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1.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575940

RESUMO

The development and testing of new antimicrobial peptides (AMPs) represent an important milestone toward the development of new antimicrobial drugs that can inhibit the growth of pathogens and multidrug-resistant microorganisms such as Pseudomonas aeruginosa, Gram-negative bacteria. Most AMPs achieve these goals through mechanisms that disrupt the normal permeability of the cell membrane, which ultimately leads to the death of the pathogenic cell. Here, we developed a unique combination of a membrane penetrating peptide and peptides prone to amyloidogenesis to create hybrid peptide: "cell penetrating peptide + linker + amyloidogenic peptide". We evaluated the antimicrobial effects of two peptides that were developed from sequences with different propensities for amyloid formation. Among the two hybrid peptides, one was found with antibacterial activity comparable to antibiotic gentamicin sulfate. Our peptides showed no toxicity to eukaryotic cells. In addition, we evaluated the effect on the antimicrobial properties of amino acid substitutions in the non-amyloidogenic region of peptides. We compared the results with data on the predicted secondary structure, hydrophobicity, and antimicrobial properties of the original and modified peptides. In conclusion, our study demonstrates the promise of hybrid peptides based on amyloidogenic regions of the ribosomal S1 protein for the development of new antimicrobial drugs against P. aeruginosa.


Assuntos
Proteínas Amiloidogênicas/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas Ribossômicas/genética , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/farmacologia , Proteínas Amiloidogênicas/ultraestrutura , Antibacterianos/efeitos adversos , Humanos , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/patogenicidade , Proteínas Ribossômicas/farmacologia , Proteínas Ribossômicas/ultraestrutura
2.
Science ; 373(6556)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385369

RESUMO

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen Pseudomonas aeruginosa, analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identified numerous metabolic- and virulence-related transcriptional states that emerged dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm just several micrometers away, underscoring the importance of the microenvironment. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.


Assuntos
Pseudomonas aeruginosa/genética , Transcriptoma , Biofilmes/crescimento & desenvolvimento , Proteínas de Fímbrias/genética , Flagelina/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Hibridização in Situ Fluorescente , Fenótipo , Plâncton/genética , Plâncton/crescimento & desenvolvimento , Plâncton/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Piocinas/biossíntese , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , Análise Espaço-Temporal , Virulência/genética
3.
Molecules ; 26(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34443400

RESUMO

The search for coatings that extend the useful life of biomedical devices has been of great interest, and titanium has been of great relevance due to its innocuousness and low reactivity. This study contributes to the investigation of Ti/Ag films in different configurations (monolayer and multilayer) deposited by magnetron sputtering. The sessile droplet technique was applied to study wettability; greater film penetrability was obtained when Ag is the external layer, conferring high efficiency in cell adhesion. The morphological properties were characterized by SEM, which showed porous nuclei on the surface in the Ag coating and crystals embedded in the Ti film. The structural properties were studied by XRD, revealing the presence of TiO2 in the anatase crystalline phase in a proportion of 49.9% and the formation of a silver cubic network centered on the faces. Tafel polarization curves demonstrated improvements in the corrosion current densities of Ag/Ti/Ag/Ti/Ag/Ti/Ag/Ti and Ti/Ag compared to the Ag coating, with values of 0.1749, 0.4802, and 2.044 nA.m-2, respectively. Antimicrobial activity was evaluated against the bacteria Pseudomonas aeruginosa and Bacillus subtilis and the yeasts Candida krusei and Candida albicans, revealing that the Ti/Ag and Ag/Ti/Ag/Ti/Ag/Ti/Ag/Ti coatings exhibit promise in biomedical material applications.


Assuntos
Materiais Revestidos Biocompatíveis/química , Prata/farmacologia , Titânio/farmacologia , Molhabilidade/efeitos dos fármacos , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/patogenicidade , Candida/efeitos dos fármacos , Candida/patogenicidade , Materiais Revestidos Biocompatíveis/farmacologia , Humanos , Teste de Materiais , Pichia/efeitos dos fármacos , Pichia/patogenicidade , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Prata/química , Propriedades de Superfície , Titânio/química
4.
FASEB J ; 35(9): e21797, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383981

RESUMO

Pseudomonas aeruginosa is a frequent cause of hospital-acquired lung infections characterized by hyperinflammation, antibiotic resistance, and high morbidity/mortality. Here, we show that the genetic ablation of one cAMP-phosphodiesterase 4 subtype, PDE4B, is sufficient to protect mice from acute lung injury induced by P aeruginosa infection as it reduces pulmonary and systemic levels of pro-inflammatory cytokines, as well as pulmonary vascular leakage and mortality. Surprisingly, despite dampening immune responses, bacterial clearance in the lungs of PDE4B-KO mice is significantly improved compared to WT controls. In wildtypes, P aeruginosa-infection produces high systemic levels of several cytokines, including TNF-α, IL-1ß, and IL-6, that act as cryogens and render the animals hypothermic. This, in turn, diminishes their ability to clear the bacteria. Ablation of PDE4B curbs both the initial production of acute response cytokines, including TNF-α and IL-1ß, as well as their downstream signaling, specifically the induction of the secondary-response cytokine IL-6. This synergistic action protects PDE4B-KO mice from the deleterious effects of the P aeruginosa-induced cytostorm, while concurrently improving bacterial clearance, rather than being immunosuppressive. These benefits of PDE4B ablation are in contrast to the effects resulting from treatment with PAN-PDE4 inhibitors, which have been shown to increase bacterial burden and dissemination. Thus, PDE4B represents a promising therapeutic target in settings of P aeruginosa lung infections.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/microbiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Hipotermia/metabolismo , Hipotermia/microbiologia , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/patogenicidade , Animais , Citocinas/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores da Fosfodiesterase 4/farmacologia , Infecções por Pseudomonas/microbiologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445684

RESUMO

The shape and transparency of the cornea are essential for clear vision. However, its location at the ocular surface renders the cornea vulnerable to pathogenic microorganisms in the external environment. Pseudomonas aeruginosa and Staphylococcus aureus are two such microorganisms and are responsible for most cases of bacterial keratitis. The development of antimicrobial agents has allowed the successful treatment of bacterial keratitis if the infection is diagnosed promptly. However, no effective medical treatment is available after progression to corneal ulcer, which is characterized by excessive degradation of collagen in the corneal stroma and can lead to corneal perforation and corneal blindness. This collagen degradation is mediated by both infecting bacteria and corneal fibroblasts themselves, with a urokinase-type plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP) cascade playing a central role in collagen destruction by the host cells. Bacterial factors stimulate the production by corneal fibroblasts of both uPA and pro-MMPs, released uPA mediates the conversion of plasminogen in the extracellular environment to plasmin, and plasmin mediates the conversion of secreted pro-MMPs to the active form of these enzymes, which then degrade stromal collagen. Bacterial factors also stimulate expression by corneal fibroblasts of the chemokine interleukin-8 and the adhesion molecule ICAM-1, both of which contribute to recruitment and activation of polymorphonuclear neutrophils, and these cells then further stimulate corneal fibroblasts via the secretion of interleukin-1. At this stage of the disease, bacteria are no longer necessary for collagen degradation. In this review, we discuss the pivotal role of corneal fibroblasts in corneal ulcer associated with infection by P. aeruginosa or S. aureus as well as the development of potential new modes of treatment for this condition.


Assuntos
Úlcera da Córnea/metabolismo , Fibroblastos/metabolismo , Ceratite/microbiologia , Animais , Colágeno/metabolismo , Córnea/metabolismo , Córnea/fisiologia , Substância Própria/metabolismo , Úlcera da Córnea/etiologia , Úlcera da Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/fisiopatologia , Fibrinolisina/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , Plasminogênio/metabolismo , Ativadores de Plasminogênio/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
6.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445336

RESUMO

Pseudomonas aeruginosa (Pae) is an opportunistic pathogen showing a high intrinsic resistance to a wide variety of antibiotics. It causes nosocomial infections that are particularly detrimental to immunocompromised individuals and to patients suffering from cystic fibrosis. We provide a snapshot on regulatory RNAs of Pae that impact on metabolism, pathogenicity and antibiotic susceptibility. Different experimental approaches such as in silico predictions, co-purification with the RNA chaperone Hfq as well as high-throughput RNA sequencing identified several hundreds of regulatory RNA candidates in Pae. Notwithstanding, using in vitro and in vivo assays, the function of only a few has been revealed. Here, we focus on well-characterized small base-pairing RNAs, regulating specific target genes as well as on larger protein-binding RNAs that sequester and thereby modulate the activity of translational repressors. As the latter impact large gene networks governing metabolism, acute or chronic infections, these protein-binding RNAs in conjunction with their cognate proteins are regarded as global post-transcriptional regulators.


Assuntos
Pseudomonas aeruginosa/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Interferência de RNA/fisiologia , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo
7.
Nat Commun ; 12(1): 4613, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326342

RESUMO

R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes. PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans , Filogenia , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/genética , Virulência , Fatores de Virulência/genética
8.
Am J Respir Cell Mol Biol ; 65(4): 378-389, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34102087

RESUMO

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity-mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as to Pseudomonas aeruginosa infection. Moreover, in vivo administration of flagellin decreased the severity of P. aeruginosa-induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by using ex vivo precision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.


Assuntos
Flagelina/farmacologia , Inflamação/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Animais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Flagelina/imunologia , Flagelina/metabolismo , Imunidade Inata/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Transdução de Sinais/efeitos dos fármacos , Suínos
9.
Nat Commun ; 12(1): 4024, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188051

RESUMO

Pseudomonas aeruginosa can cause nosocomial infections, especially in ventilated or cystic fibrosis patients. Highly pathogenic isolates express the phospholipase ExoU, an effector of the type III secretion system that acts on plasma membrane lipids, causing membrane rupture and host cell necrosis. Here, we use a genome-wide screen to discover that ExoU requires DNAJC5, a host chaperone, for its necrotic activity. DNAJC5 is known to participate in an unconventional secretory pathway for misfolded proteins involving anterograde vesicular trafficking. We show that DNAJC5-deficient human cells, or Drosophila flies knocked-down for the DNAJC5 orthologue, are largely resistant to ExoU-dependent virulence. ExoU colocalizes with DNAJC5-positive vesicles in the host cytoplasm. DNAJC5 mutations preventing vesicle trafficking (previously identified in adult neuronal ceroid lipofuscinosis, a human congenital disease) inhibit ExoU-dependent cell lysis. Our results suggest that, once injected into the host cytoplasm, ExoU docks to DNAJC5-positive secretory vesicles to reach the plasma membrane, where it can exert its phospholipase activity.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Pseudomonas aeruginosa/patogenicidade , Animais , Membrana Celular/patologia , Infecção Hospitalar/microbiologia , Drosophila melanogaster/genética , Genoma Bacteriano/genética , Proteínas de Choque Térmico HSP40/genética , Humanos , Proteínas de Membrana/genética , Chaperonas Moleculares/metabolismo , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo III/metabolismo
10.
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
11.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068733

RESUMO

Wound infections are considered a major cause for wound-associated morbidity. There is a high demand for alternative, robust, and affordable methods that can provide relatable and reproducible results when testing topical treatments, both in research and in the pharmaceutical industry. Here we present an ex vivo wound infection model using porcine skin and a burn wounding method, allowing for the efficacy evaluation of topical antimicrobial formulations. Utilizing this model, we demonstrate the potential of topical treatments after infecting the wounds with clinically significant bacteria, P. aeruginosa and S. aureus. We show that the method is compatible with several analytical tools used to analyze infection and antimicrobial effects. Both bacterial strains successfully infected the wound surface, as well as deeper regions of the tissue. Quantification of viable bacteria on the wound surface and in the tissue, longitudinal measurements of bioluminescence, fluorescence microscopy, and scanning electron microscopy were used to confirm the effects of antibacterial treatments. Furthermore, we show that biofilms are formed on the wound surface, indicating that the demonstrated method mirrors typical in vivo infections.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Queimaduras/tratamento farmacológico , Infecção dos Ferimentos/tratamento farmacológico , Administração Tópica , Animais , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Queimaduras/patologia , Modelos Animais de Doenças , Composição de Medicamentos/métodos , Humanos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Suínos , Cicatrização/efeitos dos fármacos , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/patologia
12.
Int J Mol Sci ; 22(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063704

RESUMO

Carnivorous plants are exemplary natural sources of secondary metabolites with biological activity. However, the therapeutic antimicrobial potential of these compounds is limited due to intrinsic resistance of selected bacterial pathogens, among which Pseudomonas aeruginosa represents an extreme example. The objective of the study was to overcome the intrinsic resistance of P. aeruginosa by combining silver nanoparticles (AgNPs) with secondary metabolites from selected carnivorous plant species. We employed the broth microdilution method, the checkerboard titration technique and comprehensive phytochemical analyses to define interactions between nanoparticles and active compounds from carnivorous plants. It has been confirmed that P. aeruginosa is resistant to a broad range of secondary metabolites from carnivorous plants, i.e., naphthoquinones, flavonoids, phenolic acids (MBC = 512 µg mL-1) and only weakly sensitive to their mixtures, i.e., extracts and extracts' fractions. However, it was shown that the antimicrobial activity of extracts and fractions with a significant level of naphthoquinone (plumbagin) was significantly enhanced by AgNPs. Our studies clearly demonstrated a crucial role of naphthoquinones in AgNPs and extract interaction, as well as depicted the potential of AgNPs to restore the bactericidal activity of naphthoquinones towards P. aeruginosa. Our findings indicate the significant potential of nanoparticles to modulate the activity of selected secondary metabolites and revisit their antimicrobial potential towards human pathogenic bacteria.


Assuntos
Planta Carnívora/química , Nanopartículas Metálicas/química , Extratos Vegetais/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/efeitos adversos , Antibacterianos/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Naftoquinonas/efeitos adversos , Naftoquinonas/química , Naftoquinonas/farmacologia , Extratos Vegetais/química , Pseudomonas aeruginosa/patogenicidade , Metabolismo Secundário/efeitos dos fármacos , Prata/química , Espectrofotometria Ultravioleta
13.
Nucleic Acids Res ; 49(12): 6756-6770, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139014

RESUMO

The ability to fine tune global gene expression in response to host environment is critical for the virulence of pathogenic bacteria. The host temperature is exploited by the bacteria as a cue for triggering virulence gene expression. However, little is known about the mechanism employed by Pseudomonas aeruginosa to response to host body temperature. CspA family proteins are RNA chaperones that modulate gene expression. Here we explored the functions of P. aeruginosa CspA family proteins and found that CspC (PA0456) controls the bacterial virulence. Combining transcriptomic analyses, RNA-immunoprecipitation and high-throughput sequencing (RIP-Seq), we demonstrated that CspC represses the type III secretion system (T3SS) by binding to the 5' untranslated region of the mRNA of exsA, which encodes the T3SS master regulatory protein. We further demonstrated that acetylation at K41 of the CspC reduces its affinity to nucleic acids. Shifting the culture temperature from 25°C to 37°C or infection of mouse lung increased the CspC acetylation, which derepressed the expression of the T3SS genes, resulting in elevated virulence. Overall, our results identified the regulatory targets of CspC and revealed a regulatory mechanism of the T3SS in response to temperature shift and host in vivo environment.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Pseudomonas aeruginosa/genética , Transativadores/genética , Sistemas de Secreção Tipo III/genética , Células A549 , Acetilação , Animais , Proteínas de Bactérias/biossíntese , Humanos , Camundongos , Pneumonia Bacteriana/microbiologia , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Transativadores/biossíntese , Virulência
14.
Nucleic Acids Res ; 49(12): 6925-6940, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34151378

RESUMO

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.


Assuntos
RNA Helicases DEAD-box/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Aclimatação , Adenosina Trifosfatases/metabolismo , Animais , Temperatura Baixa , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/classificação , RNA Helicases DEAD-box/fisiologia , Endorribonucleases/metabolismo , Mariposas/microbiologia , Filogenia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , RNA/metabolismo , Estabilidade de RNA , Análise de Sequência de RNA , Virulência
15.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34047769

RESUMO

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.


Assuntos
Nadadeiras de Animais/metabolismo , Movimento Celular , Inflamação/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Cicatrização , Infecção dos Ferimentos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Nadadeiras de Animais/lesões , Nadadeiras de Animais/microbiologia , Nadadeiras de Animais/patologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Inflamação/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Microscopia de Fluorescência , Neutrófilos/microbiologia , Comunicação Parácrina , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais , Fatores de Tempo , Infecção dos Ferimentos/genética , Infecção dos Ferimentos/microbiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
16.
mBio ; 12(3)2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947765

RESUMO

Antimicrobial resistance is a serious medical threat, particularly given the decreasing rate of discovery of new treatments. Although attempts to find new treatments continue, it has become clear that merely discovering new antimicrobials, even if they are new classes, will be insufficient. It is essential that new strategies be aggressively pursued. Toward that end, the search for treatments that can mitigate bacterial virulence and tilt the balance of host-pathogen interactions in favor of the host has become increasingly popular. In this review, we will discuss recent progress in this field, with a special focus on synthetic small molecule antivirulents that have been identified from high-throughput screens and on treatments that are effective against the opportunistic human pathogen Pseudomonas aeruginosa.


Assuntos
Antibacterianos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Biofilmes/efeitos dos fármacos , Descoberta de Drogas/métodos , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , Infecções por Pseudomonas/microbiologia , Percepção de Quorum/efeitos dos fármacos , Virulência
17.
Methods Mol Biol ; 2321: 111-120, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34048011

RESUMO

Infection is the leading cause of death and prolonged hospitalization in severely burned patients that survive the acute phase of injury. Here we describe a murine model of severe burn injury followed by subsequent postburn infection, both local and systemic, that leads to sepsis. A detailed description of the full-thickness scald burn procedure is provided, followed by description of infection with two common burn-associated nosocomial pathogens, Pseudomonas aeruginosa and Staphylococcus aureus.


Assuntos
Queimaduras/microbiologia , Infecções por Pseudomonas/microbiologia , Infecções Estafilocócicas/microbiologia , Infecção dos Ferimentos/microbiologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pseudomonas aeruginosa/patogenicidade , Sepse/microbiologia , Staphylococcus aureus/patogenicidade
18.
mBio ; 12(2)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879591

RESUMO

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA.IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.


Assuntos
Nitrito Redutases/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Fatores de Virulência/genética , Amônia/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans , Linhagem Celular , Modelos Animais de Doenças , Drosophila melanogaster , Ferredoxinas/metabolismo , Biblioteca Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Nitrito Redutases/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade
19.
Endocrinol Diabetes Metab ; 4(2): e00225, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33855224

RESUMO

Introduction: The most frequently prescribed empirical antibiotic agents for mild and moderate diabetic foot infections (DFIs) are amino-penicillins and second-generation cephalosporins that do not cover Pseudomonas spp. Many clinicians believe they can predict the involvement of Pseudomonas in a DFI by visual and/or olfactory clues, but no data support this assertion. Methods: In this prospective observational study, we separately asked 13 experienced (median 11 years) healthcare workers whether they thought the Pseudomonas spp. would be implicated in the DFI. Their predictions were compared with the results of cultures of deep/intraoperative specimens and/or the clinical remission of DFI achieved with antibiotic agents that did not cover Pseudomonas. Results: Among 221 DFI episodes in 88 individual patients, intraoperative tissue cultures grew Pseudomonas in 22 cases (10%, including six bone samples). The presence of Pseudomonas was correctly predicted with a sensitivity of 0.32, specificity of 0.84, positive predictive value of 0.18 and negative predictive value 0.92. Despite two feedbacks of the interim results and a 2-year period, the clinicians' predictive performance did not improve. Conclusion: The combined visual and olfactory performance of experienced clinicians in predicting the presence of Pseudomonas in a DFI was moderate, with better specificity than sensitivity, and did not improve over time. Further investigations are needed to determine whether clinicians should use a negative prediction of the presence of Pseudomonas in a DFI, especially in settings with a high prevalence of pseudomonal DFIs.


Assuntos
Competência Clínica , Pé Diabético/microbiologia , Osteomielite/microbiologia , Médicos , Infecções por Pseudomonas , Pseudomonas aeruginosa/patogenicidade , Olfato/fisiologia , Pé Diabético/patologia , Pé Diabético/cirurgia , Previsões , Humanos , Osteomielite/patologia , Osteomielite/cirurgia , Estudos Prospectivos , Pseudomonas aeruginosa/isolamento & purificação
20.
Front Immunol ; 12: 654649, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33868297

RESUMO

Extracellular traps released by neutrophils (NETs) are essential for the clearance of Pseudomonas aeruginosa. Alkaline protease (AprA) secreted by P. aeruginosa negatively correlates with clinical improvement. Moreover, anti-AprA in patients with cystic fibrosis (CF) can help identify patients with aggressive forms of chronic infection. However, the mechanism underlying the clinical outcomes remains unclear. We demonstrated that aprA deficiency in P. aeruginosa decreased the bacterial burden and reduced lung infection. AprA degraded NET components in vitro and in vivo but did not affect NET formation. Importantly, antibodies induced by AprA acted as an agonist and directly enhanced the degrading activities of AprA. Moreover, antisera from patients with P. aeruginosa infection exhibited antibody-dependent enhancement (ADE) similar to that of the antibodies we prepared. Our further investigations showed that the interaction between AprA and the specific antibodies might make the enzyme active sites better exposed, and subsequently enhance the recognition of substrates and accelerate the degradation. Our findings revealed that AprA secreted by P. aeruginosa may aggravate infection by destroying formed NETs, an effect that was further enhanced by its antibodies.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Endopeptidases/imunologia , Armadilhas Extracelulares/imunologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Endopeptidases/genética , Endopeptidases/metabolismo , Armadilhas Extracelulares/enzimologia , Feminino , Interações Hospedeiro-Patógeno/imunologia , Humanos , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/patologia , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade
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