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1.
Article in English | MEDLINE | ID: mdl-29133563

ABSTRACT

The search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identified in vitro to be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPC mRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23-231) as the substrate and PrPSc seeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90-231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPC globular domain. J8 also reduced the fibrillation of mouse rPrP23-231 seeded with in vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPC on the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPC content on the cell surface.


Subject(s)
Chalcones/pharmacology , Drugs, Investigational/pharmacology , Neurons/drug effects , Oxadiazoles/pharmacology , Prion Proteins/antagonists & inhibitors , Animals , Binding Sites , Cell Line, Tumor , Chalcones/chemical synthesis , Cloning, Molecular , Drugs, Investigational/chemical synthesis , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Kinetics , Mice , Molecular Docking Simulation , Neurons/metabolism , Neurons/pathology , Oxadiazoles/chemical synthesis , Prion Proteins/chemistry , Prion Proteins/genetics , Prion Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Thermodynamics
2.
J Appl Microbiol ; 116(5): 1322-33, 2014 May.
Article in English | MEDLINE | ID: mdl-24447276

ABSTRACT

AIMS: To use the phage display technique to develop peptides with the capability to neutralize the cytotoxicity induced by Stx1 and Stx2 toxins produced by Shiga toxin-producing Escherichia coli (STEC). METHODS AND RESULTS: The phage display technique permitted the development of three peptides, named PC7-12, P12-26 and PC7-30, which bind to the globotriaosylceramide (Gb3) receptor for Shiga toxins produced by STEC. Moreover, these peptides were capable of competing efficiently with the Shiga toxins for binding to Gb3. The peptides described herein partially inhibited the Stx-induced cytotoxicity of cell-free filtrates of STEC O157 : H7 and purified Stx toxins in Vero cells. The inhibition of lethality induced by Stx toxins in mice indicated that peptide PC7-30 inhibited the lethality caused by Stx1 (2LD50) in mice. CONCLUSIONS: The phage display technique permitted the development of peptides that inhibited the cytotoxicity induced by Stx toxins in vitro. Peptide PC7-30 inhibited the lethality of Stx1 in vivo; this molecule would be a promising candidate for the development of therapeutic agents for STEC-related diseases in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of Gb3, the common receptor for Stx1 and Stx2, may contribute to the development of efficient neutralizers for both toxins, and our approach would be an interesting alternative for the development of therapeutic molecules for the treatment of diseases caused by STEC strains.


Subject(s)
Peptides/pharmacology , Shiga Toxin 1/antagonists & inhibitors , Shiga Toxin 2/antagonists & inhibitors , Animals , Chlorocebus aethiops , Humans , Mice , Peptide Library , Peptides/chemistry , Peptides/metabolism , Shiga Toxin 1/toxicity , Shiga Toxin 2/toxicity , Shiga-Toxigenic Escherichia coli/metabolism , Trihexosylceramides/metabolism , Vero Cells
3.
Braz. j. med. biol. res ; 45(5): 417-424, May 2012. ilus, tab
Article in English | LILACS | ID: lil-622765

ABSTRACT

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.


Subject(s)
Adult , Animals , Humans , Adhesins, Bacterial/physiology , Bacterial Adhesion/physiology , Epithelial Cells/microbiology , Escherichia coli Infections/microbiology , Escherichia coli/physiology , Sepsis/microbiology , Adhesins, Bacterial/genetics , Adhesins, Bacterial/ultrastructure , Bacterial Adhesion/genetics , Chlorocebus aethiops , Epithelial Cells/ultrastructure , Escherichia coli/genetics , Escherichia coli/ultrastructure , Genotype , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Polymerase Chain Reaction , Vero Cells
4.
Braz J Med Biol Res ; 45(5): 417-24, 2012 May.
Article in English | MEDLINE | ID: mdl-22488222

ABSTRACT

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.


Subject(s)
Adhesins, Bacterial/physiology , Bacterial Adhesion/physiology , Epithelial Cells/microbiology , Escherichia coli Infections/microbiology , Escherichia coli/physiology , Sepsis/microbiology , Adhesins, Bacterial/genetics , Adhesins, Bacterial/ultrastructure , Adult , Animals , Bacterial Adhesion/genetics , Chlorocebus aethiops , Epithelial Cells/ultrastructure , Escherichia coli/genetics , Escherichia coli/ultrastructure , Genotype , Humans , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Polymerase Chain Reaction , Vero Cells
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