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1.
J Med Microbiol ; 70(4)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33909550

RESUMO

Introduction. Macrophages polarization is essential in infection control. Llipopolysaccharide (LPS) plays an essential role in host innate immune system-pathogen interaction. The LPS structure of Pseudomonas aeruginosa modifies in the adaptation of this pathogen to biofilm-related chronic infection.Gap statement. There have been several studies on LPS induced polarization of human and mouse macrophages with different results. And it was reported that the lipid A structure of the LPS derived from biofilm-forming Pseudomonas aeruginosa strain PAO1 was modified.Aim. This study aimed to investigate the effect and the involved pathway of LPS from biofilm-forming PAO1 on human and murine macrophage polarization.Methodology. LPS was isolated from biofilm-forming and planktonic PAO1 and quantified. Then the LPS was added to PMA-differentiated human macrophage THP-1 cells and Raw264.7 murine macrophage cells. The expression of iNOS, Arg-1, IL4, TNF-α, CCL3, and CCL22 was analysed in the different cell lines. The expression of TICAM-1 and MyD88 in human THP-1 macrophages was quantified by Western blot. PAO1 infected macrophages at different polarization states, and the intracellular bacterial growth in macrophages was evaluated.Results. LPS from biofilm-forming PAO1 induced more marked hyperinflammatory responses in THP-1 and Raw264.7 macrophages than LPS derived from planktonic PAO1, and these responses were related to the up-regulation of MyD88. Intracellular growth of PAO1 was significantly increased in THP-1 macrophages polarized by LPS from biofilm-forming PAO1, but decreased both in THP-1 and Raw264.7 macrophages polarized by LPS from planktonic PAO1.Conclusion. The presented in vitro study indicates that LPS derived from biofilm-forming PAO1 induces enhanced M1 polarization in human and murine macrophage cell lines than LPS from planktonic PAO1.


Assuntos
Biofilmes/crescimento & desenvolvimento , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Pseudomonas aeruginosa/química , Animais , Western Blotting , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Macrófagos/patologia , Camundongos , Microscopia de Fluorescência , Pseudomonas aeruginosa/fisiologia , Células RAW 264.7 , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Células THP-1
2.
Molecules ; 26(4)2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578646

RESUMO

Pyocyanin was the first natural phenazine described. The molecule is synthesized by about 95% of the strains of Pseudomonas aeruginosa. From discovery up to now, pyocyanin has been characterised by a very rich and avant-garde history, which includes its use in antimicrobial therapy, even before the discovery of penicillin opened the era of antibiotic therapy, as well as its use in electric current generation. Exhibiting an exuberant blue colour and being easy to obtain, this pigment is the subject of the present review, aiming to narrate its history as well as to unveil its mechanisms and suggest new horizons for applications in different areas of engineering, biology and biotechnology.


Assuntos
Biotecnologia/métodos , Cor , Pseudomonas aeruginosa/química , Piocianina/química
3.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540888

RESUMO

Macrophages are essential immune cells of the innate immune system. They participate in the development and regulation of inflammation. Macrophages play a fundamental role in fighting against bacterial infections by phagocytosis of bacteria, and they also have a specific role in immunomodulation by secreting pro-inflammatory cytokines. In bacterial infection, macrophages decrease the serum iron concentration by removing iron from the blood, acting as one of the most important regulatory cells of iron homeostasis. We examined whether the Gram-positive and Gram-negative cell wall components from various bacterial strains affect the cytokine production and iron transport, storage and utilization of THP-1 monocytes in different ways. We found that S. aureus lipoteichoic acid (LTA) was less effective in activating pro-inflammatory cytokine expression that may related to its effect on fractalkine production. LTA-treated cells increased iron uptake through divalent metal transporter-1, but did not elevate the expression of cytosolic and mitochondrial iron storage proteins, suggesting that the cells maintained iron efflux via the ferroportin iron exporter. E. coli and P. aeruginosa lipopolysaccharides (LPSs) acted similarly on THP-1 cells, but the rates of the alterations of the examined proteins were different. E. coli LPS was more effective in increasing the pro-inflammatory cytokine production, meanwhile it caused less dramatic alterations in iron metabolism. P. aeruginosa LPS-treated cells produced a smaller amount of pro-inflammatory cytokines, but caused remarkable elevation of both cytosolic and mitochondrial iron storage proteins and intracellular iron content compared to E. coli LPS. These results prove that LPS molecules from different bacterial sources alter diverse molecular mechanisms in macrophages that prepossess the outcome of the bacterial infection.


Assuntos
Parede Celular/química , Citocinas/metabolismo , Escherichia coli/química , Ferro/metabolismo , Lipopolissacarídeos/farmacologia , Pseudomonas aeruginosa/química , Staphylococcus aureus/química , Células THP-1/metabolismo , Ácidos Teicoicos/farmacologia , Transporte Biológico , Receptor 1 de Quimiocina CX3C/biossíntese , Receptor 1 de Quimiocina CX3C/genética , Quimiocina CX3CL1/metabolismo , Citocinas/biossíntese , Citosol/metabolismo , Ferritinas/biossíntese , Ferritinas/genética , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Hepcidinas/biossíntese , Hepcidinas/genética , Humanos , Mitocôndrias/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Oxirredutases/biossíntese , Oxirredutases/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/genética , Células THP-1/efeitos dos fármacos
4.
Arch Biochem Biophys ; 699: 108764, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33460582

RESUMO

The interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with calcium ATPase from sarcoplasmic reticulum (SR) was studied by means of different approaches, such as enzyme activity, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and molecular docking simulations. The ATP hydrolysis activity was fully inhibited by incubation with dirhamnolipid (diRL) up to 0.1 mM concentration, corresponding to a surfactant concentration below membrane solubilization threshold. Surfactant-protein interaction induced conformational changes in the protein observed by an increase in the accessibility of tryptophan residues to the aqueous phase and by changes in the secondary structure of the protein as seen by fluorescence and FTIR spectroscopy. As a consequence, the protein become more unstable and denatured at lower temperatures, as seen by enzyme activity and DSC studies. Finally, these results were explained at molecular level throughout molecular docking simulations. It is concluded that there is a specific dirhamnolipid-protein interaction not related to the surface activity of the surfactant but to the particular physicochemical properties of the biosurfactant molecule.


Assuntos
Glicolipídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tensoativos/metabolismo , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glicolipídeos/química , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Secundária de Proteína/efeitos dos fármacos , Pseudomonas aeruginosa/química , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Tensoativos/química
5.
Biochim Biophys Acta Gen Subj ; 1865(1): 129756, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33010351

RESUMO

BACKGROUND: Crotonase superfamily members exhibit great catalytic diversity towards various acyl-CoA substrates. A common CoA moiety binding pattern is usually observed in this family, understanding the substrate-binding mechanism would facilitate the rational engineering of crotonases for improved properties. METHODS: We applied X-ray crystallography to investigate a putative enoyl-CoA hydratase/isomerase OdaA in Pseudomonas aeruginosa. Thermal shift assay (TSA) were performed to explore the binding of OdaA with CoA thioester substrates. Furthermore, we performed molecular dynamics (MD) simulations to elucidate the dynamics of its CoA-binding site. RESULTS: We solved the crystal structures of the apo and CoA-bound OdaA. Thermal shift assay (TSA) showed that CoA thioester substrates bind to OdaA with a different degree. MD simulations demonstrated that the C-terminal alpha helix underwent a structural transition and a hinge region would associate with this conformational change. CONCLUSIONS: TSA in combination with MD simulations elucidate that the dynamics of C-terminal alpha helix in CoA-binding, and a hinge region play an important role in conformational change. GENERAL SIGNIFICANCE: Those results help to extend our knowledge about the nature of crotonases and would be informative for future mechanistic studies and industry applications.


Assuntos
Enoil-CoA Hidratase/química , Pseudomonas aeruginosa/enzimologia , Cristalografia por Raios X , Enoil-CoA Hidratase/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Conformação Proteica em alfa-Hélice , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo
6.
Chemistry ; 26(34): 7657-7671, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32297355

RESUMO

A series of homoleptic and heteroleptic bismuth(III) flavonolate complexes derived from six flavonols of varying substitution have been synthesised and structurally characterised. The complexes were evaluated for antibacterial activity towards several problematic Gram-positive (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE)) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. The cell viability of COS-7 (monkey kidney) cells treated with the bismuth flavonolates was also studied to determine the effect of the complexes on mammalian cells. The heteroleptic complexes [BiPh(L)2 ] (in which L=flavonolate) showed good antibacterial activity towards all of the bacteria but reduced COS-7 cell viability in a concentration-dependent manner. The homoleptic complexes [Bi(L)3 ] exhibited activity towards the Gram-positive bacteria and showed low toxicity towards the mammalian cell line. Bismuth uptake studies in VRE and COS-7 cells treated with the bismuth flavonolate complexes indicated that Bi accumulation is influenced by both the substitution of the flavonolate ligands and the degree of substitution at the bismuth centre.


Assuntos
Antibacterianos/farmacologia , Bismuto/química , Complexos de Coordenação/química , Escherichia coli/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Escherichia coli/química , Bactérias Gram-Positivas/química , Humanos , Staphylococcus aureus Resistente à Meticilina/química , Pseudomonas aeruginosa/química , Staphylococcus aureus/química
7.
PLoS One ; 15(3): e0229494, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32126100

RESUMO

Pseudomonas aeruginosa is a virulent pathogen that has become more threatening with the emergence of multidrug resistance. The aspartate transcarbamoylase (ATCase) of this organism is a dodecamer comprised of six 37 kDa catalytic chains and six 45 kDa chains homologous to dihydroorotase (pDHO). The pDHO chain is inactive but is necessary for ATCase activity. A stoichiometric mixture of the subunits associates into a dodecamer with full ATCase activity. Unlike other known ATCases, the P. aeruginosa catalytic chain does not spontaneously assemble into a trimer. Chemical-crosslinking and size-exclusion chromatography showed that P. aeruginosa ATCase is monomeric which accounts for its lack of catalytic activity since the active site is a composite comprised of residues from adjacent monomers in the trimer. Circular dichroism spectroscopy indicated that the ATCase chain adopts a structure that contains secondary structure elements although neither the ATCase nor the pDHO subunits are very stable as determined by a thermal shift assay. Formation of the complex increases the melting temperature by about 30°C. The ATCase is strongly inhibited by all nucleotide di- and triphosphates and exhibits extreme cooperativity. Previous studies suggested that the regulatory site is located in an 11-residue extension of the amino end of the catalytic chain. However, deletion of the extensions did not affect catalytic activity, nucleotide inhibition or the assembly of the dodecamer. Nucleotides destabilized the dodecamer which probably accounts for the inhibition and apparent cooperativity of the substrate saturation curves. Contrary to previous interpretations, these results suggest that P. aeruginosa ATCase is not allosterically regulated by nucleotides.


Assuntos
Aspartato Carbamoiltransferase/química , Aspartato Carbamoiltransferase/metabolismo , Di-Hidro-Orotase/química , Di-Hidro-Orotase/metabolismo , Pseudomonas aeruginosa/enzimologia , Motivos de Aminoácidos , Aspartato Carbamoiltransferase/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Dicroísmo Circular , Di-Hidro-Orotase/genética , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Termodinâmica
8.
Chemistry ; 26(28): 6247-6256, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32166806

RESUMO

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Lipopolissacarídeos/química , Lipídeos de Membrana/química , Pseudomonas aeruginosa/química , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Hemólise , Lipopolissacarídeos/metabolismo , Lipídeos de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína
9.
Nucleic Acids Res ; 48(4): 2156-2172, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31925429

RESUMO

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive. Here, we focus on the H-NS family protein MvaT from Pseudomonas aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strength on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DNA/química , Transativadores/química , Proteínas de Bactérias/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Concentração Osmolar , Domínios Proteicos/genética , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Transativadores/genética
10.
J Biol Chem ; 295(2): 504-516, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31771981

RESUMO

Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating ß-1,4-linked disaccharide of N-acetylglucosamine and N-acetylmuramic acid appended with a highly conserved stem peptide. In Gram-negative bacteria, PG is assembled in the cytoplasm and exported into the periplasm where it undergoes considerable maturation, modification, or degradation depending on the growth phase or presence of environmental stressors. These modifications serve important functions in diverse processes, including PG turnover, cell elongation/division, and antibiotic resistance. Conventional methods for analyzing PG composition are complex and time-consuming. We present here a streamlined MS-based method that combines differential analysis with statistical 1D annotation approaches to quantitatively compare PGs produced in planktonic- and biofilm-cultured Pseudomonas aeruginosa We identified a core assembly of PG that is present in high abundance and that does not significantly differ between the two growth states. We also identified an adaptive PG assembly that is present in smaller amounts and fluctuates considerably between growth states in response to physiological changes. Biofilm-derived adaptive PG exhibited significant changes compared with planktonic-derived PG, including amino acid substitutions of the stem peptide and modifications that indicate changes in the activity of amidases, deacetylases, and lytic transglycosylases. The results of this work also provide first evidence of de-N-acetylated muropeptides from P. aeruginosa The method developed here offers a robust and reproducible workflow for accurately determining PG composition in samples that can be used to assess global PG fluctuations in response to changing growth conditions or external stimuli.


Assuntos
Biofilmes , Peptidoglicano/metabolismo , Plâncton/fisiologia , Pseudomonas aeruginosa/fisiologia , Biofilmes/crescimento & desenvolvimento , Parede Celular/química , Parede Celular/metabolismo , Glicômica , Humanos , Espectrometria de Massas , Peptidoglicano/química , Plâncton/química , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química
11.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117394, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31351419

RESUMO

This study reports the utilization of engineered molecular networks between bacteriophage (or phage) and gold nanoparticles (AuNPs) prepared ablating a high purity gold target in water by nanosecond laser source. Gold colloids are assembled with P9b phage clone, displaying the specific peptide (QRKLAAKLT), able to bind P. aeruginosa. The single components and assembled systems were characterized by spectroscopic and electronic techniques, such as the conventional optical absorption and micro-Raman spectroscopies as well as the Dynamic Light Scattering (DLS) and Scanning Transmission Electron Microscopy (STEM) techniques. The performance of the AuNPs-phage assembly as substrate for Surface-Enhanced Raman Spectroscopy (SERS) was tested against the detection of the characteristics Raman vibrational features of the Pseudomonas aeruginosa bacteria.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Ouro/química , Nanopartículas Metálicas/química , Sondas Moleculares/química , Análise Espectral Raman/métodos , Bacteriófagos/química , Bacteriófagos/metabolismo , Sondas Moleculares/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo
12.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117417, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31362188

RESUMO

To ensure the food security and protect public health, development of rapid and reliable approaches to detecting foodborne pathogens is of great significance. In this study, polydopamine-polyethyleneimine (PDA-PEI) copolymer dots are prepared via the self-polymerization of dopamine and cross-linking with branched PEI at room temperature. The PDA-PEI copolymer dots are very stable against photobleaching, extreme pH, as well as high ionic strength. They are used as a fluorescent probe to fabricate a biosensor for rapid and sensitive detection and quantification of Pseudomonas aeruginosa (P. aeruginosa). In the biosensor, dual-aptamers of P. aeruginosa are used to label PDA-PEI copolymer dots. Compared to single aptamer labeled PDA-PEI dots, the dual-aptamers labeled PDA-PEI dots endow the biosensor with enhanced sensitivity for target pathogen. The fluorescence biosensor demonstrates a wide linear response to P. aeruginosa in the concentration range of 101-107 cfu mL-1 with acceptable selectivity. The limit of detection is calculated to be 1 cfu mL-1. The whole detection process can be finished in 1.5 h. The feasibility of the fabricated biosensor is verified by successful determination of P. aeruginosa in skim milk, orange juice, and popsicle samples. The biosensor provides an alternative and attractive platform for rapid and sensitive detection of bacteria in food products.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Indóis/química , Polietilenoimina/química , Polímeros/química , Pseudomonas aeruginosa , Animais , Técnicas de Tipagem Bacteriana/métodos , Sucos de Frutas e Vegetais/microbiologia , Leite/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/isolamento & purificação , Pontos Quânticos , Espectrometria de Fluorescência/métodos
13.
Methods Appl Fluoresc ; 8(1): 014007, 2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31791032

RESUMO

Many molecular processes within a cell are carried out by molecular machines built from a large number of proteins organized by their protein-protein interactions (PPIs). Exploring PPIs in their cellular context is critical to better understand the proteins functions. Förster resonance energy transfer measured by fluorescence lifetime imaging (FLIM-FRET) enables to monitor PPIs and to map their spatial organization in a living cell with high spatial and temporal specificity. But both the accurate measurement and the interpretation of multi-exponential FLIM-FRET data associated to mixtures of interacting and non-interacting proteins are difficult. Here we show that a simple diagram plot can find interesting visualization properties by clustering pixels with similar decay signatures. FLIM diagram plot can be used to provide valuable information about stoichiometry and binding mode in PPIs, even in the presence of large differences in protein expression levels of the different interacting partners. The proposed FLIM diagram plot is a useful visual approach for a more straightforward interpretation of complex lifetime data. This approach was applied for revealing critical features of PPIs in live Pseudomonas aeruginosa.


Assuntos
Proteínas de Bactérias/química , Transferência Ressonante de Energia de Fluorescência , Imagem Óptica , Pseudomonas aeruginosa/química , Proteínas de Bactérias/biossíntese , Ligação Proteica , Pseudomonas aeruginosa/citologia , Fatores de Tempo
14.
Nat Commun ; 10(1): 5567, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804467

RESUMO

SPOR domains are widely present in bacterial proteins that recognize cell-wall peptidoglycan strands stripped of the peptide stems. This type of peptidoglycan is enriched in the septal ring as a product of catalysis by cell-wall amidases that participate in the separation of daughter cells during cell division. Here, we document binding of synthetic denuded glycan ligands to the SPOR domain of the lytic transglycosylase RlpA from Pseudomonas aeruginosa (SPOR-RlpA) by mass spectrometry and structural analyses, and demonstrate that indeed the presence of peptide stems in the peptidoglycan abrogates binding. The crystal structures of the SPOR domain, in the apo state and in complex with different synthetic glycan ligands, provide insights into the molecular basis for recognition and delineate a conserved pattern in other SPOR domains. The biological and structural observations presented here are followed up by molecular-dynamics simulations and by exploration of the effect on binding of distinct peptidoglycan modifications.


Assuntos
Parede Celular/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Peptidoglicano/química , Domínios Proteicos , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Sequência de Carboidratos , Parede Celular/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Simulação de Dinâmica Molecular , Peptidoglicano/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo
15.
Mol Cells ; 42(12): 850-857, 2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31722511

RESUMO

The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa , has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand-binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Pseudomonas aeruginosa/química , Fatores de Transcrição/química , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Fatores de Transcrição/metabolismo
16.
Nat Commun ; 10(1): 4927, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666527

RESUMO

Raman optical spectroscopy promises label-free bacterial detection, identification, and antibiotic susceptibility testing in a single step. However, achieving clinically relevant speeds and accuracies remains challenging due to weak Raman signal from bacterial cells and numerous bacterial species and phenotypes. Here we generate an extensive dataset of bacterial Raman spectra and apply deep learning approaches to accurately identify 30 common bacterial pathogens. Even on low signal-to-noise spectra, we achieve average isolate-level accuracies exceeding 82% and antibiotic treatment identification accuracies of 97.0±0.3%. We also show that this approach distinguishes between methicillin-resistant and -susceptible isolates of Staphylococcus aureus (MRSA and MSSA) with 89±0.1% accuracy. We validate our results on clinical isolates from 50 patients. Using just 10 bacterial spectra from each patient isolate, we achieve treatment identification accuracies of 99.7%. Our approach has potential for culture-free pathogen identification and antibiotic susceptibility testing, and could be readily extended for diagnostics on blood, urine, and sputum.


Assuntos
Antibacterianos/uso terapêutico , Bactérias/classificação , Infecções Bacterianas/diagnóstico , Aprendizado Profundo , Análise Espectral Raman/métodos , Bactérias/química , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Técnicas de Tipagem Bacteriana , Candida/química , Candida/classificação , Enterococcus/química , Enterococcus/classificação , Escherichia coli/química , Escherichia coli/classificação , Humanos , Klebsiella/química , Klebsiella/classificação , Modelos Logísticos , Staphylococcus aureus Resistente à Meticilina/química , Staphylococcus aureus Resistente à Meticilina/classificação , Testes de Sensibilidade Microbiana , Redes Neurais de Computação , Análise de Componente Principal , Proteus mirabilis/química , Proteus mirabilis/classificação , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/classificação , Salmonella enterica/química , Salmonella enterica/classificação , Análise de Célula Única , Staphylococcus aureus/química , Staphylococcus aureus/classificação , Streptococcus/química , Streptococcus/classificação , Máquina de Vetores de Suporte
17.
Colloids Surf B Biointerfaces ; 184: 110517, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31605948

RESUMO

The development of environmental friendly new procedures for the synthesis of metallic nanoparticles is one of the main objectives of nanotechnology. Plants, algae, fungi and bacteria for the production of nanomaterials are viable alternatives due to their low cost, the absence of toxic waste production and their highly energy efficiency. It is also known that biosynthesized silver nanoparticles (AgNPs) show higher biocompatibility compared to the chemically-synthesized ones. In previous results, biosynthesized AgNPs were obtained from the supernatant of Pseudomonas aeruginosa, and they showed a bigger antimicrobial activity against different bacterial species compared to the chemically-synthesized ones. The aim of this work was to analyze the capping of biosynthesized AgNPs using techniques such as transmission electron microscopy (TEM), infrared spectroscopy (IR), and protein identification through mass spectrometry (MS) in order to identify the compounds responsible for their formation, stability and biocompatibility. The TEM images showed that AgNPs were surrounded by an irregular coverage. The IR spectrum showed that this coverage was composed of carbohydrates and/or proteins. Different proteins were identified in the capping associated to biosynthesized AgNPs. Some proteins seem to be important for their formation (Alkyl hydroperoxide reductase and Azurin) and stabilization (Outer membrane protein OprG and Glycine zipper 2 T M domain-containing protein). The proteins identified with the capability to interact with some biomolecules can be responsible for the biocompatibility and may be responsible for the bigger antimicrobial activity than AgNPs have previously shown. These results are pioneers in the identification of proteins in the capping of biosynthesized AgNPs.


Assuntos
Proteínas de Bactérias/química , Materiais Biocompatíveis/metabolismo , Nanopartículas Metálicas/química , Pseudomonas aeruginosa/química , Prata/metabolismo , Proteínas de Bactérias/metabolismo , Materiais Biocompatíveis/química , Tamanho da Partícula , Pseudomonas aeruginosa/metabolismo , Prata/química , Propriedades de Superfície
18.
Int J Mol Sci ; 20(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627455

RESUMO

Solute binding proteins (SBPs) form a heterogeneous protein family that is found in all kingdoms of life. In bacteria, the ligand-loaded forms bind to transmembrane transporters providing the substrate. We present here the SBP repertoire of Pseudomonas aeruginosa PAO1 that is composed of 98 proteins. Bioinformatic predictions indicate that many of these proteins have a redundant ligand profile such as 27 SBPs for proteinogenic amino acids, 13 proteins for spermidine/putrescine, or 9 proteins for quaternary amines. To assess the precision of these bioinformatic predictions, we have purified 17 SBPs that were subsequently submitted to high-throughput ligand screening approaches followed by isothermal titration calorimetry studies, resulting in the identification of ligands for 15 of them. Experimentation revealed that PA0222 was specific for γ-aminobutyrate (GABA), DppA2 for tripeptides, DppA3 for dipeptides, CysP for thiosulphate, OpuCC for betaine, and AotJ for arginine. Furthermore, RbsB bound D-ribose and D-allose, ModA bound molybdate, tungstate, and chromate, whereas AatJ recognized aspartate and glutamate. The majority of experimentally identified ligands were found to be chemoattractants. Data show that the ligand class recognized by SPBs can be predicted with confidence using bioinformatic methods, but experimental work is necessary to identify the precise ligand profile.


Assuntos
Proteínas de Bactérias/química , Pseudomonas aeruginosa/química , Calorimetria , Quimiotaxia , Biologia Computacional , Ligantes , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais
19.
Nanoscale ; 11(43): 20809-20819, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31657419

RESUMO

Mapping the dielectric constant at the nanoscale of samples showing a complex topography, such as non-planar nanocomposite materials or single cells, poses formidable challenges to existing nanoscale dielectric microscopy techniques. Here we overcome these limitations by introducing Scanning Dielectric Force Volume Microscopy. This scanning probe microscopy technique is based on the acquisition of electrostatic force approach curves at every point of a sample and its post-processing and quantification by using a computational model that incorporates the actual measured sample topography. The technique provides quantitative nanoscale images of the local dielectric constant of the sample with unparalleled accuracy, spatial resolution and statistical significance, irrespectively of the complexity of its topography. We illustrate the potential of the technique by presenting a nanoscale dielectric constant map of a single bacterial cell, including its small-scale appendages. The bacterial cell shows three characteristic equivalent dielectric constant values, namely, εr,bac1 = 2.6 ± 0.2, εr,bac2 = 3.6 ± 0.4 and εr,bac3 = 4.9 ± 0.5, which enable identifying different dielectric properties of the cell wall and of the cytoplasmatic region, as well as, the existence of variations in the dielectric constant along the bacterial cell wall itself. Scanning Dielectric Force Volume Microscopy is expected to have an important impact in Materials and Life Sciences where the mapping of the dielectric properties of samples showing complex nanoscale topographies is often needed.


Assuntos
Capacitância Elétrica , Microscopia de Força Atômica/métodos , Pseudomonas aeruginosa/química , Parede Celular/química , Microesferas , Nanotecnologia , Pseudomonas aeruginosa/metabolismo , Dióxido de Silício/química , Propriedades de Superfície
20.
Proc Natl Acad Sci U S A ; 116(44): 22275-22281, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31611393

RESUMO

Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.


Assuntos
Fibrose Cística/microbiologia , Pseudomonas aeruginosa/ultraestrutura , Ribossomos/química , Motivos de Aminoácidos , Aminoglicosídeos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Farmacorresistência Bacteriana , Humanos , Mutação , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/ultraestrutura
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