Browsing by Author "Nayak, Manoswini"

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Purification, crystallization and preliminary X-ray analysis of a thermostable direct haemolysin from Grimontia hollisae
(2011-02-01)
Wang, Yu-Kuo
;
Huang, Sheng-Cih
;
Wu, Yi-Fang
;
Chen, Yu-Ching
;
Chen, Wen-Hung
Vibrio hollisae, a halophilic species recently reclassified as Grimontia hollisae, is a causative agent of gastroenteritis and septicaemia. One important pathogenic Vibrio factor, thermostable direct haemolysin (TDH), has been purified and crystallized in two crystal forms using the vapour-diffusion method. The crystals belonged to an orthorhombic space group, with unit-cell parameters a = 104.8, b = 112.4, c = 61.3 angstrom and a = 122.9, b = 123.3, c = 89.8 angstrom. The crystals contained either four or eight molecules per asymmetric unit, with predicted solvent contents of 49.4 and 46.3% and Matthews coefficients (V(M)) of 2.4 and 2.3 angstrom(3) Da(-1), respectively. These crystals were suitable for structure determination, which would yield structural details related to the cytotoxicity and oligomeric structure of this pore-forming toxin.
Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties
(2011)
Wang, Yu-Kuo
;
Huang, Sheng-Cih
;
Wu, Yi-Fang
;
Chen, Yu-Ching
;
Lin, Yen-Ling
Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 degrees C but is reactivated by additional heating above 80 degrees C. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/T59I/S63T) triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDHWT. Circular dichroism measurements of Gh-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a beta-sheet to alpha-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.
The Thermostable Direct Hemolysin from Grimontia hollisae Causes Acute Hepatotoxicity In Vitro and In Vivo
(2013-02-21)
Lin, Yan-Ren
;
Chen, Yao-Li
;
Wang, Keh-Bin
;
Wu, Yi-Fang
;
Wang, Yu-Kuo
Background: G. hollisae thermostable direct hemolysin (Gh-TDH) is produced by most strains of G. hollisae. This toxin has been reported to be absorbed in the intestines in humans. Secondary liver injury might be caused by venous return of the toxin through the portal system. We aimed to firstly analyze the in vitro and in vivo hepatotoxicity of Gh-TDH. Methods: Liver cells (primary human non-cancer cell and FL83B mouse cells) were treated and mice (BALB/c) were fed with this toxin to investigate its hepatotoxicity. Morphological examination and cytotoxicity assays using liver cells were also performed. Fluorescein isothiocyanate-conjugated toxin was used to analyze the localization of this protein in liver cells. Mice were subjected to liver function measurements and liver biopsies following toxin treatment and wild-type bacterial infection. PET (positron emission tomography)/CT (computed tomography) images were taken to assess liver metabolism during acute injury and recovery. Results: The effect of hepatotoxicity was dose and time dependent. Cellular localization showed that the toxin was initially located around the cellular margins and subsequently entered the nucleus. Liver function measurements and liver biopsies of the mice following treatment with toxin or infection with wild-type Grimontia hollisae showed elevated levels of transaminases and damage to the periportal area, respectively. The PET/CT images revealed that the reconstruction of the liver continued for at least one week after exposure to a single dose of the toxin or bacterial infection. Conclusions: The hepatotoxicity of Gh-TDH was firstly demonstrated. The damage was located in the periportal area of the liver, and the liver became functionally insufficient.