Fisheries and Aquatic Sciences

The Korean Society of Fisheries and Aquatic Science (한국수산과학회)
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The responsibility of C-terminal domain in the thermolabile haemolysin activity of Vibrio parahaemolyticus and inhibition treatments by Phellinus sp. extracts

  • Tran Thi Huyen (Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City) ;
  • Ha Phuong Trang (Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City) ;
  • Nguyen Thi-Ngan (Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City) ;
  • Bui Dinh-Thanh (Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City) ;
  • Le Pham Tan Quoc (Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City) ;
  • Trinh Ngoc Nam (Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City)
  • Received : 2022.10.07
  • Accepted : 2023.01.19
  • Published : 2023.03.31
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Abstract

The thermolabile haemolysin (tlh) of Vibrio parahaemolyticus (Vptlh) from V. parahaemolyticus is a multiple-function enzyme, initially describes as a haemolytic factor activated by lecithin and phospholipase A2 enzymatic activity (Shinoda, 1991; Vazquez-Morado, 2021; Yanagase et al., 1970). Until now, the tlh structure has hypothesized including N-terminal and C-terminal domain, but what domain of the Vptlh structure does the haemolytic activity has not been refined yet. In this study, a 450-bp VpTLH nucleotide sequence of the entire Vptlh gene encoded the C-terminal domain cloned firstly to examine its responsibility in the activity of the Vptlh. The C-terminal domain fused with a 6-His-tag named the His-tag-VpC-terminal domain was expressed successfully in soluble form in the BL21 (DE3) PlysS cell. Remarkably, both expression and purification results confirmed a high agreement in the molecular weight of the His-tag-VpC-terminal domain was 47 kDa. This work showed the His-tag-VpC-terminal domain lysed the erythrocyte membranes in the blood agar and the phosphate buffered saline (0.9%) media without adding the lecithin substrate of the phospholipase enzyme. Haemolysis occurred at all tested diluted concentrations of His-tag-VpC-terminal domain (p < 0.05), providing evidence for the independent haemolytic activity of the His-tag-VpC-terminal domain. The content of 100 ㎍ of the His-tag-VpC-terminal domain brought the highest haemolytic activity of 80% compared to that in the three remaining contents. Significantly, the His-tag-VpC-terminal domain demonstrated not to involve the phospholipase activity in Luria-Bertani agar supplemented with 1% (vol/vol) egg yolk emulsion. All results proved the vital responsibility of the His-tag-VpC-terminal domain in causing the haemolytic activity without the required activation by the phospholipase enzyme. Raw extracts of Phellinus igniarus and Phellinus pipi at 10-1 mg/mL inhibited the haemolytic activity of the His-tag-VpC-terminal domain from 67.7% to 87.42%, respectively. Hence applying the His-tag-VpC-terminal domain as a simple biological material to evaluate quickly potential derivatives against the Vptlh in vivo conditions will accessible and more advantageous than using the whole of the Vptlh.

Keywords

Acknowledgement

Tran Thi Huyen conceived and designed the experiments, performed the experiments, analyzed the data, prepared Figs and/ or Tables, authored or reviewed drafts of the paper and approved the final draft. Phuong-Trang Ha, Bui Dinh Thanh performed the experiments, analyzed the data, prepared Figs and/or Tables. Nguyen Thi Ngan conceived and designed the extracts experiments from phellinus sp. Trinh Ngoc Nam, Le Pham Tan Quoc critically reviewed the manuscript and approved the final draft. All authors read and approved the final manuscript.

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