RESUMEN
The rise in the antibiotic resistance rate of Helicobacter pylori has led to an increasing eradication failure of this carcinogenic bacterial pathogen worldwide. This underlines the need for alternative antibacterial strategies against H. pylori infection. Antimicrobial photodynamic therapy (aPDT) is a promising non-pharmacological antibacterial technology. In this study, the selective killing activities of three benzylidene cyclopentanone (BCP) photosensitizers (Y1, P1 and P3) towards H. pylori over normal human gastric epithelial GES-1 cells were evaluated and the ex vivo photodynamic inactivation effect was preliminarily assessed on twelve H. Pylor-infected mice. Results showed that under the irradiation of 24 J/cm2 532 nm laser, Y1, P1 and P3 at 2.5 µM induced a 3-log10 reduction of H. pylori CFU (99.9% killing). Confocal images showed that P3, unlike Y1 and P1, could not be uptaken by GES-1 cells. P3 at 2.5 to 20 µM showed not significant (p > 0.05) phototoxicity to GES-1 cells, nevertheless, Y1 and P1 under the same concentrations exhibited remarkable phototoxicity to GES-1 cells. In the co-culture of H. pylori and GES-1 cells, P3 at 2.5 µM led to a complete eradication of H. pylori under the irradiation of 24 J/cm2 532 nm laser. While for the GES-1 cells, no significant (p > 0.05) phototoxicity was observed under the same aPDT dosage. The ex vivo experiments showed that P3 mediated aPDT resulted in 82.4% to 100% reduction of H. pylori CFU without damaging the gastric mucosa. To sum up, P3 is a promising anti-H. pylori photosensitizer with the ability to selectively photo-inactivate H. pylori while sparing normal gastric tissues.
Asunto(s)
Ciclopentanos/química , Helicobacter pylori/efectos de los fármacos , Rayos Láser , Fármacos Fotosensibilizantes/farmacología , Animales , Compuestos de Bencilideno/química , Cationes/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Ciclopentanos/metabolismo , Ciclopentanos/farmacología , Ciclopentanos/uso terapéutico , Modelos Animales de Enfermedad , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Mucosa Gástrica/efectos de la radiación , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/patología , Helicobacter pylori/efectos de la radiación , Helicobacter pylori/ultraestructura , Humanos , Ratones , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
Helicobacter pylori is a bacterium known mainly of its ability to cause persistent inflammations of the human stomach, resulting in peptic ulcer diseases and gastric cancers. Continuous exposure of this bacterium to antibiotics has resulted in high detection of multidrug-resistant strains and difficulties in obtaining a therapeutic effect. The purpose of the present study was to determine the usability of bacterial cellulose (BC) chemisorbed with 3-bromopyruvate (3-BP) or sertraline (SER) to act against lawn H. pylori biofilms. The characterization of BC carriers was made using a N2 adsorption/desorption analysis, tensile strength test, and scanning electron microscopy (SEM) observations. Determination of an antimicrobial activity was performed using a modified disk-diffusion method and a self-designed method of testing antibacterial activity against biofilm microbial forms. In addition, bacterial morphology was checked by SEM. It was found that BC disks were characterized by a high cross-linking and shear/stretch resistance. Growth inhibition zones for BC disks chemisorbed with 2 mg of SER or 3-BP were equal to 26.5-27.5 mm and 27-30 mm, respectively. The viability of lawn biofilm H. pylori cells after a 4-h incubation with 2 mg SER or 3-BP chemisorbed on BC disks was ≥4 log lower, suggesting their antibacterial effect. SEM observations showed a number of morphostructural changes in H. pylori cells exposed to these substances. Concluding, SER and 3-BP chemisorbed on BC carriers presented a promising antibacterial activity against biofilm H. pylori cells in in vitro conditions.
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Celulosa/metabolismo , Piruvatos/metabolismo , Sertralina/metabolismo , Biopelículas/crecimiento & desarrollo , Helicobacter pylori/crecimiento & desarrollo , Helicobacter pylori/metabolismo , Helicobacter pylori/ultraestructura , Pruebas de Sensibilidad MicrobianaRESUMEN
Toxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins' biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5'-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment.
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Helicobacter pylori/citología , Helicobacter pylori/efectos de los fármacos , Péptidos/farmacología , Sistemas Toxina-Antitoxina , Adenosina Trifosfato/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Helicobacter pylori/ultraestructura , Peróxido de Hidrógeno/toxicidad , Espacio Intracelular/metabolismo , Cinética , Potenciales de la Membrana/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Peptidoglicano/metabolismoRESUMEN
The VirB/D type IV secretion system (T4SS) plays an essential role in materials transport between host cells and pathogenic Helicobacter pylori and is considered the major pathogenic mediator of H. pylori-associated gastric disease. VirB8, an inner membrane protein that interacts with many other proteins, is a crucial component for secretory function. Here, we present a crystal structure of the periplasmic domain of CagV, the VirB8 counterpart in the H. pylori Cag-T4SS. The structure reveals a fold similar to that of other VirB8 members except for the absence of the α5 helix, a discontinuous ß1 strand, a larger angle between the α2 and α3 helices, a more hydrophobic surface groove, but exhibits a different dimer interface. Whether the dimerization occurs in solution was proved by mutagenesis, size-exclusion chromatography and cross-linking assays. Unlike the classical dimerization mode, the interface of the CagV dimer is principally formed by several hydrogen bonds, which indicates instability of dimerization. The structure here demonstrates the difference in dimerization among VirB8 homologues and indicates the considerable compositional and functional diversity of them in T4SS. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6IQT.
Asunto(s)
Infecciones por Helicobacter/microbiología , Helicobacter pylori/química , Proteínas de la Membrana/ultraestructura , Conformación Proteica , Cristalografía por Rayos X , Helicobacter pylori/patogenicidad , Helicobacter pylori/ultraestructura , Interacciones Huésped-Patógeno/genética , Humanos , Proteínas de la Membrana/química , Periplasma/química , Periplasma/ultraestructura , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína/genética , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/genéticaRESUMEN
Helicobacter pylori colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. H. pylori secretes a pore-forming toxin called vacuolating cytotoxin A (VacA), which contains two domains (p33 and p55) and assembles into oligomeric structures. Using single-particle cryo-electron microscopy, we have determined low-resolution structures of a VacA dodecamer and heptamer, as well as a 3.8-Å structure of the VacA hexamer. These analyses show that VacA p88 consists predominantly of a right-handed beta-helix that extends from the p55 domain into the p33 domain. We map the regions of p33 and p55 involved in hexamer assembly, model how interactions between protomers support heptamer formation, and identify surfaces of VacA that likely contact membrane. This work provides structural insights into the process of VacA oligomerization and identifies regions of VacA protomers that are predicted to contact the host cell surface during channel formation.
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Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Helicobacter pylori/química , Microscopía por Crioelectrón/métodos , Infecciones por Helicobacter/microbiología , Helicobacter pylori/ultraestructura , Humanos , Modelos Moleculares , Conformación Proteica , Multimerización de ProteínaRESUMEN
Human gastric pathogen Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by H. pylori and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed ß-helix made up of two domains with extensive domain-domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Toxinas Bacterianas/química , Helicobacter pylori/ultraestructura , Complejos Multiproteicos/ultraestructura , Multimerización de Proteína , Microscopía por Crioelectrón , Estructura Cuaternaria de ProteínaRESUMEN
The urea channel of Helicobacter pylori (HpUreI) is an ideal drug target for preventing gastric cancer but incomplete understanding of its gating mechanism has hampered development of inhibitors for the eradication of H. pylori. Here, we present the cryo-EM structures of HpUreI in closed and open conformations, both at a resolution of 2.7 Å. Our hexameric structures of this small membrane protein (~21 kDa/protomer) resolve its periplasmic loops and carboxyl terminus that close and open the channel, and define a gating mechanism that is pH dependent and requires cooperativity between protomers in the hexamer. Gating is further associated with well-resolved changes in the channel-lining residues that modify the shape and length of the urea pore. Site-specific mutations in the periplasmic domain and urea pore identified key residues important for channel function. Drugs blocking the urea pore based on our structures should lead to a new strategy for H. pylori eradication.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Infecciones por Helicobacter/microbiología , Helicobacter pylori/ultraestructura , Proteínas de Transporte de Membrana/ultraestructura , Proteínas Bacterianas/química , Microscopía por Crioelectrón , Infecciones por Helicobacter/genética , Helicobacter pylori/química , Helicobacter pylori/genética , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Transporte de Membrana/química , Conformación Proteica , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologíaRESUMEN
BACKGROUND: Antibiotic-resistant H. pylori was increasingly found in infected individuals, which resulted in treatment failure and required alternative therapeutic strategies. Daphnetin, a coumarin-derivative compound, has multiple pharmacological activities. METHODS: The mechanism of daphnetin on H. pylori was investigated focusing on its effect on cell morphologies, transcription of genes related to virulence, adhesion, and cytotoxicity to human gastric epithelial (GES-1) cell line. RESULTS: Daphnetin showed good activities against multidrug resistant (MDR) H. pylori clinical isolates, with minimal inhibitory concentration (MIC) values ranging from 25 to 100 µg/mL. In addition, daphnetin exposure resulted in H. pylori morphological changes. Moreover, daphnetin caused increased translocation of phosphatidylserine (PS), DNA damage, and recA expression, and RecA protein production vs. control group. Of great importance, daphnetin significantly decreased H. pylori adhesion to GES-1 cell line vs. control group, which may be related to the reduced expression of colonization related genes (e.g., babA and ureI). CONCLUSIONS: These results suggested that daphnetin has good activity against MDR H. pylori. The mechanism(s) of daphnetin against H. pylori were related to change of membrane structure, increase of DNA damage and PS translocation, and decrease of H. pylori attachment to GES-1 cells.
Asunto(s)
Antibacterianos/farmacología , Helicobacter pylori/efectos de los fármacos , Umbeliferonas/farmacología , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Transformada , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Claritromicina/farmacología , Daño del ADN , Células Epiteliales/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Helicobacter pylori/genética , Helicobacter pylori/ultraestructura , Humanos , Metronidazol/farmacología , Pruebas de Sensibilidad Microbiana , Umbeliferonas/químicaRESUMEN
The variability of Helicobacter pylori morphology and the heterogeneity of virulence factors expressed by these bacteria play a key role as a driving force for adaptation to the hostile stomach environment. The aim of the study was to determine the relationship between the presence of certain genes encoding virulence factors and H. pylori morphology. One reference and 13 clinical H. pylori strains with a known virulence profile (vacA, cagA, babA2, dupA, and iceA) were used in this study. Bacteria were cultured for 1 h and 24 h in stressogenic culture conditions, i.e., serum-free BHI broths at suboptimal conditions (room temperature and atmosphere, without shaking). H. pylori cell morphology was observed by light and scanning electron microscopy. The vacA polymorphism and the cagA and babA2 presence were positively correlated with the reduction in cell size. Exposure to short-time stressogenic conditions caused more intense transformation to coccoid forms in highly pathogenic H. pylori type I strains (35.83% and 47.5% for type I s1m2 and I s1m1, respectively) than in intermediate-pathogenic type III (8.17%) and low pathogenic type II (9.92%) strains. The inverse relationship was observed for the number of rods, which were more common in type III (46.83%) and II (48.42%) strains than in type I s1m2 (19.25%) or I s1m1 (6.58%) strains. Our results suggest that there is a close relationship between the presence of virulence genes of H. pylori strains and their adaptive morphological features.
Asunto(s)
Proteínas Bacterianas/genética , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Factores de Virulencia/genética , Adhesinas Bacterianas/genética , Gastritis/microbiología , Genes Bacterianos , Genotipo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/ultraestructura , Humanos , Microscopía Electrónica de Rastreo , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Virulencia/genéticaRESUMEN
Helicobacter pylori has an impressive ability to persist chronically in the human stomach. Similar characteristics are associated with biofilm formation in other bacteria. The H. pylori biofilm process, however, is poorly understood. To gain insight into this mode of growth, we carried out comparative transcriptomic analysis between H. pylori biofilm and planktonic cells, using the mouse-colonizing strain SS1. Optimal biofilm formation was obtained with a low concentration of serum and 3 days of growth, conditions that caused both biofilm and planktonic cells to be â¼80% coccoid. Transcriptome sequencing (RNA-seq) analysis found that 8.18% of genes were differentially expressed between biofilm and planktonic cell transcriptomes. Biofilm-downregulated genes included those involved in metabolism and translation, suggesting these cells have low metabolic activity. Biofilm-upregulated genes included those whose products were predicted to be at the cell envelope, involved in regulating a stress response, and surprisingly, genes related to formation of the flagellar apparatus. Scanning electron microscopy visualized flagella that appeared to be a component of the biofilm matrix, supported by the observation that an aflagellated mutant displayed a less robust biofilm with no apparent filaments. We observed flagella in the biofilm matrix of additional H. pylori strains, supporting that flagellar use is widespread. Our data thus support a model in which H. pylori biofilm involves a multigene stress-biased response and that flagella play an important role in H. pylori biofilm formation.IMPORTANCE Biofilms, communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances, pose a substantial health risk and are key contributors to many chronic and recurrent infections. Chronicity and recalcitrant infections are also common features associated with the ulcer-causing human pathogen H. pylori However, relatively little is known about the role of biofilms in H. pylori pathogenesis, as well as the biofilm structure itself and the genes associated with this mode of growth. In the present study, we found that H. pylori biofilm cells highly expressed genes related to cell envelope and stress response, as well as those encoding the flagellar apparatus. Flagellar filaments were seen in high abundance in the biofilm. Flagella are known to play a role in initial biofilm formation, but typically are downregulated after that state. H. pylori instead appears to have coopted these structures for nonmotility roles, including a role building a robust biofilm.
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Biopelículas/crecimiento & desarrollo , Flagelos/metabolismo , Regulación Bacteriana de la Expresión Génica , Helicobacter pylori/fisiología , Estrés Fisiológico , Flagelos/ultraestructura , Perfilación de la Expresión Génica , Helicobacter pylori/crecimiento & desarrollo , Helicobacter pylori/ultraestructura , Redes y Vías Metabólicas/genética , Microscopía Electrónica de Rastreo , Análisis de Secuencia de ARNRESUMEN
INTRODUCTION: There is no consensus on the benefit of performing a systematic complementary technique for the diagnosis of Helicobacter pylori infection. In our laboratory, a cresyl violet was carried out systematically until July 2014; since that date, a cresyl violet or immunohistochemistry is only made on request. We evaluated the value of cresyl violet staining of gastric biopsies to diagnose H. pylori infection by comparing a period of systematic staining to a time when it was made on demand. MATERIAL AND METHODS: We retrospectively studied the gastric biopsy of 786 consecutive patients from April to November 2014, taken in the absence of focal endoscopic lesion. During the first period, hematoxylin-eosin and cresyl violet were performed on all biopsies. During the second period, hematoxylin-eosin was performed and then, if necessary, cresyl violet or immunohistochemistry. All hematoxylin-eosin stained slides were revised to identify H. pylori. We performed immunohistochemistry in cases of active chronic gastritis without H. pylori identified on hematoxylin-eosin or cresyl violet. RESULTS: We have shown that gastric biopsy performed in the absence of focal mucosal lesion are normal in 55% of cases. The percentage of H. pylori infection was similar in both groups. In cases of active chronic gastritis, H. pylori infection is visible, in most cases, on hematoxylin-eosin (94%). Immunohistochemistry should be prescribed only in case of chronic active gastritis without H. pylori identified on standard staining, with bacteria rare or atypically located. CONCLUSION: In our experiment, H. pylori is present only in case of active gastritis (33% of the biopsies in our series) and being almost always identifiable on the standard staining with H-E (in 94% of the cases), it is not It is not necessary to systematically perform, on all gastric biopsies, a complementary histo- or immunohistochemical technique.
Asunto(s)
Benzoxazinas , Biopsia/métodos , Colorantes , Mucosa Gástrica/patología , Gastritis/patología , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/aislamiento & purificación , Técnicas para Inmunoenzimas , Coloración y Etiquetado/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad Crónica , Eosina Amarillenta-(YS) , Femenino , Mucosa Gástrica/microbiología , Gastritis/microbiología , Gastroscopía , Infecciones por Helicobacter/patología , Helicobacter pylori/ultraestructura , Hematoxilina , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Procedimientos Innecesarios , Adulto JovenRESUMEN
Objectives: Because of the rise in antimicrobial resistance, an inexpensive, diet-based treatment against Helicobacter pylori infection would be of great interest. The present study was performed to assess the in vitro effects of ellagic acid against clinical H. pylori strains that were resistant to antibiotics used for therapy and also to observe the morphological structure following treatment with ellagic acid. The effectiveness of ellagic acid in eradicating H. pylori infection in a murine (C57BL/6) infection model, one of the standard inbred mouse lines often used for experimental infection, was also assessed. Methods: A total of 55 strains were screened. The agar dilution method was used to determine the susceptibility of isolates to test compounds. Transmission electron microscopy was used to observe the morphology following treatment with ellagic acid. The antibacterial activity of ellagic acid in an H. pylori SS1-infected mouse model and its effect on gastric mucosal injury were determined by histology and PCR. Results: Ellagic acid inhibited the growth of all 55 of the H. pylori strains tested. The MIC of ellagic acid ranged from 5 to 30 mg/L, showing its bactericidal properties in vitro. Ellagic acid also demonstrated anti-H. pylori efficacy in eradication of this organism in an in vivo model, as well as restitution and repair of H. pylori-induced gastric mucosal damage. Conclusions: The present study paves the way for the preventive and therapeutic use of ellagic acid against H. pylori infection and, thus, ellagic acid can be considered a promising antibacterial agent against H. pylori-associated gastroduodenal diseases in humans.
Asunto(s)
Ácido Elágico/farmacología , Mucosa Gástrica/patología , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Animales , Antibacterianos/farmacología , Modelos Animales de Enfermedad , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/ultraestructura , Humanos , India , Masculino , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Organismos Libres de Patógenos EspecíficosRESUMEN
BACKGROUND AND AIM: Infection by Helicobacter pylori is implicated in a wide range of upper gastrointestinal diseases. Owing to the rapid emergence of antibiotic-resistant strains of H. pylori, the development of novel treatment modalities for antibiotic-resistant H. pylori infection is a key priority. Blue light-emitting diodes (LED) may represent a unique option owing to their antimicrobial effect. In this study, we aimed to evaluate the anti-proliferative effect of blue LED against antibiotic-resistant H. pylori. METHODS: Ten antibiotic-resistant strains and one sensitive H. pylori strain were used in this study. After irradiation by blue LED along time course, the viability of H. pylori was evaluated by enumerating colony forming units. Morphological changes in H. pylori were observed using a scanning electron microscope. Reductase activity was measured as an indicator of bacterial cellular activity. Total reactive oxygen species was monitored using fluorescence intensity and fluorescence microscope imaging. RESULTS: After irradiation by blue LED, the numbers of H. pylori in all the strains were significantly reduced compared with control group. The H. pylori exhibited a short rod-shaped morphology after irradiation; no such change was observed in H. pylori not exposed to blue LED. Re-irradiation of surviving strain after the initial irradiation also exhibited the same anti-proliferation effect. After blue LED irradiation, bacterial cellular activity was lower, and total reactive oxygen species production was significantly higher in blue LED group, compared with that in control. CONCLUSIONS: Blue LED could be a new treatment to eradicate infection with antibiotic-resistant H. pylori.
Asunto(s)
Helicobacter pylori/efectos de la radiación , Láseres de Semiconductores , Recuento de Colonia Microbiana , Farmacorresistencia Bacteriana , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/enzimología , Helicobacter pylori/ultraestructura , Microscopía Electroquímica de Rastreo , Microscopía Fluorescente , Oxidorreductasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The venom peptide bicarinalin, previously isolated from the ant Tetramorium bicarinatum, is an antimicrobial agent with a broad spectrum of activity. In this study, we investigate the potential of bicarinalin as a novel agent against Helicobacter pylori, which causes several gastric diseases. First, the effects of synthetic bicarinalin have been tested against Helicobacter pylori: one ATCC strain, and forty-four isolated from stomach ulcer biopsies of Peruvian patients. Then the cytoxicity of bicarinalin on human gastric cells and murine peritoneal macrophages was measured using XTT and MTT assays, respectively. Finally, the preventive effect of bicarinalin was evaluated by scanning electron microscopy using an adherence assay of H. pylori on human gastric cells treated with bicarinalin. This peptide has a potent antibacterial activity at the same magnitude as four antibiotics currently used in therapies against H. pylori. Bicarinalin also inhibited adherence of H. pylori to gastric cells with an IC50 of 0.12 µg·mL-1 and had low toxicity for human cells. Scanning electron microscopy confirmed that bicarinalin can significantly decrease the density of H. pylori on gastric cells. We conclude that Bicarinalin is a promising compound for the development of a novel and effective anti-H. pylori agent for both curative and preventive use.
Asunto(s)
Venenos de Hormiga/farmacología , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Helicobacter pylori/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Helicobacter pylori/ultraestructura , Humanos , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Células RAW 264.7RESUMEN
Uptake, intracellular trafficking and pathologic effects of VacA toxin from Helicobacter pylori have been widely investigated in vitro. However, no systematic analysis investigated VacA intracellular distribution and fate in H. pylori-infected human gastric epithelium in vivo, using ultrastructural immunocytochemistry that combines precise toxin localization with analysis of the overall cell ultrastructure and intercompartimental/interorganellar relationships. By immunogold procedure, in this study we investigated gastric biopsies taken from dyspeptic patients to characterize the overall toxin's journey inside human gastric epithelial cells in vivo. Endocytic pits were found to take up VacA at sites of bacterial adhesion, leading to a population of peripheral endosomes, which in deeper (juxtanuclear) cytoplasm enlarged and fused each other to form large VacA-containing vacuoles (VCVs). These directly opened into endoplasmic reticulum (ER) cisternae, which in turn enveloped mitochondria and contacted the Golgi apparatus. In all such organelles we found toxin molecules, often coupled with structural damage. These findings suggest direct toxin transfer from VCVs to other target organelles such as ER/Golgi and mitochondria. VacA-induced cytotoxic changes were associated with the appearance of auto(phago)lysosomes containing VacA, polyubiquitinated proteins, p62/SQSTM1 protein, cathepsin D, damaged mitochondria and bacterial remnants, thus leading to persistent cell accumulation of degradative products.
Asunto(s)
Proteínas Bacterianas/metabolismo , Helicobacter pylori/fisiología , Estómago/microbiología , Adulto , Anciano , Epitelio/microbiología , Femenino , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/ultraestructura , Humanos , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Estómago/patología , Estómago/ultraestructura , Vesículas Transportadoras/microbiología , VacuolasRESUMEN
The Helicobacter pylori flagellum is a complex rotatory nanomachine fundamental for the bacterium's survival in the human stomach. Protein FlgE is a component of the hook, a flexible junction exposed on the cell surface. In the H. pylori genome two different genes are present in different positions coding for hypothetical FlgE. The first protein, FlgE1, is the actual component of the flagellum hook, whilst the second, FlgE2, shares only 26% of the sequence identity with the other and its physiological function is still undefined. We have cloned, purified and crystallized FlgE2, whose structure, determined by the single-wavelength anomalous diffraction method, shows that in overall organization, the protein is composed of three distinct domains, two of them relatively similar to those of FlgE from other Gram-negative bacteria, whilst the third is peculiar to H. pylori. The crystal structure, along with the detected interaction with the regulatory cap protein FlgD, suggests a complementary function of FlgE1 and FlgE2 in the H. pylori flagellum, possibly typical of polar flagella, confirming the role of both proteins in the flagellar hook organization. Although some general features are shared with other Gram-negative bacteria, the presence of two different hook proteins indicates that the molecular organization of H. pylori flagellum has its own peculiarities. DATABASE: Atomic coordinates and structural factors have been deposited in the Protein Data Bank as 5NPY.
Asunto(s)
Proteínas Bacterianas/química , Flagelos/química , Helicobacter pylori/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Cristalografía por Rayos X , Flagelos/ultraestructura , Helicobacter pylori/genética , Helicobacter pylori/ultraestructura , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
Nearly half of the global population are carriers of Helicobacter pylori (H. pylori), a Gram-negative bacterium that persists in the healthy human stomach. H. pylori can be a pathogen and causes development of peptic ulcer disease in a certain state of the macroorganism. It is well established that H. pylori infection is the main cause of chronic gastritis and peptic ulcer disease (PUD). Decontamination of the gastric mucosa with various antibiotics leads to H. pylori elimination and longer remission in this disease. However, the reasons for repeated detection of H. pylori in recurrent PUD after its successful eradication remain unclear. The reason for the redetection of H. pylori in recurrent PUD can be either reinfection or ineffective anti-Helicobacter therapy. The administration of antibacterial drugs can lead not only to the emergence of resistant strains of microorganisms, but also contribute to the conversion of H. pylori into the resting (dormant) state. The dormant forms of H. pylori have been shown to play a potential role in the development of relapses of PUD. The paper discusses morphological H. pylori forms, such as S-shaped, C-shaped, U-shaped, and coccoid ones. The authors proposes the classification of H. pylori according to its morphological forms and viability.
Asunto(s)
Mucosa Gástrica/microbiología , Gastritis Atrófica/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Úlcera Péptica/microbiología , Gastritis Atrófica/epidemiología , Infecciones por Helicobacter/epidemiología , Helicobacter pylori/aislamiento & purificación , Helicobacter pylori/fisiología , Helicobacter pylori/ultraestructura , Humanos , Microscopía Electrónica , Úlcera Péptica/epidemiología , Prevalencia , RecurrenciaRESUMEN
Romanowsky-Giemsa (RG) stains were devised during the 19th century for identifying plasmodia parasites in blood smears. Later, RG stains became standard procedures for hematology and cytology. Numerous attempts have been made to apply RG staining to formalin-fixed paraffin-embedded tissue sections, with varied success. Most published work on this topic described RG staining methods in which sections were overstained, then subjected to acid differentiation; unfortunately, the differentiation step often caused inconsistent staining outcomes. If staining is performed under optimal conditions with control of dye concentration, pH, solution temperature and staining time, no differentiation is required. We used RG and 0.002 M buffer, pH 42, for staining and washing sections. All steps were performed at room temperature. After staining and air drying, sections were washed in 96-100% ethanol to remove extraneous stain. Finally, sections were washed in xylene and mounted using DPX. Staining results were similar to routine hemalum and eosin (H & E) staining. Nuclei were blue; intensity depended largely on chromatin density. RNA-rich sites were purple. Collagen fibers, keratin, muscle cells, erythrocytes and white matter of the central nervous system were stained pinkish and reddish hues. Cartilage matrix, mast cell granules and areas of myxomatous degeneration were purple. Sulfate-rich mucins were stained pale blue, while those lacking sulfate groups were unstained. Deposits of hemosiderin, lipofuscin and melanin were greenish, and calcium deposits were blue. Helicobacter pylori bacteria were violet to purple. The advantages of the method are its close similarity to H & E staining and technical simplicity. Hemosiderin, H. pylori, mast cell granules, melanin and specific granules of different hematopoietic cells, which are invisible or barely distinguishable by H & E staining, are visualized. Other advantages over previous RG stains include shorter staining time and avoidance of acetone.
Asunto(s)
Colorantes Azulados/química , Eosina Amarillenta-(YS)/química , Técnicas de Preparación Histocitológica/métodos , Formaldehído , Helicobacter pylori/ultraestructura , Humanos , Intestino Delgado/ultraestructura , Adhesión en ParafinaRESUMEN
The Helicobacterpylori bacterium is one of the main causes of chronic gastritis, peptic ulcers, and even gastric cancer. It affects an average of half of the world population. Its difficult eradication depends upon multi-drug therapy. Since its classification as a group 1 carcinogenic by International Agency for Research on Cancer (IARC), the importance of H. pylori eradication has obtained a novel meaning. There is considerable interest in alternative therapies for the eradication of H. pylori using compounds from a wide range of natural products. In the present study, we investigated the antibacterial property of the isocoumarin paepalantine against H. pylori and it exhibited significant anti-H. pylori activity at a minimum inhibitory concentration (MIC) of 128 µg/mL and at a minimum bactericidal concentration (MBC) of 256 µg/mL. The scanning electron microscopy (SEM) revealed significant morphological changes of the bacterial cell as a response to a sub-MIC of paepalantine, suggesting a penicillin-binding protein (PBP) inhibition. Computational studies were carried out in order to study binding modes for paepalantine in PBP binding sites, exploring the active and allosteric sites. The data from the present study indicates that paepalantine exhibits significant anti-H. pylori activity, most likely by inhibiting membrane protein synthesis.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Isocumarinas/química , Isocumarinas/farmacología , Helicobacter pylori/ultraestructura , Humanos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Proteínas de Unión a las Penicilinas/químicaRESUMEN
It remains unclear why gastric disease does not develop in all cases of Helicobacter pylori infection. In this study, we analyzed whether simian virus 5 (SV5) enhanced adherence of H. pylori to adenocarcinoma epithelial cells (AGS). H. pylori in AGS (harboring SV5) and SV5-infected Vero cells, and an agglutination of H. pylori mixed with SV5 were observed by light microscopy, scanning and transmission electron microscopies. The adherent rate of H. pylori to SV5-infected Vero cells and treated with an anti-SV5 antibody was determined. H. pylori adhered to the surface of AGS cells near SV5 particles, as shown by scanning and transmission electron microscopies. The adherence of H. pylori to SV5-infected Vero cells was significantly enhanced compared with that to Vero cells. In contrast, the adherence of H. pylori to Vero cells was decreased by treatment with the anti-SV5 antibody. Agglutination of H. pylori mixed with SV5 was observed by scanning and transmission electron microscopies. Agglutination did not occur when SV5 was treated with the anti-SV5 antibody before mixing. These findings demonstrated that SV5 enhanced the adherence of H. pylori to host cells, suggesting that a persistently infected virus may be a factor enhancing the pathogenicity of H. pylori in humans.