RESUMO
Bacterial phospholipases and sphingomyelinases are lipolytic esterases that are structurally and evolutionarily heterogeneous. These enzymes play crucial roles as virulence factors in several human and animal infectious diseases. Some bacterial phospholipases C (PLCs) have both phosphatidylcholinesterase and sphingomyelinase C activities. Among them, Listeria monocytogenes PlcB, Clostridium perfringens PLC, and Pseudomonas aeruginosa PlcH are the most deeply understood. In silico predictions of substrates docking with these three bacterial enzymes provide evidence that they interact with different substrates at the same active site. This review discusses structural aspects, substrate specificity, and the mechanism of action of those bacterial enzymes on target cells and animal infection models to shed light on their roles in pathogenesis.
Assuntos
Esfingomielina Fosfodiesterase/metabolismo , Esfingomielina Fosfodiesterase/fisiologia , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/fisiologia , Animais , Clostridium perfringens/enzimologia , Clostridium perfringens/patogenicidade , Humanos , Listeria monocytogenes/enzimologia , Listeria monocytogenes/patogenicidade , Fosfolipases , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Fosfolipases Tipo C/genéticaRESUMO
The GlcNAcstatin is a potent inhibitor of O-glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase, which has been related with type II diabetes and neurodegenerative disorders. Herein, hybrid quantum mechanics/molecular mechanics, molecular dynamics simulations, and potential of mean force were employed to study the interactions established between GlcNAcstatin and a bacterial O-GlcNAcase enzyme from Clostridium perfringens. The results reveal that the imidazole nitrogen atom of GlcNAcstatin has shown a better interaction with the active site of Clostridium perfringens in its protonated form, which is compatible with a substrate-assisted reaction mechanism involving two conserved aspartate residues (297 and 298). Furthermore, the quantum mechanics/molecular mechanics-molecular dynamics simulations appointed a strong interaction between Asp401, Asp298, and Asp297 residues and the GlcNAcstatin inhibitor, which is in accordance with experimental data. Lastly, these results may contribute to understand the molecular mechanism of inhibition of Clostridium perfringens by GlcNAcstatin inhibitor and, consequently, this study might be useful to design new molecules with more interesting inhibitory activity.
Assuntos
Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Imidazóis/química , beta-N-Acetil-Hexosaminidases/química , Acetilglucosamina/farmacologia , Domínio Catalítico , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/enzimologia , Imidazóis/farmacologia , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Termodinâmica , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
The substitution of serine and threonine residues in nucleocytoplasmic proteins with 2-acetamido-2-deoxy-ß-D-glucopyranose (O-GlcNAc) residues is an essential post-translational modification found in many multicellular eukaryotes. O-glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase (O-GlcNAcase) hydrolyzes O-GlcNAc residues from post-translationally modified serine/threonine residues of nucleocytoplasmic protein. O-GlcNAc has been implicated in several disease states such as cancer, Alzheimer's disease, and type II diabetes. For this paper, a model of the human O-GlcNAcase (hOGA) enzyme based on the X-ray structures of bacterial Clostridium perfringens (CpNagJ) and Bacteroides thetaiotaomicrometer (BtOGA) homologues has been generated through molecular homology modeling. In addition, molecular docking, molecular dynamics (MD) simulations, and Linear Interaction Energy (LIE) were employed to determine the bind for derivatives of two potent inhibitors: O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-methyl-R-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NAG-thiazoline), with hOGA. The results show that the binding free energy calculations using the Linear Interaction Energy (LIE) are correlated with inhibition constant values. Therefore, the model of the human O-GlcNAcase (hOGA) obtained here may be used as a target for rational design of new inhibitors.
Assuntos
Acetilglucosamina/análogos & derivados , Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Oximas/química , Fenilcarbamatos/química , Tiazóis/química , beta-N-Acetil-Hexosaminidases/química , Acetilglucosamina/química , Proteínas de Bactérias/antagonistas & inibidores , Bacteroides/química , Bacteroides/enzimologia , Sítios de Ligação , Clostridium perfringens/química , Clostridium perfringens/enzimologia , Cristalografia por Raios X , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Cinética , Ligantes , Ligação Proteica , Conformação Proteica , Homologia Estrutural de Proteína , Termodinâmica , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.
Assuntos
Infecções por Clostridium/microbiologia , Infecções por Clostridium/veterinária , Clostridium perfringens/enzimologia , Clostridium perfringens/genética , Microbiologia Ambiental , Polimorfismo Genético , Fosfolipases Tipo C/genética , Animais , Bovinos , Clostridium/genética , Clostridium/metabolismo , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/patogenicidade , Cães , Filogenia , Coelhos , Análise de Sequência de Proteína , Fosfolipases Tipo C/metabolismo , VirulênciaRESUMO
Histotoxic strains of Clostridium perfringens cause human gas gangrene, a devastating infection during which potent tissue-degrading toxins are produced and secreted. Although this pathogen only grows in anaerobic-nutrient-rich habitats such as deep wounds, very little is known regarding how nutritional signals influence gas gangrene-related toxin production. We hypothesize that sugars, which have been used throughout history to prevent wound infection, may represent a nutritional signal against gas gangrene development. Here we demonstrate, for the first time, that sugars (sucrose, glucose) inhibited the production of the main protein toxins, PLC (alpha-toxin) and PFO (theta-toxin), responsible for the onset and progression of gas gangrene. Transcription analysis experiments using plc-gusA and pfoA-gusA reporter fusions as well as RT-PCR analysis of mRNA transcripts confirmed that sugar represses plc and pfoA expression. In contrast an isogenic C. perfringens strain that is defective in CcpA, the master transcription factor involved in carbon catabolite response, was completely resistant to the sugar-mediated inhibition of PLC and PFO toxin production. Furthermore, the production of PLC and PFO toxins in the ccpA mutant strain was several-fold higher than the toxin production found in the wild type strain. Therefore, CcpA is the primary or unique regulatory protein responsible for the carbon catabolite (sugar) repression of toxin production of this pathogen. The present results are analyzed in the context of the role of CcpA for the development and aggressiveness of clostridial gas gangrene and the well-known, although poorly understood, anti-infective and wound healing effects of sugars and related substances.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Clostridium perfringens/metabolismo , Regulação para Baixo , Gangrena Gasosa/microbiologia , Glucose/metabolismo , Proteínas Hemolisinas/metabolismo , Sacarose/metabolismo , Fosfolipases Tipo C/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Clostridium perfringens/enzimologia , Clostridium perfringens/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas/genética , Humanos , Fosfolipases Tipo C/genéticaRESUMO
O-Glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase (O-GlcNAcase) hydrolyzes O-linked 2-acetamido-2-deoxy-ß-D-glucopyranoside (O-GlcNAc) residues from post-translationally modified serine/threonine residues of nucleocytoplasmic protein. The chemical process involves substrate-assisted catalysis, where two aspartate residues have been identified as the two key catalytic residues of O-GlcNAcase. In this report, the first step of the catalytic mechanism used by O-GlcNAcase involving substrate-assisted catalysis has been studied using a hybrid quantum mechanical/molecular mechanical (QM/MM) Molecular Dynamics (MD) calculations. The free energy profile shows that the formation of the oxazoline intermediate in the O-GlcNAcase catalytic reaction takes place by means of a stepwise mechanism. The first step would be a cyclization of the acetomide group, which seems to be dependent on the proton transfer from a conserved aspartate, Asp298 in Clostridium perfringens O-GlcNAcase. From this new intermediate, a proton is transferred from the azoline ring to another conserved aspartate (Asp297) thus forming the oxazoline ion and departure of the aglycone. In addition, averaged values of protein-substrate interaction energy along the reaction path shows that, in fact, the transition states present the highest binding affinities. A deeper analysis of the binding contribution of the individual residues shows that Asp297, Asp298, and Asp401 are basically responsible of the stabilization of these complexes. These results would explain why O-(2-acetamido-2deoxy-d-glucopyranosylidene)amino-N-phenycarbamate (PUGNAc), 1,2-dideoxy-2'-methyl-α-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NAG-thiazoline), and GlcNAcstatin derivatives are potent inhibitors of this enzyme, resembling the two transition states of the O-GlcNAcase catalytic reaction path. These results may be useful to rational design compounds with more interesting inhibitory activity.
Assuntos
Clostridium perfringens/enzimologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo , Clostridium perfringens/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , Oxazóis/metabolismo , Teoria Quântica , Especificidade por Substrato , Termodinâmica , beta-N-Acetil-Hexosaminidases/químicaRESUMO
The enzyme O-glycoprotein 2-acetamino-2-deoxy-beta-d-glucopyranosidase (O-GlcNAcase) is responsible for the removal of N-acetylglucosamine moieties from 2-acetamido-2-deoxy-beta-D-glucopyranose (O-GlcNAc) residues of serine/threonine residues of modified proteins. We herein present results of hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations applied to the study of the interactions established between a bacterial Clostridium perfringens homologue (CpNagJ) and PUGNAc, a potent known inhibitor of this enzyme. Electrostatic binding free energy and energy term decomposition have been computed for the wild-type CpNagJ and several mutants: D297N, D298N, Y335F, N390A, N396A, D401A, and W490A. The theoretical results have been compared with recently experimental data based on crystallographic and mutation studies on the same system. Our results reveal that, first, there is a strong interaction between Asp401, Asp298, and Asp297 residues and the PUGNAc inhibitor; and, second, the electrostatic substrate binding free energy is higher in wild-type, N390A and W490A mutants than in D297N, D298N, Y335F, N396A, and D401A ones, in accordance with the experimental results. Finally, both our theoretical predictions and the experimental data are compatible with a substrate-assisted reaction mechanism, involving two conserved aspartate residues.
Assuntos
Acetilglucosamina/análogos & derivados , Clostridium perfringens/enzimologia , Simulação de Dinâmica Molecular , Oximas/química , Fenilcarbamatos/química , beta-N-Acetil-Hexosaminidases/química , Acetilglucosamina/química , Substituição de Aminoácidos , Cristalografia por Raios X , Mutagênese Sítio-Dirigida , Ligação Proteica , Teoria Quântica , Eletricidade Estática , Termodinâmica , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
In order to explore how molecules are linked to the membrane surface in larval Taenia solium, whole cysticerci were incubated in the presence of phospholipase C from Clostridium perfringens (PLC). Released material was collected and analyzed in polyacrylamide gels with sodium dodecyl sulfate. Two major bands with apparent molecular weights of 180 and 43 kDa were observed. Western blot of released material and localization assays in cysticerci tissue sections using antibodies against five known surface glycoproteins of T. solium cysticerci indicated that only one, previously called GP1, was released. Similar localization studies using the lectins wheat-germ-agglutinin and Concanavalin A showed that N-acetyl-D-glucosamine, N-acetylneuraminic, sialic acid, alphamethyl-D-mannoside, D-manose/glucose, and N-acetyl-D-glucosamine residues are abundantly present on the surface. On the other hand, we find that treatment with PLC releases molecules from the surface; they do not reveal Cross Reacting Determinant (CRD), suggesting a novel anchor to the membrane for the glycoprotein GP1.
Assuntos
Clostridium perfringens/enzimologia , Cysticercus/metabolismo , Glicoproteínas de Membrana/metabolismo , Taenia solium/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Western Blotting , Cysticercus/citologia , Cysticercus/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Histocitoquímica , Glicoproteínas de Membrana/química , Microscopia Eletrônica , Músculo Esquelético/parasitologia , SuínosRESUMO
In order to reduce the toxicity of Clostridium perfringens fermentation broths used in vaccine preparation, we developed two-phase aqueous systems for removal of toxin-activating proteases. Removal of the proteases inhibits the conversion of protoxin to active toxin. In order to establish the conditions under which the phase separation occurs, binodal curves, formed by poly(ethylene glycol) (PEG) and sodium citrate, were investigated at different values of pH and PEG molar mass. A 2(4)-experimental design was used to evaluate the influence of PEG molar mass and concentration, citrate concentration and pH on protease partition coefficient, removal.factor and protease removal yield. It has been found that simultaneous increase in PEG molar mass and decrease in citrate concentration remarkably improved the removal factor, whereas the protease removal yield showed an opposite trend. The best conditions for the system under consideration (removal factor of 2.69 and yield of 116%) were obtained at pH 8.0 using PEG molar mass of 8000 g mol(-1) and concentrations of PEG and citrate of 24 and 15%, respectively.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Técnicas de Química Analítica/métodos , Clostridium perfringens/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Transição de Fase , Proteínas de Ligação ao Cálcio/isolamento & purificação , Citratos , Clostridium perfringens/crescimento & desenvolvimento , Clostridium perfringens/metabolismo , Meios de Cultura , Fermentação , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Polietilenoglicóis , Citrato de SódioRESUMO
Beta toxin of C. chauvoei has desoxiribonuclease (DNase) activity which is regarded as one of its virulence factors. The production of DNase was detected in strains isolated from bovines, using as controls C. chauvoei ATCC 10092, and C. perfringens Type A and C. septicum, both laboratory isolates. The enzyme activity was made evident on a DNA substrate observing the macroscopic degradation. A simple methodology was developed using a commercial medium for DNase test, with the incorporation of sterile horse serum. Each strain was streaked on the surface of the medium, incubated in anaerobic atmosphere at 37 degrees C for 48 hours. The plates were revealed with HCI 1 N. The appearance of a clear and transparent zone around and under the microbial growing was considered a positive reaction. Enzyme activity was detected in 10 of 12 strains and also in the controls. The serum addition to the commercial basal medium allows the optimum development of the microorganism showing the enzymatic digestion zone.