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1.
Int J Mol Sci ; 24(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36675051

RESUMO

The bacterial heat-shock response is regulated by the alternative sigma factor, σ32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose-inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH (σ32) regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden change in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurred without arabinose induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased five-fold within 15 min of arabinose addition. Transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many genes essential for P. aeruginosa growth are induced by RpoH. Other stress response genes induced by RpoH are involved in damaged nucleic acid repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. Phenotypic analysis of P. aeruginosa ∆rpoH (PBAD-rpoH) showed that it is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also had increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside sensitivity of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes, such as aminoglycoside acetyl transferases, that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient-limiting conditions.


Assuntos
Pseudomonas aeruginosa , Regulon , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Choque Térmico/metabolismo , Arabinose , Fator sigma/genética , Fator sigma/metabolismo , Aminoglicosídeos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transcrição Genética
2.
Commun Biol ; 6(1): 16, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609683

RESUMO

Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in cancer, and therapies that incorporate bacteria have been tried in various types of cancer. We previously demonstrated that cupredoxin azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa, enters human cancer cells and induces apoptotic death1-4. However, the physiological interactions between P. aeruginosa and humans and their role in tumor homeostasis are largely unknown. Here, we show that P. aeruginosa upregulated azurin secretion in response to increasing numbers of and proximity to cancer cells. Conversely, cancer cells upregulated aldolase A secretion in response to increasing proximity to P. aeruginosa, which also correlated with enhanced P. aeruginosa adherence to cancer cells. Additionally, we show that cancer patients had detectable P. aeruginosa and azurin in their tumors and exhibited increased overall survival when they did, and that azurin administration reduced tumor growth in transgenic mice. Our results suggest host-bacterial symbiotic mutualism acting as a diverse adjunct to the host defense system via inter-kingdom communication mediated by the evolutionarily conserved proteins azurin and human aldolase A. This improved understanding of the symbiotic relationship of bacteria with humans indicates the potential contribution to tumor homeostasis.


Assuntos
Azurina , Neoplasias , Camundongos , Animais , Humanos , Azurina/genética , Azurina/metabolismo , Azurina/farmacologia , Pseudomonas aeruginosa/metabolismo , Frutose-Bifosfato Aldolase , Neoplasias/genética , Fenômenos Fisiológicos Celulares
3.
BMC Microbiol ; 23(1): 6, 2023 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-36617571

RESUMO

The control of cellular zinc (Zn) concentrations by dedicated import and export systems is essential for the survival and virulence of Pseudomonas aeruginosa. The transcription of its many Zn transporters is therefore tightly regulated by a known set of transcription factors involved in either the import or the export of Zn. In this work, we show that the Zur protein, a well-known repressor of Zn import, plays a dual role and functions in both import and export processes. In a situation of Zn excess, Zur represses Zn entry, but also activates the transcription of czcR, a positive regulator of the Zn export system. To achieve this, Zur binds at two sites, located by DNA footprinting in the region downstream the czcR transcription start site. In agreement with this regulation, a delay in induction of the efflux system is observed in the absence of Zur and Zn resistance is reduced. The discovery of this regulation highlights a new role of Zur as global regulator of Zn homeostasis in P. aeruginosa disclosing an important link between Zur and zinc export.


Assuntos
Pseudomonas aeruginosa , Zinco , Zinco/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/genética
4.
Cells ; 12(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36611990

RESUMO

Pseudomonas aeruginosa is one of the most virulent opportunistic Gram-negative bacterial pathogens in humans. It causes many acute and chronic infections with morbidity and mortality rates as high as 40%. P. aeruginosa owes its pathogenic versatility to a large arsenal of cell-associated and secreted virulence factors which enable this pathogen to colonize various niches within hosts and protect it from host innate immune defenses. Induction of cytotoxicity in target host cells is a major virulence strategy for P. aeruginosa during the course of infection. P. aeruginosa has invested heavily in this strategy, as manifested by a plethora of cytotoxins that can induce various forms of cell death in target host cells. In this review, we provide an in-depth review of P. aeruginosa cytotoxins based on their mechanisms of cytotoxicity and the possible consequences of their cytotoxicity on host immune responses.


Assuntos
Infecções por Pseudomonas , Humanos , Virulência , Fatores de Virulência/metabolismo , Citotoxinas , Pseudomonas aeruginosa/metabolismo
5.
J Med Chem ; 66(2): 1380-1425, 2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36634346

RESUMO

We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.


Assuntos
Acinetobacter baumannii , Inibidores da Topoisomerase II , Humanos , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/química , Pseudomonas aeruginosa/metabolismo , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Escherichia coli/metabolismo , Benzotiazóis , Testes de Sensibilidade Microbiana , DNA Girase/metabolismo
6.
Bioorg Med Chem ; 79: 117167, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36682225

RESUMO

Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence pathways in P. aeruginosa, including quorum sensing (QS) and Type IV pili (TFP). We show that auranofin acts via multiple targets, one of which being Vfr. Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/metabolismo , Auranofina/farmacologia , Auranofina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Fatores de Virulência/metabolismo , Fatores de Virulência/farmacologia , Fatores de Virulência/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Biofilmes , Percepção de Quorum , Proteínas de Bactérias/farmacologia
7.
Int J Mol Sci ; 24(2)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36674894

RESUMO

Chemosensory pathways and two-component systems are important bacterial signal transduction systems. In the human pathogen Pseudomonas aeruginosa, these systems control many virulence traits. Previous studies showed that inorganic phosphate (Pi) deficiency induces virulence. We report here the abundance of chemosensory and two-component signaling proteins of P. aeruginosa grown in Pi deficient and sufficient media. The cellular abundance of chemoreceptors differed greatly, since a 2400-fold difference between the most and least abundant receptors was observed. For many chemoreceptors, their amount varied with the growth condition. The amount of chemoreceptors did not correlate with the magnitude of chemotaxis to their cognate chemoeffectors. Of the four chemosensory pathways, proteins of the Che chemotaxis pathway were most abundant and showed little variation in different growth conditions. The abundance of chemoreceptors and solute binding proteins indicates a sensing preference for amino acids and polyamines. There was an excess of response regulators over sensor histidine kinases in two-component systems. In contrast, ratios of the response regulators CheY and CheB to the histidine kinase CheA of the Che pathway were all below 1, indicative of different signaling mechanisms. This study will serve as a reference for exploring sensing preferences and signaling mechanisms of other bacteria.


Assuntos
Proteínas de Bactérias , Pseudomonas aeruginosa , Humanos , Histidina Quinase/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Proteínas de Transporte/metabolismo , Quimiotaxia/fisiologia , Transdução de Sinais
8.
J Med Chem ; 66(1): 553-576, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36548006

RESUMO

Rising infection rates with multidrug-resistant pathogens calls for antibiotics with novel modes of action. Herein, we identify the inner membrane protein TonB, a motor of active uptake in Gram-negative bacteria, as a novel target in antimicrobial therapy. The interaction of the TonB box of outer membrane transporters with TonB is crucial for the internalization of essential metabolites. We designed TonB box peptides and coupled them with synthetic siderophores in order to facilitate their uptake into bacteria in up to 32 synthetic steps. Three conjugates repressed the growth of Pseudomonas aeruginosa cells unable to produce their own siderophores, with minimal inhibitory concentrations between 0.1 and 0.5 µM. The transporters mediating uptake of these compounds were identified as PfeA and PirA. The study illustrates a variant of cellular suicide where a transporter imports its own inhibitor and demonstrates that artificial siderophores can import cargo with molecular weights up to 4 kDa.


Assuntos
Antibacterianos , Sideróforos , Sideróforos/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Membrana/metabolismo , Transporte Biológico , Proteínas de Membrana Transportadoras/metabolismo
9.
Appl Environ Microbiol ; 89(1): e0171422, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36533942

RESUMO

Glutaredoxins (Grxs), ubiquitous redox enzymes belonging to the thioredoxin family, catalyze the reduction of thiol-disulfide exchange reactions in a glutathione-dependent manner. A Pseudomonas aeruginosa ΔgrxD mutant exhibited hypersensitivity to oxidative stress-generating agents, such as paraquat (PQ) and cumene hydroperoxide (CHP). In vitro studies showed that P. aeruginosa GrxD acts as an electron donor for organic hydroperoxide resistance enzyme (Ohr) during CHP degradation. The ectopic expression of iron-sulfur cluster ([Fe-S]) carrier proteins, including ErpA, IscA, and NfuA, complements the function of GrxD in the ΔgrxD mutant under PQ toxicity. Constitutively high expression of iscR, nfuA, tpx, and fprB was observed in the ΔgrxD mutant. These results suggest that GrxD functions as a [Fe-S] cluster carrier protein involved in [Fe-S] cluster maturation. Moreover, the ΔgrxD mutant demonstrates attenuated virulence in a Drosophila melanogaster host model. Altogether, the data shed light on the physiological role of GrxD in oxidative stress protection and virulence of the human pathogen, P. aeruginosa. IMPORTANCE Glutaredoxins (Grxs) are ubiquitous disulfide reductase enzymes. Monothiol Grxs, containing a CXXS motif, play an essential role in iron homeostasis and maturation of [Fe-S] cluster proteins in various organisms. We now establish that the human pathogen Pseudomonas aeruginosa GrxD is crucial for bacterial virulence, maturation of [Fe-S] clusters and facilitation of Ohr enzyme activity. GrxD contains a conserved signature monothiol motif (C29GFS), in which C29 is essential for its function in an oxidative stress protection. Our findings reveal the physiological roles of GrxD in oxidative stress protection and virulence of P. aeruginosa.


Assuntos
Glutarredoxinas , Pseudomonas aeruginosa , Animais , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Virulência , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Drosophila melanogaster/metabolismo , Estresse Oxidativo , Ferro/metabolismo
10.
Eur J Med Chem ; 247: 115025, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36549118

RESUMO

In this work, ß-thiogalactoside mimetics bearing 1,1-diarylmethylene or benzophenone aglycons have been prepared and assayed for their affinity towards LecA, a lectin and virulence factor from Pseudomonas aeruginosa involved in bacterial adhesion and biofilm formation. The hit compound presents higher efficiency than previously described monovalent inhibitors and the crystal structure confirmed the occurrence of additional contacts between the aglycone and the protein surface. The highest affinity (160 nM) was obtained for a divalent ligand containing two galactosides. The monovalent high affinity compound (Kd = 1 µM) obtained through structure-activity relationship (SAR) showed efficient antibiofilm activity with no associated bactericidal activity.


Assuntos
Adesinas Bacterianas , Pseudomonas aeruginosa , Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Galactosídeos/química , Relação Estrutura-Atividade , Biofilmes , Antibacterianos/farmacologia , Antibacterianos/metabolismo
11.
Chemosphere ; 313: 137471, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36493888

RESUMO

It is a well-established fact that aerobic denitrifying strains are profoundly affected by antibiotics, but bacterium performing simultaneous aerobic denitrification and antibiotic degradation is hardly reported. Here, a typical aerobic denitrifying bacterium Pseudomonas aeruginosa PCN-2 was discovered to be capable of sulfamethoxazole (SMX) degradation. The results showed that nitrate removal efficiency was decreased from 100% to 88.12%, but the resistance of strain PCN-2 to SMX stress was enhanced with the increment of SMX concentration from 0 to 100 mg/L. Transcriptome analysis revealed that the down-regulation of energy metabolism pathways rather than the denitrifying functional genes was responsible for the suppressed nitrogen removal, while the up-regulation of antibiotic resistance pathways (e.g., biofilm formation, multi-drug efflux system, and quorum sensing) ensured the survival of bacterium and the carrying out of aerobic denitrification. Intriguingly, strain PCN-2 could degrade SMX during aerobic denitrification. Seven metabolites were identified by the UHPLC-MS, and three degradation pathways (which includes a new pathway that has never been reported) was proposed combined with the expressions of drug metabolic genes (e.g., cytP450, FMN, ALDH and NAT). This work provides a mechanistic understanding of the metabolic adaption of strain PCN-2 under SMX stress, which provided a broader idea for the treatment of SMX-containing wastewater.


Assuntos
Desnitrificação , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Sulfametoxazol/farmacologia , Sulfametoxazol/metabolismo , Aerobiose , Nitrogênio/metabolismo , Perfilação da Expressão Gênica
12.
Biochem Biophys Res Commun ; 642: 107-112, 2023 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-36566561

RESUMO

Avoidance of harmful substances is survival strategy used cross invertebrates and vertebrates. For example, the nematode Caenorhabditis elegans evolves a sufficient avoidance response to pathogenic bacteria. Despite G protein has been found to exert neural plasticity for avoidance behaviours in C. elegans, the function of Gi/o and Gq subunit signalling in experience-dependent aversive behaviour remains unclear. In this study, we show that EGL-30/Gq coupled with EGL-8/UNC-13 regulates aversive behaviour of C. elegans to pathogenic bacterium Pseudomonas aeruginosa PA01 via acetylcholine and its receptor nAChR. Pyocyanin, a toxin secreted from P. aeruginosa, acts as a signal molecule to trigger aversive behaviour. ODR-3 and ODR-7 in AWA and AWC neurons function as upstream of EGL-30 to induce experience-dependent aversive behaviour to P. aeruginosa, respectively. These results suggested that a novel signalling pathway to regulate a behavioural response.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Pseudomonas aeruginosa/metabolismo , Aprendizagem da Esquiva , Proteínas de Caenorhabditis elegans/metabolismo , Transdução de Sinais/fisiologia
13.
Sci Rep ; 12(1): 21444, 2022 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-36509824

RESUMO

We previously demonstrated that P. aeruginosa isolates that persisted in children with cystic fibrosis (CF) despite inhaled tobramycin treatment had increased anti-Psl antibody binding in vitro compared to those successfully eradicated. We aimed to validate these findings by directly visualizing P. aeruginosa in CF sputum. This was a prospective observational study of children with CF with new-onset P. aeruginosa infection who underwent inhaled tobramycin eradication treatment. Using microbial identification passive clarity technique (MiPACT), P. aeruginosa was visualized in sputum samples obtained before treatment and classified as persistent or eradicated based on outcomes. Pre-treatment isolates were also grown as biofilms in vitro. Of 11 patients enrolled, 4 developed persistent infection and 7 eradicated infection. P. aeruginosa biovolume and the number as well as size of P. aeruginosa aggregates were greater in the sputum of those with persistent compared with eradicated infections (p < 0.01). The amount of Psl antibody binding in sputum was also greater overall (p < 0.05) in samples with increased P. aeruginosa biovolume. When visualized in sputum, P. aeruginosa had a greater biovolume, with more expressed Psl, and formed more numerous, larger aggregates in CF children who failed eradication therapy compared to those who successfully cleared their infection.


Assuntos
Fibrose Cística , Infecções por Pseudomonas , Criança , Humanos , Pseudomonas aeruginosa/metabolismo , Fibrose Cística/complicações , Fibrose Cística/tratamento farmacológico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/complicações , Tobramicina/uso terapêutico , Tobramicina/metabolismo , Escarro
14.
Microb Cell Fact ; 21(1): 262, 2022 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-36528623

RESUMO

BACKGROUND: Pyocyanin, a specific extracellular secondary metabolite pigment produced by Pseudomonas aeruginosa, exhibits redox activity and has toxic effects on mammalian cells, making it a new and potent alternative for treating cancer. Breast cancer (BC) treatment is now defied by acquired and de novo resistance to chemotherapy, radiation, or targeted therapies. Therefore, the anticancer activity of purified and characterized pyocyanin was examined against BC in our study. RESULTS: The maximum production of pyocyanin (53 µg/ml) was achieved by incubation of the highest pyocyanin-producing P. aeruginosa strain (P32) in pH-adjusted peptone water supplemented with 3% cetrimide under shaking conditions at 37 °C for 3 days. The high purity of the extracted pyocyanin was proven by HPLC against standard pyocyanin. The stability of pyocyanin was affected by the solvent in which it was stored. Therefore, the purified pyocyanin extract was lyophilized to increase its shelf-life up to one year. Using the MTT assay, we reported, for the first time, the cytotoxic effect of pyocyanin against human breast adenocarcinoma (MCF-7) with IC50 = 15 µg/ml while it recorded a safe concentration against human peripheral blood mononuclear cells (PBMCs). The anticancer potential of pyocyanin against MCF-7 was associated with its apoptotic and necrotic activities which were confirmed qualitatively and quantitively using confocal laser scanning microscopy, inverted microscopy, and flow cytometry. Caspase-3 measurements, using real-time PCR and western blot, revealed that pyocyanin exerted its apoptotic activity against MCF-7 through caspase-3 activation. CONCLUSION: Our work demonstrated that pyocyanin may be an ideal anticancer candidate, specific to cancer cells, for treating MCF-7 by its necrotic and caspase-3-dependent apoptotic activities.


Assuntos
Adenocarcinoma , Neoplasias da Mama , Animais , Humanos , Feminino , Piocianina/metabolismo , Piocianina/farmacologia , Pseudomonas aeruginosa/metabolismo , Caspase 3/metabolismo , Células MCF-7 , Leucócitos Mononucleares/metabolismo , Neoplasias da Mama/tratamento farmacológico , Mamíferos/metabolismo
15.
Molecules ; 27(24)2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36558064

RESUMO

An anti-biofilm that can inhibit the matrix of biofilm formation is necessary to prevent recurrent and chronic Pseudomonas aeruginosa infection. This study aimed to design compounds with a new mechanism through competitive inhibitory activity against phosphomannomutase/phosphoglucomutase (PMM/PGM), using in vitro assessment and a computational (in silico) approach. The active site of PMM/PGM was assessed through molecular redocking using L-tartaric acid as the native ligand and other small molecules, such as glucaric acid, D-sorbitol, and ascorbic acid. The docking program set the small molecules to the active site, showing a stable complex formation. Analysis of structural similarity, bioavailability, absorption, distribution, metabolism, excretion, and toxicity properties proved the potential application of ligands as an anti-biofilm. In vitro assessment with crystal violet showed that the ligands could reach up to 95.87% inhibition at different concentrations. The nitrocellulose membrane and scanning electron microscopic visualization showed that the untreated P. aeruginosa biofilm was denser than the ligand-treated biofilm.


Assuntos
Fosfoglucomutase , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Ligantes , Fosfoglucomutase/química , Fosfoglucomutase/metabolismo , Domínio Catalítico , Biofilmes , Antibacterianos/farmacologia
16.
Int J Mol Sci ; 23(24)2022 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-36555423

RESUMO

Antimicrobial resistance is an old and silent pandemic. Resistant organisms emerge in parallel with new antibiotics, leading to a major global public health crisis over time. Antibiotic resistance may be due to different mechanisms and against different classes of drugs. These mechanisms are usually found in the same organism, giving rise to multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria. One resistance mechanism that is closely associated with the emergence of MDR and XDR bacteria is the efflux of drugs since the same pump can transport different classes of drugs. In Gram-negative bacteria, efflux pumps are present in two configurations: a transmembrane protein anchored in the inner membrane and a complex formed by three proteins. The tripartite complex has a transmembrane protein present in the inner membrane, a periplasmic protein, and a porin associated with the outer membrane. In Pseudomonas aeruginosa, one of the main pathogens associated with respiratory tract infections, four main sets of efflux pumps have been associated with antibiotic resistance: MexAB-OprM, MexXY, MexCD-OprJ, and MexEF-OprN. In this review, the function, structure, and regulation of these efflux pumps in P. aeruginosa and their actions as resistance mechanisms are discussed. Finally, a brief discussion on the potential of efflux pumps in P. aeruginosa as a target for new drugs is presented.


Assuntos
Antibacterianos , Proteínas de Membrana Transportadoras , Proteínas de Membrana Transportadoras/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/metabolismo
17.
Nat Commun ; 13(1): 7631, 2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36494359

RESUMO

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.


Assuntos
Alginatos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Alginatos/metabolismo , Ácidos Hexurônicos/metabolismo , Proteínas de Bactérias/metabolismo , Ácido Glucurônico/metabolismo , Biofilmes , Polímeros/metabolismo
18.
Int J Mol Sci ; 23(23)2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36498910

RESUMO

Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial infections. Its ability to survive under different conditions relies on a complex regulatory network engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use the available resources. P. aeruginosa&nbsp;PA3973 encodes an uncharacterized TetR family transcriptional regulator. In this study, we applied a transcriptome profiling (RNA-seq), genome-wide identification of binding sites using ChIP-seq, as well as the phenotype analyses to unravel the biological role of PA3973. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes in the mRNA level of 648 genes. Concomitantly, ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome. A 13 bp sequence motif was indicated as the binding site of PA3973. The PA3973 regulon encompasses the PA3972-PA3971 genes encoding a probable acyl-CoA dehydrogenase and a thioesterase. In vitro analysis showed PA3973 binding to PA3973p. Accordingly, the lack of PA3973 triggered increased expression of PA3972 and PA3971. The ∆PA3972-71 PAO1161 strain demonstrated impaired growth in the presence of stress-inducing agents hydroxylamine or hydroxyurea, thus suggesting the role of PA3972-71 in pathogen survival upon stress. Overall our results showed that TetR-type transcriptional regulator PA3973 has multiple binding sites in the P. aeruginosa genome and influences the expression of diverse genes, including PA3972-PA3971, encoding proteins with a proposed role in stress response.


Assuntos
Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulon/genética , Sítios de Ligação
19.
ACS Appl Mater Interfaces ; 14(46): 51763-51775, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36373472

RESUMO

Postcataract endophthalmitis (PCE), a devastating complication following cataract surgeries, is one of the most crucial diseases causing irreversible eye blindness. Pseudomonas aeruginosa (PA), a multiple-drug-resistance (MDR) pathogen, always leads to uncontrolled infection and severe inflammation in PCE that can be difficult to treat by antibiotics. Therefore, it is urgent to develop new feasible strategies composed of both antibacterial and anti-inflammatory capabilities. Here, we report a multifunctional non-antibiotic nanoplatform (Ga-mSiO2-BFN) comprised of clinically approved gallium, mesoporous silica, and bromfenac (BFN) as a co-modified release system to simultaneously eradicate MDR-PA infection and cure inflammation for PCE. The released gallium ions can disrupt bacterial iron metabolism. Meanwhile, the simultaneously released BFN can suppresses the inflammation both postoperation and postinfection of PCE. In the PCE rabbit model, the slit-lamp dispersion and retro-illumination micrograph, ophthalmic clinical grading, and etiological histopathology analysis demonstrated that Ga-mSiO2-BFN could eradicate the MDR infection and alleviate the secondary inflammation from MDR-PA infection. Moreover, both cellular biocompatibility and in vivo animal model application verified the biocompatibility. A potential antibacterial mechanism implicated in the antibacterial action was demonstrated by comprehensive assays of iron antagonism evolutionary curve, colony autofluorescence, polymerase chain reaction, and electron microscopy, showing a repressing siderophore peptide pyoverdine, pyoverdine synthetase D, and interfering with bacterial DNA synthesis. All composites of our nanoplatform were FDA approved, making the Ga-mSiO2-BFN as a potentially promising therapeutic approach for treating MDR-PA in PCE accompanying satisfactory prognosis and prospects for clinical translations.


Assuntos
Catarata , Endoftalmite , Gálio , Infecções por Pseudomonas , Animais , Coelhos , Pseudomonas aeruginosa/metabolismo , Gálio/farmacologia , Gálio/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Endoftalmite/tratamento farmacológico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antibacterianos/metabolismo , Bactérias/metabolismo , Ferro/metabolismo , Inflamação/tratamento farmacológico , Catarata/tratamento farmacológico
20.
Structure ; 30(12): 1626-1636.e4, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36379213

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for thousands of deaths every year in the United States. P. aeruginosa virulence factor production is mediated by quorum sensing, a mechanism of bacterial cell-cell communication that relies on the production and detection of signal molecules called autoinducers. In P. aeruginosa, the transcription factor receptor RhlR is activated by a RhlI-synthesized autoinducer. We recently showed that RhlR-dependent transcription is enhanced by a physical interaction with the enzyme PqsE via increased affinity of RhlR for promoter DNA. However, the molecular basis for complex formation and how complex formation enhanced RhlR transcriptional activity remained unclear. Here, we report the structure of ligand-bound RhlR in complex with PqsE. Additionally, we determined the structure of the complex bound with DNA, revealing the mechanism by which RhlR-mediated transcription is enhanced by PqsE, thereby establishing the molecular basis for RhlR-dependent virulence factor production in P. aeruginosa.


Assuntos
Pseudomonas aeruginosa , Percepção de Quorum , Percepção de Quorum/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
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