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Published on: September 15, 2023
Molecular identification and functional characterization of a tumor necrosis factor (TNF) gene in Crassostrea
Fufa Qu1, Zhiming Xiang2, Yang Zhang2
1Key Laboratory of Tropical Marine Bio-resources and Ecology and Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510275, China; Department of Biological and Environmental Engineering, Changsha University, Changsha 410022, China.
Abstract:
Tumor necrosis factor superfamily (TNFSF) represents a group of multifunctional inflammatory cytokines that have been shown to participate in a variety of pathological and immunological process. However, the functions of these proteins in oyster are still poorly understood. In the present study, an oyster TNF homolog (named ChTNF) was identified from a cDNA library of Crassostrea hongkongensis. The complete cDNA of ChTNF was 2457bp in length containing an open reading frame (ORF) of 1044bp, a 5'-untranslated region (UTR) of 381bp and a 3'-UTR of 1032bp. The deduced ChTNF protein consisted of 347 amino acids with a characteristic transmembrane domain and a typical TNF homology domain (THD). Quantitative real-time PCR analysis revealed that ChTNF was broadly expressed in various oyster tissues and different stages of embryonic development. In addition, transcriptional analysis indicated that ChTNF transcription levels in hemocytes were increased significantly in pathogen challenge groups (Vibrio alginolyticus and Staphylococcus haemolyticus) compared to that in the control. Moreover, in vitro PAMP (lipopolysaccharide and peptidoglycan) treatments showed a stimulatory effect on the expression of ChTNF in the primary cultured hemocytes of C. hongkongensis. Finally, dual-luciferase reporter assays revealed that ChTNF could activate the NF-κB-Luc reporter in a dose-dependent manner in HEK293T cells. Altogether, these findings may provide valuable information regarding oyster TNFs and its role in innate immunity.
Insights
Researchers identified a novel oyster Tumor Necrosis Factor (TNF) homolog, ChTNF, crucial for innate immunity. ChTNF expression increases upon pathogen challenge, highlighting its role in oyster immune responses.
Area of Science:
- Marine biology
- Immunology
- Molecular biology
Background:
- The Tumor Necrosis Factor Superfamily (TNFSF) comprises key inflammatory cytokines, but their function in oysters remains largely unknown.
- Understanding oyster immune mechanisms is vital for aquaculture and marine ecosystem health.
Purpose of the Study:
- To identify and characterize an oyster TNF homolog (ChTNF) from Crassostrea hongkongensis.
- To investigate the expression patterns and immune-related functions of ChTNF in oysters.
Main Methods:
- cDNA library screening and sequencing to identify ChTNF.
- Quantitative real-time PCR (qPCR) to analyze ChTNF expression in various tissues and developmental stages.
- Pathogen challenge and in vitro PAMP treatments to assess ChTNF response.
- Dual-luciferase reporter assays to determine ChTNF's effect on NF-κB signaling.
Main Results:
- The complete cDNA of ChTNF was characterized, encoding a protein with transmembrane and TNF homology domains.
- ChTNF exhibited broad expression across oyster tissues and developmental stages.
- ChTNF transcription significantly increased in hemocytes following challenge with Vibrio alginolyticus and Staphylococcus haemolyticus.
- In vitro treatments with LPS and peptidoglycan stimulated ChTNF expression in oyster hemocytes.
- ChTNF dose-dependently activated the NF-κB-Luc reporter in HEK293T cells.
Conclusions:
- This study identifies and functionally characterizes an oyster TNF homolog, ChTNF.
- ChTNF plays a significant role in the oyster's innate immune response, particularly against bacterial pathogens.
- ChTNF activation of NF-κB signaling provides insights into oyster immune pathways.
