Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by COILS. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of Delta escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

Fucoidan protects against dopaminergic neuron death in vivo and in vitro

Parkinson's disease is a neurodegenerative disorder of uncertain pathogenesis characterized by a loss of dopaminergic neurons in substantia nigra pars compacta, and can be modeled by the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Oxidative stress may contribute to MPTP- and Parkinson's disease-related neurodegeneration. Fucoidan is a sulfated polysaccharide extracted from brown seaweeds which possesses a wide variety of biological activities including potent antioxidative effects. Here we investigated the effect of fucoidan treatment on locomoter activities of animals, striatal dopamine and its metabolites and survival of nigral dopaminergic neurons in MPTP-induced animal model of Parkinsonism in C57/BL mice in vivo and on the neuronal damage induced by 1-methyl-4-phenylpyridinium (MPP+) in vitro, and to study the possible mechanisms. When administered prior to MPTP, fucoidan reduced behavioral deficits, increased striatal dopamine and its metabolites levels, reduced cell death, and led to a marked increase in tyrosine hydroxylase expression relative to mice treated with MPTP alone. Furthermore, we found that fucoidan inhibited MPTP-induced lipid peroxidation and reduction of antioxidant enzyme activity. In addition, pre-treatment with fucoidan significantly protected against MPP+-induced damage in MN9D cells. Taken together, these findings suggest that fucoidan has protective effect in MPTP-induced neurotoxicity in this model of Parkinson's disease via its antioxidative activity. (C) 2009 Elsevier B.V. All rights reserved.

Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation

It has been suggested that endothelial apoptosis is a primary lesion in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). We tested this hypothesis by examining the phenotypic signatures of endothelial microparticles (EMP) in TTP patients. In addition, the effect of TTP plasma on microvascular endothelial cells (MVEC) in culture was further delineated. EMP released by endothelial cells (EC) express markers of the parent EC; EMP released in activation carry predominantly CD54 and CD62E, while those in apoptosis CD31 and CD105. We investigated EMP release in vitro and in TTP patients. Following incubation of MVEC with TTP plasma, EMP and EC were analysed by flow cytometry for the expression of CD31, CD51, CD54, CD62E, CD105, CD106 and von Willebrand factor (VWF) antigen. EMP were also analysed in 12 TTP patients. In both EC and EMP, CD62E and CD54 expression were increased 3- to 10-fold and 8- to 10-fold respectively. However, CD31 and CD105 were reduced 40-60% in EC but increased twofold in EMP. VWF expression was found in 55 +/- 15% of CD62E(+) EMP. Markers of apoptosis were negative. In TTP patients, CD62E(+) and CD31(+)/CD42b(-) EMP were markedly elevated, and preceded and correlated well with a rise in platelet counts and a fall in lactate dehydrogenase. CD62E(+) EMP (60 +/- 20%) co-expressed VWF and CD62E. The ratio of CD31(+)/42b(-) to CD62E(+) EMP exhibited a pattern consistent with activation. In conclusion, our studies indicate endothelial activation in TTP. EMP that co-express VWF and CD62E could play a role in the pathogenesis of TTP.