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1.
J Bacteriol ; 197(6): 1051-64, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25561716

RESUMO

Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program.


Assuntos
Colágeno/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Metaloproteases/metabolismo , Vibrio cholerae/metabolismo , Motivos de Aminoácidos , Colágeno/química , Metaloproteases/genética , Proteínas Recombinantes de Fusão , Especificidade por Substrato
2.
Infect Immun ; 82(7): 2788-801, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24733097

RESUMO

Vibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that under laboratory growth conditions, the T2S complex was continuously expressed throughout V. cholerae growth, whereas there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure of V. cholerae to different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S. Subsequent screening of a V. cholerae genomic library suggested that σ(E) stress response, phosphate metabolism, and the second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on σ(E), we discovered that the upstream region of the T2S operon possesses both the consensus σ(E) and σ(70) signatures, and deletion of the σ(E) binding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of σ(E) stimulated transcription of T2S in wild-type and isogenic ΔrpoE strains of V. cholerae, providing additional support for the idea that the T2S complex belongs to the σ(E) regulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness of V. cholerae in different ecological niches.


Assuntos
Proteínas de Bactérias/metabolismo , Fator sigma/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Fator sigma/genética , Fatores de Tempo , Transcrição Gênica
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