RESUMO
Helicobacter pylori is an infectious agent that colonizes the gastric mucosa of half of the population worldwide. This bacterium has been recognized as belonging to group 1 carcinogen by the World Health Organization for the role in development of gastritis, peptic ulcers, and cancer. Due to the increase in resistance to antibiotics used in the anti-H. pylori therapy, the development of an effective vaccine is an alternative of great interest, which remains a challenge. Therefore, a rational, strategic, and efficient vaccine design against H. pylori is necessary where the use of the most current bioinformatics tools could help achieve it. In this study, immunoinformatics approach was used to design a novel multiepitope oral vaccine against H. pylori. Our multiepitope vaccine is composed of cholera toxin subunit B (CTB) that is used as a mucosal adjuvant to enhance vaccine immunogenicity for oral immunization. CTB fused to 11 epitopes predicted of pathogenic (UreB170-189, VacA459-478, CagA1103-1122, GGT106-126, NapA30-44, and OipA211-230) and colonization (HpaA33-52, FlaA487-506, FecA437-456, BabA129-149, and SabA540-559) proteins from H. pylori. CKS9 peptide (CKSTHPLSC) targets epithelial microfold cells to enhance vaccine uptake from the gut barrier. All sequences were joined to each other by proper linkers. The vaccine was modeled and validated to achieve a high-quality three-dimensional structure. The vaccine design was evaluated as nonallergenic, antigenic, soluble, and with an appropriate molecular weight and isoelectric point. Our results suggest that our newly designed vaccine could serve as a promising anti-H. pylori vaccine candidate.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Epitopos/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Administração Oral , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/química , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/química , Toxina da Cólera/administração & dosagem , Toxina da Cólera/imunologia , Biologia Computacional , Epitopos/administração & dosagem , Infecções por Helicobacter/imunologia , Helicobacter pylori/química , Humanos , Camundongos , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
The Helicobacter pylori cytotoxin-associated gene A (CagA) is known for causing gastroduodenal diseases, such as atrophic gastritis and peptic ulcerations. Furthermore Helicobacter pylori CagA positive strains has been reported as one of the main risk factors for gastric cancer (Parsonnet et al., 1997). Structural variations in the CagA structure can alter its affinity with the host proteins, inducing differences in the pathogenicity of H. pylori. CagA N-terminal region is characterized for be conserved among all H. pylori strains since the C-terminal region is characterized by an intrinsically disorder behavior. We generated complete structural models of CagA using different conformations of the C-terminal region for two H. pylori strains. These models contain the same EPIYA (ABC1C2) motifs but different level of pathogenicity: gastric cancer and duodenal ulcer. Using these structural models we evaluated the pathogenicity level of the H. pylori strain, based on the affinity of the interaction with SHP-2 and Grb2 receptors and on the number of interactions with the EPIYA motif. We found that the main differences in the interaction was due to the contributions of certain types of energies from each strain and not from the total energy of the molecule. Specifically, the electrostatic energy, helix dipole energy, Wander Waals clashes, torsional clash, backbone clash and cis bond energy allowed a separation between severe and mild pathology for the interaction of only CagA with SHP2.
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Proteína Adaptadora GRB2/química , Helicobacter pylori/patogenicidade , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Termodinâmica , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Úlcera Duodenal/etiologia , Proteína Adaptadora GRB2/metabolismo , Helicobacter pylori/química , Simulação de Acoplamento Molecular , Análise de Componente Principal , Ligação Proteica , Conformação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Neoplasias Gástricas/etiologiaRESUMO
BACKGROUND: Helicobacter pylori is an important factor in the development of diseases such as ulcer and gastric cancer. This bacterium uses a periplasmic transporter, UreI, to deliver urea to the intracelullar space, where later it is transformed into ammonia by the cytoplasmic enzyme urease to survive the acidic condition of the human stomach. The UreI transporter presents a pH-dependent activity, where this pH-dependence remains unknown at a structural level. Althought the existance of several protonable residues in the periplasmic loops are related to the pH-dependent activity, we find interesting to have a clear view of the conformational changes involved in this phenomena through a molecular dynamic study. RESULTS: Molecular dynamic simulations of the UreI transporter at three different pH conditions were performed, revealing two main pH-dependent conformations, which we present as the open and close states. We find that salt bridges between the periplasmic loops are crucial interactions that stabilize these conformations. Besides, a cooperative behaviour exists between the six subunits of the system that is necessary to fulfill the activity of this transporter. CONCLUSIONS: We found different pH-dependent conformations of the urea transporter UreI from Helicobacter pylori, which are related to salt-bridge interactions in the periplasmic regions. The behaviour of every channel in the system is not independent, given the existance of a cooperative behaviour through the formation of salt-bridges between the subunits of the hexameric system. We believe that our results will be related to the generation of new eradication therapies using this transporter as an attractive target, denoting that the knowledge of the possible pH-dependent conformations adopted for this transporter are important for the development of rational drug design approximations.
Assuntos
Proteínas de Bactérias/química , Helicobacter pylori/metabolismo , Proteínas de Membrana Transportadoras/química , Helicobacter pylori/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Periplasma/metabolismo , Conformação Proteica , SaisRESUMO
BACKGROUND: Mongolian gerbils that are experimentally infected with Helicobacter pylori develop a chronic inflammation that is similar to natural infections in humans. The aim of this study was to compare the antigens of H. pylori cagPAI+ and cagPAI- strains that are expressed during Meriones unguiculatus colonization. MATERIALS AND METHODS: We identified H. pylori cagPAI+ and cagPAI- strain antigens via Western blotting of samples from Mongolian gerbils that were subjected to unique, mixed, and sequential bacterial infections. RESULTS: The antigens from the J99/CG3 (cagPAI+) strain had a lower molecular weight than the antigens from the 251F/CG3 (cagPAI-) strain. There were fewer identified antigens in the single unique infections compared with the mixed and sequential infections. The number of recognized antigens that had a frequency of recognition >60% was higher for the simultaneous and sequential infection groups compared with the single infection group. A 57-kDa antigen was present in >60% of the samples and four of the five experimental groups. Antigens specific to each bacterial strain were identified; the 190- and 158-kDa antigens appear to be specific for cagPAI-, and the 70-kDa antigen appears to be specific for cagPAI+. CONCLUSIONS: In this study, we identified antigens that are common and specific to the H. pylori cagPAI+ and cagPAI- strains.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Imunoglobulina G/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Western Blotting , Modelos Animais de Doenças , Ilhas Genômicas , Gerbillinae , Infecções por Helicobacter/microbiologia , Helicobacter pylori/química , Helicobacter pylori/fisiologia , Humanos , Imunoglobulina G/sangue , Masculino , Peso Molecular , CoelhosRESUMO
In order to identify novel inhibitors of the Helicobacter pylori nickel response regulator (HpNikR) an integrative protocol was performed for half a million compounds retrieved from the ZINC database. We firstly implement a structure-based virtual screening to build a library of potential inhibitors against the HpNikR using a docking analysis (AutoDock Vina). The library was then used to perform a hierarchical clustering of docking poses, based on protein-contact footprints calculation from the multiple conformations given by the AutoDock Vina software, and the drug-protein interaction analyses to identify and remove potential promiscuous compounds likely interacting with human proteins, hence causing drug side effects. 250 drug-like compounds were finally proposed as non-promicuous potential inhibitors for HpNikR. These compounds target the DNA-binding sites of HpNikR so that HpNikR-compound binding could be able to mimic key interactions in the DNA-protein recognition process. HpNikR inhibitors with promising potential against H. pylori could also act against other human bacterial pathogens due to the conservation of targeting motif of NikR involved in DNA-protein interaction.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Simulação por Computador , Proteínas de Ligação a DNA/antagonistas & inibidores , Descoberta de Drogas/métodos , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Rodaminas/química , Bibliotecas de Moléculas Pequenas/química , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Bases de Dados Factuais , Infecções por Helicobacter/microbiologia , Helicobacter pylori/química , Helicobacter pylori/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Níquel/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Rodaminas/farmacologia , Rodaminas/uso terapêutico , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Colonization of the gastric mucosa by Helicobacter pylori is one of the most important causes of acute and chronic gastric pathologies in humans. Achieving the growth of H. pylori in liquid media is of great importance in the development of clinical studies. In this study, we developed a sequential optimization strategy based on statistical models to improve the conditions of liquid culture of H. pylori. MATERIALS AND METHODS: Four statistical models were sequentially used. First, a Box-Behnken design was used to select the best process conditions (shaking speed, inoculum concentration, and final volume of culture). Secondly, a general factorial design was used to evaluate the influence of adding gel blocks or gel beads (shape and composition). Then a D-optimal reduce design was carried out to allow the selection of the most influential factors in increasing the cell concentration (culture media components). Finally, another Box-Behnken design was used to optimize the concentration of the culture media components previously selected. RESULTS: After 12 hours of liquid culture a concentration of 25 x 10(8) cells per mL (9.4 log(10) cells per mL) of H. pylori was obtained, compared with a predicted 32 x 10(8) (9.5 log(10) cells per mL), which means between 1 and 5 log(10) units higher than some previous reports. CONCLUSIONS: The sequential statistical approach increased the planktonic H. pylori cell culture. The final culture media and conditions were: Brain Heart Infusion, blood agarose (1.5% w/v), lamb's blood (3.18% v/v), DENT (0.11% v/v), and Vitox (0.52% v/v) at 60 rpm and 37 degrees C with filtered CO2 (5% v/v) bubbled directly into the culture media in a final volume of 76.22 mL.
Assuntos
Técnicas Bacteriológicas , Técnicas de Cultura , Helicobacter pylori/crescimento & desenvolvimento , Meios de Cultura/química , Meios de Cultura/metabolismo , Técnicas de Cultura/métodos , Helicobacter pylori/química , Helicobacter pylori/metabolismo , Modelos EstatísticosRESUMO
In the present work, the in vitro effect of a standardized extract of apple peel APPE (60% of total polyphenols; 58% of flavonoids; 30% of flavan-3-ols and procyanidins) was evaluated with regard to the viability of Helicobacter pylori. The cytotoxic effect of APPE on H. pylori was also evaluated through the resazurin assay and ATP level determination. In both assays, APPE showed an early cytotoxic effect, which was both concentration and time-dependent. Additionally, the effect of APPE on the intra and extracellular production of reactive oxygen species (ROS) was evaluated in human neutrophils stimulated by H. pylori, phorbol myristate acetate (PMA), and formyl-methionyl-leucyl-phenylalanine (fMLP). The extracellular and intracellular production of ROS was evaluated through chemiluminiscence with the isoluminol-horseradish peroxidase (HRP) and luminol-superoxide dismutase (SOD)-catalase systems, respectively. APPE showed an inhibiting effect on the multiplication of two H. pylori strains (ATCC 43504 and TX136) with a miminnum inhibitory concentration (MIC) value of 112.5 microg gallic acid equivalent (GAE)/mL. APPE inhibited the respiratory burst of neutrophils induced by H. pylori, PMA, and fMLP in concentration-dependent form. Interestingly, this effect was observed on both the interior and exterior of the neutrophil. This result suggests that apple peel polyphenols have an attenuating effect on the damage to gastric mucosa caused by neutrophil generated ROS and, particularly, when H. pylori displays its evasion mechanisms.
Assuntos
Frutas/química , Helicobacter pylori/efeitos dos fármacos , Malus/química , Neutrófilos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Explosão Respiratória/efeitos dos fármacos , Trifosfato de Adenosina/análise , Flavonoides/análise , Helicobacter pylori/química , Helicobacter pylori/crescimento & desenvolvimento , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/fisiologia , Fenóis/análise , Extratos Vegetais/química , Polifenóis , Proantocianidinas/análise , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Protein patterns of 129 Helicobacter pylori strains isolated from Korean and Colombian patients suffering from duodenal ulcer or gastric cancer were analyzed by the high-throughput methodology SELDI-TOF-MS. Eighteen statistically significant candidate biomarkers discriminating between the two clinical outcomes were selected by using the Mann-Whitney test. Three biomarker proteins were purified and identified as a neutrophil-activating protein NapA (HU HPAG1_0821), a RNA-binding protein (HPAG1_0813), and a DNA-binding histone-like protein HU, respectively (jhp0228). These novel biomarkers can be used for development of diagnostic assays predicting the evolution to gastric cancer in H. pylori-infected patients.