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1.
Cancer Sci ; 112(11): 4799-4811, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34449934

RESUMEN

Epstein-Barr virus (EBV)-associated gastric cancer belongs to 1 of the 4 subtypes of gastric cancer and accounts for 10% of total gastric cancers. However, most cases of gastric cancer have a history of Helicobacter pylori infection. Therefore, we investigated the possibility that H. pylori infection promotes the development of EBV-associated gastric cancer. H. pylori was exposed to principal EBV receptor, CD21, negative gastric epithelial cells, and then infected with EBV recombinant expressing enhanced green fluorescent protein. Changes in EBV infectivity due to prior H. pylori exposure were analyzed using flow cytometry. The treatment of gastric epithelial cells with H. pylori increased the efficiency of EBV infection. An increase was also observed when CagA-deficient, VacA-deficient, and FlaA-deficient H. pylori strains were used, but not when cag pathogenicity island-deficient H. pylori was used. The treatment of epithelial cells with H. pylori induced the expression of accessory EBV receptors, EphA2 and NMHC-IIA, and increased the efficiency of EBV infection depending on their expression levels. When gastric epithelial cells were treated with EPHA2 or NMHC-IIA siRNA, EBV infection via H. pylori attachment was decreased. The adhesion of H. pylori induced the expression of accessory EBV receptors in gastric epithelial cells and increased the efficiency of EBV infection.


Asunto(s)
Infecciones por Virus de Epstein-Barr/etiología , Infecciones por Helicobacter/complicaciones , Helicobacter pylori/fisiología , Herpesvirus Humano 4 , Neoplasias Gástricas/virología , Antígenos Bacterianos/metabolismo , Sitios de Ligazón Microbiológica/fisiología , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Proteínas Fluorescentes Verdes/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidad , Humanos , Hidroliasas/deficiencia , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Oxidorreductasas/deficiencia , ARN Interferente Pequeño/farmacología , Receptor EphA2/genética , Receptor EphA2/metabolismo , Receptores de Complemento 3d/metabolismo , Neoplasias Gástricas/microbiología
2.
Microbiology (Reading) ; 161(6): 1231-9, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25767109

RESUMEN

Helicobacter pylori growth medium is usually supplemented with horse serum (HS) or FCS. However, cyclodextrin derivatives or activated charcoal can replace serum. In this study, we purified self-growth-inhibiting (SGI) compounds from H. pylori growth medium. The compounds were recovered from porous resin, Diaion HP-20, which was added to the H. pylori growth medium instead of known supplements. These SGI compounds were also identified from 2,6-di-O-methyl-ß-cyclodextrin, which was supplemented in a pleuropneumonia-like organisms broth. The growth-inhibiting compounds were identified as lauric acid (LA) and 7-(Z)-tetradecenoic acid [7-(Z)-TDA]. Although several fatty acids had been identified in H. pylori, these specific compounds were not previously found in this species. However, we confirmed that these fatty acids were universally present in the cultivation medium of the H. pylori strains examined in this study. A live/dead assay carried out without HS indicated that these compounds were bacteriostatic; however, no significant growth-inhibiting effect was observed against other tested bacterial species that constituted the indigenous bacterial flora. These findings suggested that LA and 7-(Z)-TDA might play important roles in the survival of H. pylori in human stomach epithelial cells.


Asunto(s)
Ácidos Grasos Monoinsaturados/metabolismo , Sustancias de Crecimiento/metabolismo , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/crecimiento & desarrollo , Ácidos Láuricos/metabolismo , Medios de Cultivo/química , Ácidos Grasos Monoinsaturados/aislamiento & purificación , Sustancias de Crecimiento/aislamiento & purificación , Helicobacter pylori/metabolismo , Ácidos Láuricos/aislamiento & purificación
3.
Biomed Res ; 29(1): 9-18, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18344593

RESUMEN

H. pylori is a gram-negative bacterium associated with gastric inflammation and peptic ulcer and considered a risk factor for gastric cancer in its natural habitat. However, the energy metabolism of H. pylori in the stomach remains to be clarified. H. pylori shows rather high respiratory activity with L-proline and significantly large amounts of L-proline are present in the gastric juice from H. pylori infected patients. We constructed a disrupted mutant of the put A gene, which encodes the proline utilization A (Put A) flavin-linked enzyme, in order to examine the role of put A in the gastric colonization of H. pylori. The put A disrupted mutant, DeltaputA, was constructed by inserting a chloramphenicol resistant gene into put A. DeltaputA did not show respiratory activity using L-proline and could not incorporate L-proline into cells. DeltaputA also did not show motility in response to amino acids and did not display the swarming activity observed with the wild-type. DeltaputA had lost its ability to colonize the stomach of nude mice, an ability possessed by the wild-type. These findings indicate that put A may play an important role in H. pylori colonization on the gastric mucus layer.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proliferación Celular , Helicobacter pylori/enzimología , Helicobacter pylori/patogenicidad , Proteínas de la Membrana/metabolismo , Movimiento , Animales , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/genética , Transporte Biológico , Células Cultivadas , Resistencia al Cloranfenicol , Recuento de Colonia Microbiana , Femenino , Infecciones por Helicobacter/enzimología , Helicobacter pylori/genética , Helicobacter pylori/crecimiento & desarrollo , Procesos Heterotróficos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Mutagénesis Insercional , Proteínas Mutantes , Prolina/metabolismo , Prolina Oxidasa/deficiencia , Estómago/microbiología , Estómago/fisiopatología , Transformación Bacteriana
4.
J Bacteriol ; 190(9): 3244-55, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18245281

RESUMEN

Helicobacter pylori requires flagellar motility and chemotaxis to establish and maintain chronic infection of the human stomach. The pH gradient in the stomach mucus is essential for bacterial orientation and guides the bacterium toward a narrow layer of the mucus, suggesting that H. pylori is capable of energy sensing or taxis. In the present study, H. pylori wild-type behavior in a temporal swimming assay could be altered by electron transport inhibitors, indicating that a connection between metabolism and behavior exists. In order to elucidate mechanisms of behavioral responses of H. pylori related to energy sensing, we investigated the phenotypes of single and multiple mutants of the four proposed chemotaxis sensor proteins. All sensor mutants were motile, but they diverged in their behavior in media supporting different energy yields. One proposed intracellular sensor, TlpD, was crucial for behavioral responses of H. pylori in defined media which did not permit growth and led to reduced bacterial energy levels. Suboptimal energetic conditions and inhibition of electron transport induced an increased frequency of stops and direction changes in the wild type but not in tlpD mutants. Loss of metabolism-dependent behavior in tlpD mutants could be reversed by complementation but not by electron donors bypassing the activity of the electron transport chain, in contrast to the case for the wild type. TlpD, which apparently lacks transmembrane domains, was detected both in the bacterial cytoplasm and at the bacterial periphery. The proposed energy sensor TlpD was found to mediate a repellent tactic response away from conditions of reduced electron transport.


Asunto(s)
Proteínas Bacterianas/metabolismo , Quimiotaxis , Helicobacter pylori/fisiología , Adenosina Trifosfato/análisis , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Quimiotaxis/genética , Medios de Cultivo , Citoplasma/química , Citoplasma/metabolismo , Transporte de Electrón , Helicobacter pylori/química , Helicobacter pylori/metabolismo , Mutagénesis
5.
Curr Genet ; 47(3): 156-62, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15614489

RESUMEN

Two redundant genes, THI20 and THI21, of Saccharomyces cerevisiae encode a 2-methyl-4-amino-5-hydroxymethylpyrimidine monophosphate (HMP-P) kinase required for thiamin biosynthesis. Using functional complementation analysis with an Escherichia coli mutant strain and a defined biochemical system containing partially purified proteins for the reconstitution of thiamin monophosphate synthesis, we demonstrate that both Thi20p and Thi21p proteins also have HMP kinase activity. Although each isoform independently can synthesize HMP pyrophosphate (HMP-PP) from HMP, there is a marked difference in efficiency between the two proteins. The thi20 deletion strain grows at the same rate as the parental strain in minimal medium without thiamin, but its ability to synthesize HMP-PP from HMP is significantly decreased. We discuss the possibility that HMP is not involved in the pathway of de novo thiamin synthesis in S. cerevisiae.


Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Pirimidinas/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Tiamina Pirofosfato/biosíntesis , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
Microbiology (Reading) ; 145 ( Pt 2): 495-501, 1999 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-10075431

RESUMEN

A 1.7 kb DNA fragment isolated from an E. coli genomic library was able to complement the thiamin requirement of strains carrying the thiM, thiJ and thiD mutations. The three genes encode hydroxyethylthiazole kinase, hydroxymethylpyrimidine (HMP) kinase and phosphomethylpyrimidine (HMP-p) kinase, respectively. Sequence analysis revealed that the 1.7 kb fragment contained two ORFs of 708 bp and 801 bp. The former ORF complemented the thiM mutation and the latter ORF both the thiJ and thiD mutations. The latter ORF was cloned into the expression vector pET3a, and the encoded protein was purified through three successive column chromatographies. The purified protein was able to convert HMP to its monophosphate and the monophosphate to its pyrophosphate. These results suggest that the two distinct enzyme activities, HMP kinase and HMP-P kinase, are indeed a bifunctional enzyme encoded by a single gene, designated thiDIJ.


Asunto(s)
Escherichia coli/enzimología , Complejos Multienzimáticos/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/aislamiento & purificación , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Plásmidos/genética , Mapeo Restrictivo , Análisis de Secuencia de ADN , Tiamina/biosíntesis
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