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1.
FASEB J ; 35(10): e21899, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34569661

RESUMO

The cornea of the eye differs from other mucosal surfaces in that it lacks a viable bacterial microbiome and by its unusually high density of sensory nerve endings. Here, we explored the role of corneal nerves in preventing bacterial adhesion. Pharmacological and genetic methods were used to inhibit the function of corneal sensory nerves or their associated transient receptor potential cation channels TRPA1 and TRPV1. Impacts on bacterial adhesion, resident immune cells, and epithelial integrity were examined using fluorescent labeling and quantitative confocal imaging. TRPA1/TRPV1 double gene-knockout mice were more susceptible to adhesion of environmental bacteria and to that of deliberately-inoculated Pseudomonas aeruginosa. Supporting the involvement of TRPA1/TRPV1-expressing corneal nerves, P. aeruginosa adhesion was also promoted by treatment with bupivacaine, or ablation of TRPA1/TRPV1-expressing nerves using RTX. Moreover, TRPA1/TRPV1-dependent defense was abolished by enucleation which severs corneal nerves. High-resolution imaging showed normal corneal ultrastructure and surface-labeling by wheat-germ agglutinin for TRPA1/TRPV1 knockout murine corneas, and intact barrier function by absence of fluorescein staining. P. aeruginosa adhering to corneas after perturbation of nerve or TRPA1/TRPV1 function failed to penetrate the surface. Single gene-knockout mice showed roles for both TRPA1 and TRPV1, with TRPA1-/- more susceptible to P. aeruginosa adhesion while TRPV1-/- corneas instead accumulated environmental bacteria. Corneal CD45+/CD11c+ cell responses to P. aeruginosa challenge, previously shown to counter bacterial adhesion, also depended on TRPA1/TRPV1 and sensory nerves. Together, these results demonstrate roles for corneal nerves and TRPA1/TRPV1 in corneal resistance to bacterial adhesion in vivo and suggest that the mechanisms involve resident immune cell populations.


Assuntos
Aderência Bacteriana , Córnea , Pseudomonas aeruginosa/metabolismo , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Córnea/inervação , Córnea/metabolismo , Córnea/microbiologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Canal de Cátion TRPA1/genética , Canais de Cátion TRPV/genética
2.
ACS Infect Dis ; 7(9): 2666-2685, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34503335

RESUMO

P. aeruginosa (PA) continues to pose a threat to global public health due to its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led to an alarming shortage of effective treatments for resistant microbes, and hence there is a pressing demand for the development of novel antimicrobial interventions. The potential use of antivirulence therapeutics to tackle bacterial infections has attracted considerable attention over the past decades as they hamper the pathogenicity of target microbes with reduced selective pressure, minimizing the emergence of resistance. One such approach is to interfere with the PA pqs quorum sensing system which upon the interaction of PqsR, a Lys-R type transcriptional regulator, with its cognate signal molecules 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits and host-microbe interactions. In this study, we report the hit identification and optimization of PqsR antagonists using virtual screening coupled with whole cell assay validation. The optimized hit compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile) was found to inhibit the expression of the PA PpqsA promoter controlled by PqsR with an IC50 of 1 µM. Using isothermal titration calorimetry, a Kd of 10 nM for the PqsR ligand binding domain (PqsRLBD) was determined for 61. Furthermore, the crystal structure of 61 with PqsRLBD was attained with a resolution of 2.65 Å. Compound 61 significantly reduced levels of pyocyanin, PQS, and HHQ in PAO1-L, PA14 lab strains and PAK6085 clinical isolate. Furthermore, this compound potentiated the effect of ciprofloxacin in early stages of biofilm treatment and in Galleria mellonella infected with PA. Altogether, this data shows 61 as a potent PqsR inhibitor with potential for hit to lead optimization toward the identification of a PA QS inhibitor which can be advanced into preclinical development.


Assuntos
Pseudomonas aeruginosa , Percepção de Quorum , Proteínas de Bactérias/metabolismo , Biofilmes , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/metabolismo
3.
Biomed Res Int ; 2021: 7077344, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34471641

RESUMO

In recent years, the prevalence of resistance to aminoglycosides among clinical isolates of Pseudomonas aeruginosa is increasing. The aim of this study was to investigate the role of aminoglycoside-modifying enzymes (AMEs) in resistance to aminoglycosides in clinical isolates of P. aeruginosa. The clinical isolates were collected from different hospitals. Disk agar diffusion test was used to determine the antimicrobial resistance pattern of the clinical isolates, and the minimum inhibitory concentration of aminoglycosides was detected by microbroth dilution method. The PCR was performed for discovery of aminoglycoside-modifying enzyme-encoding genes. Among 100 screened isolates, 43 (43%) isolates were resistant to at least one tested aminoglycosides. However, 13 (13%) isolates were resistant to all tested aminoglycosides and 37 isolates were detected as multidrug resistant (MDR). The resistance rates of P. aeruginosa isolates against tested antibiotics were as follows: ciprofloxacin (41%), piperacillin-tazobactam (12%), cefepime (32%), piperacillin (26%), and imipenem (31%). However, according to the MIC method, 13%, 32%, 33%, and 37% of the isolates were resistant to amikacin, gentamicin, tobramycin, and netilmicin, respectively. The PCR results showed that AAC(6')-Ib was the most commonly (26/43, 60.4%) identified AME-encoding gene followed by AAC(6')-IIa (41.86%), APH(3')-IIb (34.8%), ANT(3 ″ )-Ia (18.6), ANT(2 ″ )-Ia (13.95%), and APH(3 ″ )-Ib (2.32%). However, APH(3')-Ib was not found in any of the studied isolates. The high prevalence of AME-encoding genes among aminoglycoside-resistant P. aeruginosa isolates in this area indicated the important role of AMEs in resistance to these antibiotics similar to most studies worldwide. Due to the transmission possibility of these genes between the Gram-negative bacteria, we need to control the prescription of aminoglycosides in hospitals.


Assuntos
Aminoglicosídeos/farmacologia , Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Aminoglicosídeos/química , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Feminino , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo
4.
Nat Microbiol ; 6(9): 1199-1210, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34413503

RESUMO

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion-site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Proteínas de Bactérias/genética , Complexos Multiproteicos/genética , Ligação Proteica , Sistemas de Secreção Tipo VI/genética
5.
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
6.
Viruses ; 13(7)2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-34372538

RESUMO

Bacterial surface structures of a proteinic nature and glycoconjugates contribute to biofilm formation and provide shields to host defense mechanisms (e.g., the complement system and phagocytosis). A loss or alteration of these molecules, leading to phage resistance, could result in fewer virulent bacteria. In this study, we evaluate the biology and phenotype changes in Pseudomonas aeruginosa PAO1 phage-resistant clones, which emerge in phage-treated biofilms. We characterize these clones for phage-typing patterns, antibiotic resistance, biofilm formation, pathogenicity, and interactions with the innate immune system. Another important question that we address is whether phage-resistant mutants are also generated incidentally, despite the phage treatment-selective pressure, as the natural adaptation of the living biofilm population. It is found that the application of different phages targeting a particular receptor selects similar phage resistance patterns. Nevertheless, this results in a dramatic increase in the population heterogeneity, giving over a dozen phage-typing patterns, compared to one of the untreated PAO1 sessile forms. We also confirm the hypothesis that "phage-resistant bacteria are more susceptible to antibiotics and host-clearance mechanisms by the immune system". These findings support phage application in therapy, although the overall statement that phage treatment selects the less virulent bacterial population should be further verified using a bigger collection of clinical strains.


Assuntos
Resistência Microbiana a Medicamentos/genética , Fagos de Pseudomonas/genética , Pseudomonas aeruginosa/virologia , Antibacterianos/farmacologia , Bacteriófagos/genética , Biofilmes/crescimento & desenvolvimento , Resistência Microbiana a Medicamentos/fisiologia , Humanos , Terapia por Fagos/métodos , Fagocitose/genética , Fenótipo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Virulência
7.
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
8.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445410

RESUMO

Development of novel therapeutics to treat antibiotic-resistant infections, especially those caused by ESKAPE pathogens, is urgent. One of the most critical pathogens is P. aeruginosa, which is able to develop a large number of factors associated with antibiotic resistance, including high level of impermeability. Gram-negative bacteria are protected from the environment by an asymmetric Outer Membrane primarily composed of lipopolysaccharides (LPS) at the outer leaflet and phospholipids in the inner leaflet. Based on a large hemi-synthesis program focusing on amphiphilic aminoglycoside derivatives, we extend the antimicrobial activity of 3',6-dinonyl neamine and its branched isomer, 3',6-di(dimethyloctyl) neamine on clinical P. aeruginosa, ESBL, and carbapenemase strains. We also investigated the capacity of 3',6-homodialkyl neamine derivatives carrying different alkyl chains (C7-C11) to interact with LPS and alter membrane permeability. 3',6-Dinonyl neamine and its branched isomer, 3',6-di(dimethyloctyl) neamine showed low MICs on clinical P. aeruginosa, ESBL, and carbapenemase strains with no MIC increase for long-duration incubation. In contrast from what was observed for membrane permeability, length of alkyl chains was critical for the capacity of 3',6-homodialkyl neamine derivatives to bind to LPS. We demonstrated the high antibacterial potential of the amphiphilic neamine derivatives in the fight against ESKAPE pathogens and pointed out some particular characteristics making the 3',6-dinonyl- and 3',6-di(dimethyloctyl)-neamine derivatives the best candidates for further development.


Assuntos
Compostos Alílicos/farmacologia , Antibacterianos/farmacologia , Framicetina/química , Bactérias Gram-Negativas/crescimento & desenvolvimento , Lipopolissacarídeos/metabolismo , Compostos Alílicos/síntese química , Compostos Alílicos/química , Antibacterianos/síntese química , Antibacterianos/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/metabolismo , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo
9.
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
10.
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
11.
Curr Microbiol ; 78(9): 3494-3504, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34279672

RESUMO

The gram-negative Pseudomonas aeruginosa is an opportunistic human pathogen that contains two different types of strains: the "classical" and the "outlier". In the "classical" strain, its bacterial subfamily I.1 lipases, such as LipA and LipC in P. aeruginosa PAO1, play critical roles in its pathogenicity. However, less is known about the subfamily I.1 lipases in the "outlier" strain, nor the evolution paths of those lipases in both types of P. aeruginosa strains. Our genome-scale investigation on I.1 lipases across different bacterial strains demonstrates the presence of one LipA-like and one new type of I.1 lipase (LipC2) in those "outlier" strains. The related genomic islands analyses further suggest that the LipC counterpart gene in the "outlier" strain was lost by gene truncation. In addition, the evolutionary analyses also indicates the horizontal LipC2 gene transfer from other gammaproteobacterial species, as well as the horizontal LipA gene transfer between two different phyla, both suggesting that the gene transfer of bacterial I.1 lipases might occur in different taxonomical levels. Our results not only provide an evidence to understand the pathogenicity among different P. aeruginosa strains, but add to the knowledge of I.1 lipase evolution in bacteria.


Assuntos
Lipase , Pseudomonas aeruginosa , Proteínas de Bactérias/genética , Transferência Genética Horizontal , Ilhas Genômicas , Humanos , Lipase/genética , Lipase/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Virulência
12.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298910

RESUMO

Bacterial S1 protein is a functionally important ribosomal protein. It is a part of the 30S ribosomal subunit and is also able to interact with mRNA and tmRNA. An important feature of the S1 protein family is a strong tendency towards aggregation. To study the amyloidogenic properties of S1, we isolated and purified the recombinant ribosomal S1 protein of Pseudomonas aeruginosa. Using the FoldAmyloid, Waltz, Pasta 2.0, and AGGRESCAN programs, amyloidogenic regions of the protein were predicted, which play a key role in its aggregation. The method of limited proteolysis in combination with high performance liquid chromatography and mass spectrometric analysis of the products, made it possible to identify regions of the S1 protein from P. aeruginosa that are protected from the action of proteinase K, trypsin, and chymotrypsin. Sequences of theoretically predicted and experimentally identified amyloidogenic regions were used to synthesize four peptides, three of which demonstrated the ability to form amyloid-like fibrils, as shown by electron microscopy and fluorescence spectroscopy. The identified amyloidogenic sites can further serve as a basis for the development of new antibacterial peptides against the pathogenic microorganism P. aeruginosa.


Assuntos
Amiloide/metabolismo , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Peptídeos/metabolismo , Proteólise , RNA Bacteriano/metabolismo
13.
Appl Environ Microbiol ; 87(17): e0082621, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34132585

RESUMO

Hexabromocyclododecanes (HBCDs) are widely used brominated flame retardants that cause antidiuretic hormone syndrome and even induce cancer. However, little information is available about the degradation mechanisms of HBCDs. In this study, genomic and proteomic analyses, reverse transcription-quantitative PCR, and gene knockout assays reveal that a cytochrome P450-encoding gene is responsible for HBCD catabolism in Pseudomonas aeruginosa HS9. The CO difference spectrum of the enzyme CYP168A1 was matched to P450 characteristics via UV visibility. We demonstrate that the reactions of debromination and hydrogenation are carried out one after another based on detection of the metabolites pentabromocyclododecanols (PBCDOHs), tetrabromocyclododecadiols (TBCDDOHs), and bromide ion. In the 18O isotope experiments, PBCD18OHs were only detected in the H218O group, proving that the added oxygen is derived from H2O, not from O2. This study elucidates the degradation mechanism of HBCDs by Pseudomonas. IMPORTANCE Hexabromocyclododecanes (HBCDs) are environmental pollutants that are widely used in industry. In this study, we identified and characterized a novel key dehalogenase, CYP168A1, that is responsible for HBCD degradation from Pseudomonas aeruginosa strain HS9. This study provides new insights into understanding biodegradation of HBCDs.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hidrocarbonetos Bromados/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/genética , Biodegradação Ambiental , Sistema Enzimático do Citocromo P-450/genética , Halogenação , Hidrogenação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo
14.
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
15.
J Bacteriol ; 203(16): e0022421, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34096777

RESUMO

The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated PRha) and found that PRha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated PBAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the PRha and PBAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in PRha and PBAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution. IMPORTANCE Heterologous gene expression and complementation constitute a valuable and widely utilized tool in bacterial genetics. The arabinose-inducible ParaBAD (PBAD) and rhamnose-inducible PrhaBAD (PRha) promoter systems are commonly used in P. aeruginosa genetics and prized for the tight control and dynamic expression ranges that can be achieved. In this study, we demonstrate that the activity of both promoters is dependent upon the cAMP-dependent transcription factor Vfr. While this poses an obvious problem for use in a vfr mutant background, the issue is more pervasive, considering that vfr transcription/synthesis and cAMP homeostasis are highly integrated into the cellular physiology of the organism and influenced by numerous global regulatory systems. Fortunately, the synthetic PTac promoter is not subject to Vfr regulatory control.


Assuntos
Arabinose/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/metabolismo , Ramnose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Repressão Catabólica , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Regulon , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
16.
Int J Biol Macromol ; 184: 760-767, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34175341

RESUMO

The 2-TM-GxN family of membrane proteins is widespread in prokaryotes and plays an important role in transport of divalent cations. The canonical signature motif, which is also a selectivity filter, has a composition of Gly-Met-Asn. Some members though deviate from this composition, however no data are available as to whether this has any functional implications. Here we report the functional and structural analysis of CmaX protein from a pathogenic Pseudomonas aeruginosa bacterium, which has a Gly-Ile-Asn signature motif. CmaX readily transports Zn2+, Mg2+, Cd2+, Ni2+ and Co2+ ions, but it does not utilize proton-symport as does ZntB from Escherichia coli. Together with the bioinformatics analysis, our data suggest that deviations from the canonical signature motif do not reveal any changes in substrate selectivity or transport and easily alter in course of evolution.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Clonagem Molecular/métodos , Pseudomonas aeruginosa/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Microscopia Crioeletrônica , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Pseudomonas aeruginosa/genética
17.
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
18.
Curr Microbiol ; 78(8): 3133-3141, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34185129

RESUMO

Pseudomonas aeruginosa is a common opportunistic pathogen that causes infections in vulnerable patients including those with metabolic disorders, hematologic diseases, and malignancies, and in those who have undergone surgery. In addition, P. aeruginosa exhibits high intrinsic resistance to numerous antibiotics and tends to form biofilms rendering it even more refractory to treatment. Among the mechanisms used by P. aeruginosa to adapt to environmental stresses are those involving small regulatory RNAs (sRNAs), which are 40-500 nucleotides long and are ubiquitous in bacteria. sRNAs play important regulatory roles in various vital processes in diverse bacteria, with their quantity and diversity of regulatory functions exceeding those of proteins. In this study, we show that deletion of the sRNA, rgsA, decreased the growth rate of P. aeruginosa. Furthermore, ΔrgsA P. aeruginosa exhibited decreased ability to resist the stress induced by exposure to different concentrations and durations of peroxides in both planktonic and biofilm growth modes compared with the wild-type strain. These results highlight the role of rgsA in the defense of P. aeruginosa against oxidative stress.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos , Proteínas de Bactérias/genética , Biofilmes , Regulação Bacteriana da Expressão Gênica , Humanos , Estresse Oxidativo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo
19.
Nucleic Acids Res ; 49(12): 7075-7087, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139006

RESUMO

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery.


Assuntos
Proteínas de Bactérias/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Biossíntese de Proteínas , Pseudomonas aeruginosa/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Motivos de Nucleotídeos , Pseudomonas aeruginosa/metabolismo , Estabilidade de RNA , RNA Mensageiro/química
20.
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
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