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1.
PLoS Pathog ; 11(12): e1005312, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26641249

RESUMEN

Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric Helicobacter species constituted a decisive evolutionary event to allow Helicobacter to colonize the hostile gastric environment, in which no other bacteria persistently thrives. This acquisition was key for the emergence of one of the most successful bacterial pathogens, H. pylori.


Asunto(s)
Proteínas Bacterianas/metabolismo , Evolución Biológica , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Cromatografía Liquida , Modelos Animales de Enfermedad , Helicobacter/genética , Helicobacter/metabolismo , Helicobacter/patogenicidad , Helicobacter pylori/metabolismo , Immunoblotting , Ratones , Datos de Secuencia Molecular , Níquel/metabolismo , Filogenia , Proteínas/metabolismo , Proteómica , Espectrometría de Masas en Tándem , Ureasa/metabolismo
2.
Infect Immun ; 78(6): 2782-92, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20368342

RESUMEN

In addition to their classical roles as carbon or nitrogen sources, amino acids can be used for bacterial virulence, colonization, or stress resistance. We found that original deamidase-transport systems impact colonization by Helicobacter pylori, a human pathogen associated with gastric pathologies, including adenocarcinoma. We demonstrated that l-asparaginase (Hp-AnsB) and gamma-glutamyltranspeptidase (Hp-gammaGT) are highly active periplasmic deamidases in H. pylori, producing ammonia and aspartate or glutamate from asparagine and glutamine, respectively. Hp-GltS was identified as a sole and specialized transporter for glutamate, while aspartate was exclusively imported by Hp-DcuA. Uptake of Gln and Asn strictly relies on indirect pathways following prior periplasmic deamidation into Glu and Asp. Hence, in H. pylori, the coupled action of periplasmic deamidases with their respective transporters enables the acquisition of Glu and Asp from Gln and Asn, respectively. These systems were active at neutral rather than acidic pH, suggesting their function near the host epithelial cells. We showed that Hp-DcuA, the fourth component of these novel deamidase-transport systems, was as crucial as Hp-gammaGT, Hp-AnsB, and Hp-GltS for animal model colonization. In conclusion, the pH-regulated coupled amino acid deamidase-uptake system represents an original optimized system that is essential for in vivo colonization of the stomach environment by H. pylori. We propose a model in which these two nonredundant systems participate in H. pylori virulence by depleting gastric or immune cells from protective amino acids such as Gln and producing toxic ammonia close to the host cells.


Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/fisiología , Asparaginasa/fisiología , Proteínas Bacterianas/fisiología , Transportadores de Ácidos Dicarboxílicos/fisiología , Helicobacter pylori/enzimología , Helicobacter pylori/patogenicidad , Factores de Virulencia/fisiología , gamma-Glutamiltransferasa/fisiología , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Aminoácidos/metabolismo , Amoníaco/metabolismo , Amoníaco/toxicidad , Animales , Asparaginasa/metabolismo , Ácido Aspártico/metabolismo , Proteínas Bacterianas/metabolismo , Recuento de Colonia Microbiana , Transportadores de Ácidos Dicarboxílicos/metabolismo , Ácido Glutámico/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Modelos Biológicos , Estómago/microbiología , Virulencia , Factores de Virulencia/metabolismo , gamma-Glutamiltransferasa/metabolismo
3.
Eukaryot Cell ; 7(5): 848-58, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18375614

RESUMEN

The genome of the type strain of Candida glabrata (CBS138, ATCC 2001) contains homologs of most of the genes involved in mating in Saccharomyces cerevisiae, starting with the mating pheromone and receptor genes. Only haploid cells are ever isolated, but C. glabrata strains of both mating types are commonly found, the type strain being MAT alpha and most other strains, such as BG2, being MATa. No sexual cycle has been documented for this species. In order to understand which steps of the mating pathway are defective, we have analyzed the expression of homologs of some of the key genes involved as well as the production of mating pheromones and the organism's sensitivity to artificial pheromones. We show that cells of opposite mating types express both pheromone receptor genes and are insensitive to pheromones. Nonetheless, cells maintain specificity through regulation of the alpha1 and alpha2 genes and, more surprisingly, through differential splicing of the a1 transcript.


Asunto(s)
Empalme Alternativo , Candida glabrata/fisiología , Genes del Tipo Sexual de los Hongos , Feromonas/metabolismo , Secuencia de Aminoácidos , Candida glabrata/efectos de los fármacos , Candida glabrata/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Haploidia , Intrones , Datos de Secuencia Molecular , Feromonas/química , Feromonas/genética , Receptores de Feromonas/genética , Receptores de Feromonas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Alineación de Secuencia
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