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MKK6 from pacific white shrimp Litopenaeus vannamei is responsive to bacterial and WSSV infection
Haoyang Li1, Sheng Wang1, Zhe Qian2
1MOE Key Laboratory of Aquatic Product Safety/State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, PR China; Institute of Aquatic Economic Animals and Guangdong Provice Key Laboratory for Aquatic Economic Animals, Sun Yat-sen University, Guangzhou, PR China; Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Sun Yat-sen University, Guangzhou, PR China.
Abstract:
p38 mitogen-actived protein kinases (MAPKs) broadly exist from yeast to mammals and participate in diverse cellular responses to various stimuli, whose activation can be induced by the MAPK kinase 6 (MKK6). In this study, a novel MKK6 homolog from Litopenaeus vannamei (LvMKK6) was cloned and characterized. The transcript of LvMKK6 was 1465bp long with an open reading frame (ORF) of 987bp that encoded a polypeptide of 328 amino acids. LvMKK6 was a both cytoplasmic- and nuclear-localized protein and its expression was up-regulated with the treatment of different stimuli including LPS, Vibrio parahaemolyticus, Staphylococcus aureus, Poly (I:C) and white spot syndrome virus (WSSV). Overexpression of LvMKK6 could lead to activate the promoter activities of several antimicrobial peptides (AMPs) such as PEN4. The further investigation demonstrated that LvMKK6 could interact with and phosphorylate Lvp38, suggesting LvMKK6 was an activator of Lvp38. Knockdown of LvMKK6 caused attenuate expression of several AMPs and resulted in the higher mortality of shrimp under V. parahaemolyticus infection, suggesting LvMKK6 could play vital roles in defense against bacterial infection. Interestingly, silencing of LvMKK6 led to the lower virus loads and suppressed viral gene (VP28) expression during WSSV challenge. In addition, overexpression of LvMKK6 promoted the promoter activities of 19 WSSV immediate-early genes such as wsv069, wsv249, wsv108 and wsv403. These results suggested that LvMKK6 could be used by WSSV. Above all, these data provided experimental evidences that participation of LvMKK6 in regulating AMPs and host defense against bacteria, as well as the immune response to WSSV infection.
Insights
A novel shrimp protein, LvMKK6, plays a dual role in immunity. It enhances defense against bacterial infections by activating antimicrobial peptides but is exploited by white spot syndrome virus (WSSV) to promote viral replication.
Area of Science:
- Immunology
- Molecular Biology
- Crustacean Biology
Background:
- Mitogen-activated protein kinases (MAPKs) are crucial signaling molecules involved in cellular responses to stimuli.
- MAPK kinase 6 (MKK6) activates p38 MAPKs.
- Understanding immune responses in shrimp is vital for aquaculture.
Purpose of the Study:
- To clone and characterize a novel MKK6 homolog from Litopenaeus vannamei (LvMKK6).
- To investigate the role of LvMKK6 in shrimp immune responses against bacterial and viral infections.
Main Methods:
- Cloning and sequencing of LvMKK6.
- Analysis of LvMKK6 expression under various stimuli (LPS, Vibrio parahaemolyticus, Staphylococcus aureus, Poly (I:C), WSSV).
- Overexpression and knockdown experiments of LvMKK6.
- Luciferase reporter assays to assess promoter activities of antimicrobial peptides and viral genes.
- Interaction and phosphorylation assays between LvMKK6 and Lvp38.
- Mortality assays and viral load determination.
Main Results:
- LvMKK6 transcript is 1465bp, encoding a 328-amino acid protein localized in both cytoplasm and nucleus.
- LvMKK6 expression is upregulated by bacterial, viral, and PAMP stimuli.
- Overexpression of LvMKK6 activates antimicrobial peptide promoters and phosphorylates Lvp38, enhancing bacterial defense.
- Knockdown of LvMKK6 increases shrimp mortality during Vibrio parahaemolyticus infection.
- Silencing LvMKK6 reduces WSSV loads and suppresses viral gene expression.
- LvMKK6 overexpression promotes WSSV immediate-early gene promoter activities, suggesting viral exploitation.
Conclusions:
- LvMKK6 is involved in regulating antimicrobial peptide production and host defense against bacterial infections.
- LvMKK6 plays a role in the immune response to WSSV infection, but appears to be exploited by the virus.
- LvMKK6 exhibits a complex role in shrimp immunity, acting as both a defense activator and a factor potentially utilized by WSSV.
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