-localize in lungs of immunocompromised patients and individuals with cystic fibrosis (CF). The antifungal activity of P. aeruginosa has been described for its filtrates. Pyoverdine and pyocyanin are the principal antifungal P. aeruginosa molecules active against A. fumigatus biofilm metabolism present in iron-limited or iron-replete planktonic P. aeruginosa culture filtrates, respectively. Using various P. aeruginosa laboratory wild-type strains (PA14, PAO1, PAK), we found antifungal activity against Aspergillus colonies on agar. Comparing 36 PA14 and 7 PAO1 mutants, we found that mutants lacking both major siderophores, pyoverdine and pyochelin, display higher antifungal activity on agar than their wild types, while quorum sensing mutants lost antifungal activity. Addition of ferric iron, but not calcium or magnesium, reduced the antifungal effects of P. aeruginosa on agar, whereas iron-poor agar enhanced antifungal effects. Antifungal activity on agar was mediated by PQS and HHQ, via MvfR. Among the MvfR downstream factors, rhamnolipids and elastase were produced in larger quantities by pyoverdine-pyochelin double mutants and showed antifungal activity on agar. In summary, antifungal factors produced by P. aeruginosa on agar differ from those produced by bacteria grown in liquid cultures, are dependent on quorum sensing, and are downregulated by the availability of ferric iron. Rhamnolipids and elastase seem to be major mediators of Pseudomonas' antifungal activity on a solid surface.


Assuntos
Infecções por Pseudomonas , Pseudomonas , Aspergillus , Biofilmes , Humanos , Pseudomonas aeruginosa , Piocianina , Percepção de Quorum
2.
F1000Res ; 10: 14, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34540201

RESUMO

Background: Pseudomonas aeruginosa, a multidrug-resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may potentially overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth, pyocyanin production, and biofilm formation of P. aeruginosa. Methods: Pseudomonas aeruginosa ATCC 10145 and clinical isolates were cultured in a royal jelly-containing medium to test the antibacterial activity. Pyocyanin production was observed by measuring the absorbance at 690 nm after 36 h culture and determined using extinction coefficient 4310 M-1 cm-1. Static microtiter plate biofilm assay performed to detect the biofilm formation, followed by scanning electron microscopy. Results: Royal jelly effectively inhibited the viability of both strains from a concentration of 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC 10145, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates. Concentrations of 12.5% and 6.125% significantly induced biofilm formation of P. aeruginosa ATCC 10145, in line with the results of the SEM analysis. Conclusions: The royal jelly concentration of 25% or higher inhibits bacterial growth; however, the subinhibitory concentration increases pyocyanin production and biofilm formation in P. aeruginosa . It is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.


Assuntos
Pseudomonas aeruginosa , Piocianina , Biofilmes , Ácidos Graxos
3.
Anal Chem ; 93(43): 14481-14488, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34661405

RESUMO

Pseudomonas aeruginosa produces a number of phenazine metabolites, including pyocyanin (PYO), phenazine-1-carboxamide (PCN), and phenazine-1-carboxylic acid (PCA). Among these, PYO has been most widely studied as a biomarker of P. aeruginosa infection. However, despite its broad-spectrum antibiotic properties and its role as a precursor in the biosynthetic route leading to other secondary phenazines, PCA has attracted less attention, partially due to its relatively low concentration and interference from other highly abundant phenazines. This challenge is addressed here by constructing a hierarchically organized nanostructure consisting of a pH-responsive block copolymer (BCP) membrane with nanopore electrode arrays (NEAs) filled with gold nanoparticles (AuNPs) to separate and detect PCA in bacterial environments. The BCP@NEA strategy is designed such that adjusting the pH of the bacterial medium to 4.5, which is above the pKa of PCA but below the pKa of PYO and PCN, ensures that PCA is negatively charged and can be selectively transported across the BCP membrane. At pH 4.5, only PCA is transported into the AuNP-filled NEAs, while PYO and PCN are blocked. Structural characterization illustrates the rigorous spatial segregation of the AuNPs in the NEA nanopore volume, allowing PCA secreted from P. aeruginosa to be quantitatively determined as a function of incubation time using square-wave voltammetry and surface-enhanced Raman spectroscopy. The strategy proposed in this study can be extended by changing the nature of the hydrophilic block and subsequently applied to detect other redox-active metabolites at a low concentration in complex biological samples and, thus, help understand metabolism in microbial communities.


Assuntos
Nanopartículas Metálicas , Nanoporos , Eletrodos , Ouro , Fenazinas , Pseudomonas aeruginosa , Piocianina
4.
Analyst ; 146(22): 6924-6934, 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34647550

RESUMO

A portable surface-enhanced Raman spectroscopy (SERS) sensor for detecting pyocyanin (PYO) in simulated wound fluid and from bacteria samples was developed. Solution-phase SERS detection protocols are designed to be compatible with two different clinical practices for wound exudate collection, namely negative pressure liquid collection and swabbing. For citrate-coated metal nanoparticles of three different compositions, i.e. gold (AuNPs), alloyed silver/gold (AgAuNPs), and silver (AgNPs), we firstly confirmed their interaction with PYO in the complex wound fluid, using fluorescence quenching experiments, which rationalized the Raman enhancement effects. We then demonstrated the Raman enhancement effects of the metal nanoparticles in the order of AgNPs > AgAuNPs > AuNPs. The limit of detection (LOD) achieved for PYO is 1.1 µM (in a linear range of 0.1-25 µM by the AgNPs), 10.9 µM (in a linear range of 5-100 µM, by the AgAuNPs), and 17.7 µM (in a linear range of 10-100 µM by the AuNPs). The AgNP and AgAuNP sensors together cover the sensitivity and dynamic range requirements for the clinical detection of wound infection, where PYO is present at a concentration of 1-50 µM. In addition, sterilized cotton swabs were used to collect wound fluid and transfer samples into AgNP solution for SERS measurements. This detection protocol was completed within 5 minutes with a LOD of 23.1 µM (in a linear range of 15-100 µM). The SERS sensing protocol was validated by its successful detection of PYO in cultured Pseudomonas aeruginosa bacteria. The findings presented in this work pave the way towards point-of-care diagnostics of wound infections.


Assuntos
Nanopartículas Metálicas , Piocianina , Ouro , Prata , Análise Espectral Raman
5.
PLoS Pathog ; 17(8): e1009425, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34460871

RESUMO

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.


Assuntos
Biofilmes/crescimento & desenvolvimento , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Vesículas Extracelulares/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Quinolonas/metabolismo , Percepção de Quorum , Sistema de Translocação de Argininas Geminadas/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , DNA Bacteriano/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Sistema de Translocação de Argininas Geminadas/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
6.
Sci Rep ; 11(1): 15639, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34341384

RESUMO

The objective of this study was to evaluate 50 [chicken meat (n = 45) and ground beef (n = 5)] Pseudomonas aeruginosa isolates to determine the expression of the lasI and rhl QS systems, related virulence factors, and the presence of N-3-oxo-dodecanoyl homoserine lactone (AHL: 3-O-C12-HSL). For the isolation and identification of P. aeruginosa, conventional culture and oprL gene-based molecular techniques were used. In relation to QS systems, eight genes consisting of four intact and four internal (lasI/R, rhlI/R) genes were analyzed with PCR assay. The two QS systems genes in P. aeruginosa isolates from ground beef (80.00%) and chicken meat (76.00%) were present at quite high levels. The 3-O-C12-HSL was detected in 14.00% of the isolates. Both biofilm formation and motility were detected in 98.00% of the isolates. Protease activity was determined in 54.00% of the isolates. Pyocyanin production was detected in 48.00% of the isolates. The las system scores strongly and positively correlated with the rhl system (p ˂ .01). PYA moderately and positively correlated with protease (p ˂ .05). In addition, there was statistically significance between lasI and protease activity (p < .10), and rhlI and twitching motility (p < .10). In conclusion, the high number of isolates having QS systems and related virulence factors are critical for public health. Pyocyanin, protease, and biofilm formation can cause spoilage and play essential role in food spoilage and food safety.


Assuntos
Carne , Pseudomonas aeruginosa , Animais , Proteínas de Bactérias/metabolismo , Biofilmes , Bovinos , Galinhas , Endopeptidases/metabolismo , Piocianina/metabolismo
7.
Molecules ; 26(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205355

RESUMO

Rottlerin is a natural product consisting of chalcone and flavonoid scaffolds, both of which have previously shown quorum sensing (QS) inhibition in various bacteria. Therefore, the unique rottlerin scaffold highlights great potential in inhibiting the QS system of Pseudomonas aeruginosa. Rottlerin analogues were synthesised by modifications at its chalcone- and methylene-bridged acetophenone moieties. The synthesis of analogues was achieved using an established five-step synthetic strategy for chalcone derivatives and utilising the Mannich reaction at C6 of the chromene to construct morpholine analogues. Several pyranochromene chalcone derivatives were also generated using aldol conditions. All the synthetic rottlerin derivatives were screened for QS inhibition and growth inhibition against the related LasR QS system. The pyranochromene chalcone structures displayed high QS inhibitory activity with the most potent compounds, 8b and 8d, achieving QS inhibition of 49.4% and 40.6% and no effect on bacterial growth inhibition at 31 µM, respectively. Both compounds also displayed moderate biofilm inhibitory activity and reduced the production of pyocyanin.


Assuntos
Acetofenonas/farmacologia , Benzopiranos/farmacologia , Produtos Biológicos/farmacologia , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Flavonoides/farmacologia , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/farmacologia
8.
Molecules ; 26(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066034

RESUMO

The chemical composition of three Citrus limon oils: lemon essential oil (LEO), lemon terpenes (LT) and lemon essence (LE), and their influence in the virulence factors production and motility (swarming and swimming) of two Pseudomonas aeruginosa strains (ATCC 27853 and a multidrug-resistant HT5) were investigated. The main compound, limonene, was also tested in biological assays. Eighty-four compounds, accounting for a relative peak area of 99.23%, 98.58% and 99.64%, were identified by GC/MS. Limonene (59-60%), γ-terpinene (10-11%) and ß-pinene (7-15%) were the main compounds. All lemon oils inhibited specific biofilm production and bacterial metabolic activities into biofilm in a dose-dependent manner (20-65%, in the range of 0.1-4 mg mL-1) of both strains. Besides, all samples inhibited about 50% of the elastase activity at 0.1 mg mL-1. Pyocyanin biosynthesis decreases until 64% (0.1-4 mg mL-1) for both strains. Swarming motility of P. aeruginosa ATCC 27853 was completely inhibited by 2 mg mL-1 of lemon oils. Furthermore, a decrease (29-55%, 0.1-4 mg mL-1) in the synthesis of Quorum sensing (QS) signals was observed. The oils showed higher biological activities than limonene. Hence, their ability to control the biofilm of P. aeruginosa and reduce the production of virulence factors regulated by QS makes lemon oils good candidates to be applied as preservatives in the food processing industry.


Assuntos
Antibacterianos/farmacologia , Citrus/química , Óleos Voláteis/farmacologia , Óleos Vegetais/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Monoterpenos Bicíclicos/química , Monoterpenos Bicíclicos/farmacologia , Biofilmes/efeitos dos fármacos , Monoterpenos Cicloexânicos/química , Monoterpenos Cicloexânicos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Limoneno/química , Limoneno/farmacologia , Óleos Voláteis/química , Elastase Pancreática/metabolismo , Óleos Vegetais/química , Pseudomonas aeruginosa/metabolismo , Piocianina/biossíntese , Transdução de Sinais/efeitos dos fármacos , Virulência , Fatores de Virulência , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/farmacologia
9.
PLoS One ; 16(6): e0253259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34115807

RESUMO

Studies of the outcome of Pseudomonas aeruginosa bacteremia (Pab) have focused mainly on antibiotic appropriateness. However, P. aeruginosa possesses many virulence factors whose roles in outcomes have not been examined in humans, except for the type III secretion system (T3SS) toxins. The purpose of this study was to examine the role of virulence factors other than the T3SS toxins. Bacterial isolates were collected from 75 patients who suffered from Pa blood stream infections. Host factors such as neutropenia, immunosuppression, comorbidities, time to effective antibiotics, source of bacteremia, and presence of multidrug resistant (MDR) isolate were studied. The isolates were analyzed for the presence of toxin genes, proteolytic activity, swimming and twitching motility, and pyocyanin production. The data were analyzed to ascertain which virulence factors correlated with poor outcomes defined as septic shock or death (SS) within 7 days. Septic shock or death occurred in 25/75 patients. Univariate analysis identified age as a host factor that exerted a significant effect on these outcomes. Ineffective antibiotics administered during the first 24 hours of treatment or MDR P. aeruginosa did not influence the frequency of SS, nor did the presence of lasB, exoA, exoS exoU, plcH genes and proteolytic activity. However, 6/8 patients infected with non-motile isolates, developed SS, p = 0.014 and 5/6 isolates that produced large amounts of pyocyanin (>18ug/ml), were associated with SS, p = 0.014. Multivariate analysis indicated that the odds ratio (OR) for development of SS with a non-motile isolate was 6.8, with a 95% confidence interval (CI) (1.37, 51.5), p = 0.030 and with high pyocyanin producing isolates, an OR of 16.9, 95% CI = (2.27, 360), p = .017. This study evaluating the role of microbial factors that significantly effect outcomes following Pa bloodstream infection suggests that P. aeruginosa strains showing high pyocyanin production and the lack of motility independently increase the risk of SS.


Assuntos
Bacteriemia/mortalidade , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Choque Séptico/etiologia , Fatores de Virulência/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/complicações , Bacteriemia/microbiologia , Feminino , Genes Bacterianos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/mortalidade , Fatores de Risco , Choque Séptico/microbiologia , Choque Séptico/mortalidade , Adulto Jovem
10.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066609

RESUMO

Pseudomonas aeruginosa (P. aeruginosa), one of the dangerous multidrug resistance pathogens, orchestrates virulence factors production through quorum sensing (QS). Since the exploration of QS inhibitors, targeting virulence to circumvent bacterial pathogenesis without causing significant growth inhibition is a promising approach to treat P. aeruginosa infections. The present study has evaluated the anti-QS and anti-infective activity of epigallocatechin-3-gallate (EGCG), a bioactive ingredient of the traditional green tea, against P. aeruginosa. EGCG showed significant inhibitory effects on the development of biofilm, protease, elastase activity, swimming, and swarming motility, which was positively related to the production of C4-AHL. The expression of QS-related and QS-regulated virulence factors genes was also evaluated. Quantitative PCR analysis showed that EGCG significantly reduced the expression of las, rhl, and PQS genes and was highly correlated with the alterations of C4-AHL production. In-vivo experiments demonstrated that EGCG treatment reduced P. aeruginosa pathogenicity in Caenorhabditis elegans (C. elegans). EGCG increased the survival of C. elegans by 23.25%, 30.04%, and 36.35% in a dose-dependent manner. The findings of this study strongly suggest that EGCG could be a potential candidate for QS inhibition as an anti-virulence compound against bacterial infection.


Assuntos
Biofilmes/crescimento & desenvolvimento , Catequina/análogos & derivados , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/efeitos dos fármacos , Acil-Butirolactonas/metabolismo , Animais , Biofilmes/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Catequina/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glicolipídeos/biossíntese , Testes de Sensibilidade Microbiana , Movimento , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Piocianina/biossíntese , Percepção de Quorum/genética
11.
Microb Biotechnol ; 14(4): 1613-1626, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34000093

RESUMO

Pseudomonas aeruginosa produces phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO), which aid its anaerobic survival by mediating electron transfer to distant oxygen. These natural secondary metabolites are being explored in biotechnology to mediate electron transfer to the anode of bioelectrochemical systems. A major challenge is that only a small fraction of electrons from microbial substrate conversion is recovered. It remained unclear whether phenazines can re-enter the cell and thus, if the electrons accessed by the phenazines arise mainly from cytoplasmic or periplasmic pathways. Here, we prove that the periplasmic glucose dehydrogenase (Gcd) of P. aeruginosa and P. putida is involved in the reduction of natural phenazines. PYO displayed a 60-fold faster enzymatic reduction than PCA; PCA was, however, more stable for long-term electron shuttling to the anode. Evaluation of a Gcd knockout and overexpression strain showed that up to 9% of the anodic current can be designated to this enzymatic reaction. We further assessed phenazine uptake with the aid of two molecular biosensors, which experimentally confirm the phenazines' ability to re-enter the cytoplasm. These findings significantly advance the understanding of the (electro) physiology of phenazines for future tailoring of phenazine electron discharge in biotechnological applications.


Assuntos
Fenazinas , Piocianina , Transporte de Elétrons , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo
12.
Vet Immunol Immunopathol ; 237: 110265, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33989854

RESUMO

Severe equine asthma is characterized by airway hyperresponsiveness, neutrophilic inflammation and structural alterations of the lower airways. In asthmatic horses with neutrophilic inflammation, there is insensitivity to corticosteroids characterized by the persistence of neutrophils within the airways with therapy. We hypothesized that hypoxia or oxidative stress in the microenvironment of the lung contributes to this insensitivity of neutrophils to corticosteroids in asthmatic horses. Blood neutrophils isolated from horses with severe asthma (N = 8) and from healthy controls (N = 8) were incubated under different cell culture conditions simulating hypoxia and oxidative stress and, in the presence, or absence of dexamethasone. The pro-inflammatory gene and protein expression of neutrophils were studied. In both groups, pyocyanin-induced oxidative stress increased the mRNA expression of IL-8, IL-1ß, and TNF-α. While IL-1ß and TNF-α were downregulated by dexamethasone under these conditions, IL-8 was not. Simulated hypoxic conditions did not enhance pro-inflammatory gene expression in neutrophils from either group of horses. In conclusion, oxidative stress but not hypoxia may contribute to corticosteroid insensitivity via a selective gene regulation pathway. Equine neutrophil responses were similar in both heathy and asthmatic horses, indicating that it is not specific to asthmatic inflammation.


Assuntos
Asma/veterinária , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Doenças dos Cavalos/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Animais , Asma/tratamento farmacológico , Asma/imunologia , Células Cultivadas , Quimiocinas/biossíntese , Quimiocinas/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Doenças dos Cavalos/imunologia , Cavalos , Hipóxia/imunologia , Hipóxia/metabolismo , Hipóxia/veterinária , Mediadores da Inflamação/metabolismo , Interleucina-17/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Neutrófilos/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/imunologia , Piocianina/farmacologia
13.
Nat Commun ; 12(1): 2103, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33833234

RESUMO

Mitochondrial diseases impair oxidative phosphorylation and ATP production, while effective treatment is still lacking. Defective complex III is associated with a highly variable clinical spectrum. We show that pyocyanin, a bacterial redox cycler, can replace the redox functions of complex III, acting as an electron shunt. Sub-µM pyocyanin was harmless, restored respiration and increased ATP production in fibroblasts from five patients harboring pathogenic mutations in TTC19, BCS1L or LYRM7, involved in assembly/stabilization of complex III. Pyocyanin normalized the mitochondrial membrane potential, and mildly increased ROS production and biogenesis. These in vitro effects were confirmed in both DrosophilaTTC19KO and in Danio rerioTTC19KD, as administration of low concentrations of pyocyanin significantly ameliorated movement proficiency. Importantly, daily administration of pyocyanin for two months was not toxic in control mice. Our results point to utilization of redox cyclers for therapy of complex III disorders.


Assuntos
Trifosfato de Adenosina/biossíntese , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/genética , Doenças Mitocondriais/tratamento farmacológico , Proteínas Mitocondriais/genética , Piocianina/farmacologia , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Drosophila melanogaster , Complexo III da Cadeia de Transporte de Elétrons/genética , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Doenças Mitocondriais/patologia , Chaperonas Moleculares/genética , Oxirredução/efeitos dos fármacos , Piocianina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra
14.
Appl Environ Microbiol ; 87(12): e0002921, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-33837019

RESUMO

The Pseudomonas aeruginosa LasR-LasI (LasR-I) quorum sensing system regulates secreted proteases that can be exploited by cheaters, such as quorum sensing receptor-defective (lasR) mutants. lasR mutants emerge in populations growing on casein as a sole source of carbon and energy. These mutants are exploitative cheaters because they avoid the substantial cost of engaging in quorum sensing. Previous studies showed that quorum sensing increases resistance to some antibiotics, such as tobramycin. Here, we show that tobramycin suppressed the emergence of lasR mutants in casein-passaged populations. Several mutations accumulated in those populations, indicating evidence of antibiotic adaptation. We found that mutations in one gene, ptsP, increased antibiotic resistance and also pleiotropically increased production of a quorum sensing-controlled phenazine, pyocyanin. When passaged on casein, ptsP mutants suppressed cheaters in a manner that was tobramycin independent. We found that the mechanism of cheater suppression in ptsP mutants relied on pyocyanin, which acts as a policing toxin by selectively blocking growth of cheaters. Thus, tobramycin suppresses lasR mutants through two mechanisms: first, through direct effects on cheaters and, second, by selecting mutations in ptsP that suppressed cheating in a tobramycin-independent manner. This work demonstrates how adaptive mutations can alter the dynamics of cooperator-cheater relationships, which might be important for populations adapting to antibiotics during interspecies competition or infections. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa is a model for understanding quorum sensing, a type of cell-cell signaling important for cooperation. Quorum sensing controls production of cooperative goods, such as exoenzymes, which are vulnerable to cheating by quorum sensing-defective mutants. Because uncontrolled cheating can ultimately cause a population to collapse, much focus has been on understanding how P. aeruginosa can control cheaters. We show that an antibiotic, tobramycin, can suppress cheaters in cooperating P. aeruginosa populations. Tobramycin suppresses cheaters directly because the cheaters are more susceptible to tobramycin than cooperators. Tobramycin also selects for mutations in a gene, ptsP, that suppresses cheaters independent of tobramycin through pleiotropic regulation of a policing toxin, pyocyanin. This work supports the idea that adaptation to antibiotics can have unexpected effects on the evolution of quorum sensing and has implications for understanding how cooperation evolves in dynamic bacterial communities.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum , Tobramicina/farmacologia , Proteínas de Bactérias/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiologia , Piocianina/metabolismo , Transativadores/genética
15.
Science ; 371(6533): 1059-1063, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674494

RESUMO

Color detection is used by animals of diverse phyla to navigate colorful natural environments and is thought to require evolutionarily conserved opsin photoreceptor genes. We report that Caenorhabditis elegans roundworms can discriminate between colors despite the fact that they lack eyes and opsins. Specifically, we found that white light guides C. elegans foraging decisions away from a blue-pigment toxin secreted by harmful bacteria. These foraging decisions are guided by specific blue-to-amber ratios of light. The color specificity of color-dependent foraging varies notably among wild C. elegans strains, which indicates that color discrimination is ecologically important. We identified two evolutionarily conserved cellular stress response genes required for opsin-independent, color-dependent foraging by C. elegans, and we speculate that cellular stress response pathways can mediate spectral discrimination by photosensitive cells and organisms-even by those lacking opsins.


Assuntos
Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/efeitos da radiação , Visão de Cores , Comportamento Alimentar , Animais , Aprendizagem da Esquiva , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Sequência Conservada , Escherichia coli , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Piocianina/toxicidade
16.
Arch Microbiol ; 203(6): 2863-2874, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33751172

RESUMO

This research aimed to study siderophores secreted from Pseudomonas sp. PDMZnCd2003, a Zn/Cd tolerant bacterium. The effects of Zn and/or Cd stress were examined in nutrient broth to achieve the actual environmental conditions. Acid and alkali supernatants and liquid-liquid extraction with ethyl acetate and butanol were carried out to obtain crude extracts containing different amounts of the metals. The bacterial growth, UV-visible spectra of the supernatants and siderophore production indicated that the production of siderophores tended to be linked to primary metabolites. Pyocyanin was produced in all treatments, while pyoverdine was induced by stress from the metals, especially Cd. FT-IR spectra showed C=O groups and sulfur functional groups that were involved in binding with the metals. LC-MS revealed that pyocyanin, 1-hydroxy phenazine, pyoverdine, and pyochelin were present in the crude extracts. S K-edge XANES spectra showed that the main sulfur species in the extracts were the reduced forms of sulfide, thiol, and disulfide, and their oxidation states were affected by coordination with Zn and/or Cd. In addition, Zn K-edge EXAFS spectra and Cd K-edge EXAFS spectra presented Zn-O and Cd-O as coordination in the first shell, in case the extracts contained less metal. Although the mix O/S ligands had chelation bonding with Zn and Cd in the other extracts. For the role of S groups in pyochelin binding with the metals, this was the first report. The results of these experiments could be extended to Pseudomonas that respond to metal contaminated environments.


Assuntos
Cádmio/farmacologia , Pseudomonas/metabolismo , Sideróforos/isolamento & purificação , Zinco/farmacologia , Nutrientes , Pseudomonas/efeitos dos fármacos , Pseudomonas/crescimento & desenvolvimento , Piocianina/biossíntese
17.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723058

RESUMO

Pseudomonas aeruginosa is an opportunistic human pathogen that develops difficult-to-treat biofilms in immunocompromised individuals, cystic fibrosis patients, and in chronic wounds. P. aeruginosa has an arsenal of physiological attributes that enable it to evade standard antibiotic treatments, particularly in the context of biofilms where it grows slowly and becomes tolerant to many drugs. One of its survival strategies involves the production of the redox-active phenazine, pyocyanin, which promotes biofilm development. We previously identified an enzyme, PodA, that demethylated pyocyanin and disrupted P. aeruginosa biofilm development in vitro. Here, we asked if this protein could be used as a potential therapeutic for P. aeruginosa infections together with tobramycin, an antibiotic typically used in the clinic. A major roadblock to answering this question was the poor yield and stability of wild-type PodA purified from standard Escherichia coli overexpression systems. We hypothesized that the insufficient yields were due to poor packing within PodA's obligatory homotrimeric interfaces. We therefore applied the protein design algorithm, AffiLib, to optimize the symmetric core of this interface, resulting in a design that incorporated five mutations leading to a 20-fold increase in protein yield from heterologous expression and purification and a substantial increase in stability to environmental conditions. The addition of the designed PodA with tobramycin led to increased killing of P. aeruginosa cultures under oxic and hypoxic conditions in both the planktonic and biofilm states. This study highlights the potential for targeting extracellular metabolites to assist the control of P. aeruginosa biofilms that tolerate conventional antibiotic treatment.


Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Oxirredutases N-Desmetilantes/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Tobramicina/farmacologia , Desenho de Fármacos , Sinergismo Farmacológico , Humanos , Oxirredutases N-Desmetilantes/farmacologia , Engenharia de Proteínas , Pseudomonas aeruginosa/fisiologia
18.
PLoS One ; 16(3): e0248014, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33662048

RESUMO

Pseudomonas aeruginosa is an environmental pathogen that can cause severe infections in immunocompromised patients. P. aeruginosa infections are typically treated with multiple antibiotics including tobramycin, ciprofloxacin, and meropenem. However, antibiotics do not always entirely clear the bacteria from the infection site, where they may remain virulent. This is because the effective antibiotic concentration and diffusion in vitro may differ from the in vivo environment in patients. Therefore, it is important to understand the effect of non-lethal sub-inhibitory antibiotic concentrations on bacterial phenotype. Here, we investigate if sub-inhibitory antimicrobial concentrations cause alterations in bacterial virulence factor production using pyocyanin as a model toxin. We tested this using the aforementioned antibiotics on 10 environmental P. aeruginosa strains. Using on-the-spot electrochemical screening, we were able to directly quantify changes in production of pyocyanin in a measurement time of 17 seconds. Upon selecting 3 representative strains to further test the effects of sub-minimum inhibitory concentration (MICs), we found that pyocyanin production changed significantly when the bacteria were exposed to 10-fold MIC of the 3 antibiotics tested, and this was strain specific. A series of biologically relevant measured pyocyanin concentrations were also used to assess the effects of increased virulence on a culture of epithelial cells. We found a decreased viability of the epithelial cells when incubated with biologically relevant pyocyanin concentrations. This suggests that the antibiotic-induced virulence also is a value worth being enclosed in regular testing of pathogens.


Assuntos
Antibacterianos/farmacologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Fatores de Virulência/metabolismo , Linhagem Celular , Humanos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/metabolismo
19.
PLoS Biol ; 19(3): e3001093, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33690640

RESUMO

Bacterial opportunistic human pathogens frequently exhibit intrinsic antibiotic tolerance and resistance, resulting in infections that can be nearly impossible to eradicate. We asked whether this recalcitrance could be driven by these organisms' evolutionary history as environmental microbes that engage in chemical warfare. Using Pseudomonas aeruginosa as a model, we demonstrate that the self-produced antibiotic pyocyanin (PYO) activates defenses that confer collateral tolerance specifically to structurally similar synthetic clinical antibiotics. Non-PYO-producing opportunistic pathogens, such as members of the Burkholderia cepacia complex, likewise display elevated antibiotic tolerance when cocultured with PYO-producing strains. Furthermore, by widening the population bottleneck that occurs during antibiotic selection and promoting the establishment of a more diverse range of mutant lineages, PYO increases apparent rates of mutation to antibiotic resistance to a degree that can rival clinically relevant hypermutator strains. Together, these results reveal an overlooked mechanism by which opportunistic pathogens that produce natural toxins can dramatically modulate the efficacy of clinical antibiotics and the evolution of antibiotic resistance, both for themselves and other members of clinically relevant polymicrobial communities.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Bactérias/genética , Burkholderia cepacia/efeitos dos fármacos , Burkholderia cepacia/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Tolerância a Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Piocianina/farmacologia
20.
World J Microbiol Biotechnol ; 37(4): 66, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33740144

RESUMO

Pseudomonas aeruginosa is one of the vulnerable opportunistic pathogens associated with nosocomial infections, cystic fibrosis, burn wounds and surgical site infections. Several studies have reported that quorum sensing (QS) systems are controlled the P. aeruginosa pathogenicity. Hence, the targeting of QS considered as an alternative approach to control P. aeruginosa infections. This study aimed to evaluate the anti-quorum sensing and antibiofilm inhibitory potential of Musa paradisiaca against Chromobacterium violaceum (ATCC 12472) and Pseudomonas aeruginosa. The methanol extract of M. paradisiacsa exhibits that better antibiofilm potential against P. aeruginosa. Then, the crude methanol extract was subjected to purify by column chromatography and collected the fractions. The mass-spectrometric analysis of a methanol extract of M. paradisiaca revealed that 1,8-cineole is the major compounds. 1, 8-cineole significantly inhibited the QS regulated violacein production in C. violaceum. Moreover, 1,8-cineole significantly inhibited the QS mediated virulence production and biofilm formation of P. aeruginosa without affecting their growth. The real-time PCR analysis showed the downregulation of autoinducer synthase and transcriptional regulator genes upon 1,8-cineole treatment. The findings of the present study strongly suggested that metabolite of M. paradisiaca impedes P. aeruginosa QS system and associated virulence productions.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Eucaliptol/química , Eucaliptol/farmacologia , Musa/química , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Alginatos/metabolismo , Biofilmes/crescimento & desenvolvimento , Chromobacterium/efeitos dos fármacos , Eucaliptol/isolamento & purificação , Expressão Gênica/efeitos dos fármacos , Glicolipídeos/biossíntese , Índia , Testes de Sensibilidade Microbiana , Extratos Vegetais/farmacologia , Polissacarídeos Bacterianos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Piocianina/biossíntese , Virulência/efeitos dos fármacos , Fatores de Virulência
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