RESUMO
Outer membrane protein U (OmpU) is a major porin from Vibrio alginolyticus and has been considered a vaccine candidate against infection by V. alginolyticus. After pre-incubated with polyclonal antibody against rOmpU, V. alginolyticus showed a 78% decrease in extracellular iron level, suggesting that interruption of OmpU could increase intracellular iron level. The mRNA expression of ompU under iron-limited conditions was determined using real-time reverse transcriptase PCR. The mRNA level of ompU was downregulated to 0.27-, 0.036- and 0.019-fold after the addition of the iron chelator 2,2'-bipyridyl for 10, 30 and 60 min, respectively. In addition, the promoter of ompU contained a ferric uptake regulator (Fur) binding site, which revealed the potential regulation of ompU by Fur and iron. Fur from V. alginolyticus was purified and used for electrophoretic mobility shift assay. The result showed that in the absence of Fe2+, purified recombinant Fur could specifically bind to the promoter DNA of ompU, while in the presence of Fe2+, the binding of Fur and the promoter DNA was suppressed. Our study preliminarily explored the function of OmpU in iron balance in V. alginolyticus, and these findings were helpful in understanding iron metabolism in V. alginolyticus.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Ferro/metabolismo , Vibrio alginolyticus/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Ligação Proteica , Vibrio alginolyticus/genéticaRESUMO
BACKGROUND: Vibrio splendidus is an aquaculture pathogen that can cause skin ulcer syndrome (SUS) in Apostichopus japonicus. HopPmaJ is a type III system effector (T3SE) that has been reported to be an important virulence factor. In this study, a gene named hop, which encodes HopPmaJ in V. splendidus was cloned and its cytotoxicity to coelomocytes and its effects on the expression of immune-related genes in A. japonicus were characterized. METHODS: Real time reverse transcription PCR (RT-PCR) was used to determine the expression of the hop gene under various conditions. To obtain the purified Hop, hop gene was conditionally expressed in Escherichia coli BL21(DE3) and was purified by GST tag. The cytotoxicity of Hop to coelomocyte was determined using MTT method, and the effect of Hop on the expression of immune-related genes was determined using real time RT-PCR. RESULTS: The deduced amino acid sequence of Hop from V. splendidus shared 84%-96% homology with those of Hops from other Vibrio spp. The expression of hop gene was induced not only by host-pathogen contact but also by high cell density. Purified recombinant Hop (rHop) showed cytotoxicity to the coelomocyte of A. japonicus. The cell viability decreased to approximately 42%, 26%, 32%, 30% and 20%, when 30, 50, 60, 80 and 100 µL of purified rHop was added, respectively. After being injected with rHop, the expression levels of immune-related genes that encode complement component (C1q) and caspase were significantly increased, and the production of reactive oxygen species were also increased in A. japonicus. CONCLUSION: Our results indicated that Hop not only contributed to the cytotoxicity to coelomocyte, but also caused immune response in A. japonicus.
Assuntos
Proteínas de Bactérias/genética , Vibrio/genética , Fatores de Virulência/genética , Sequência de Aminoácidos , Animais , Aquicultura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Imunidade Inata , Filogenia , Alinhamento de Sequência , Stichopus/imunologia , Stichopus/microbiologia , Vibrio/química , Vibrio/classificação , Vibrio/metabolismo , Vibrioses/imunologia , Vibrioses/microbiologia , Fatores de Virulência/química , Fatores de Virulência/metabolismoRESUMO
Vibrio splendidus is one of the most opportunistic marine pathogens and infects many important marine animals, including the sea cucumber Apostichopus japonicus. In this study, two genes named DLD1 and DLD2, encoding dihydrolipoamide dehydrogenase (DLD) homologues in pathogenic V. splendidus, were cloned, and conditionally expressed in Escherichia coli BL21 (DE3). The enzymatic activities of DLD1 and DLD2 showed that they both belonged to the NADH oxidase family. Both DLD1 and DLD2 were located on the outer membrane of V. splendidus as detected by whole-cell ELISA. To study the adhesion function of DLD1 and DLD2, polyclonal antibodies were prepared, and antibody block assay was performed to detect the normal function of the two proteins. DLD1 and DLD2 were determined to play important roles in adhesion to different matrices and the adhesive ability of V. splendidus reduced more than 50% when DLD1 or DLD2 was defective.
