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Published on: December 20, 2017
PcToll2 positively regulates the expression of antimicrobial peptides by promoting PcATF4 translocation into the
Jiang-Feng Lan1, Li-Juan Zhao1, Shun Wei1
1Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei, 430070, China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, Hubei, 430070, China.
Abstract:
Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog PcATF4 from Procambarus clarkii, and analyzed the likely regulatory activity of PcATF4 in PcToll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. PcToll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, PcToll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus, PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the PcATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio, the translocation of PcATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio, the expression of ALF1 and ALF2 was also decreased while the expression of PcToll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of PcATF4, instead of the routine transcription factor PcDorsal, into the nucleus participating in the immune defense against Gram-negative bacteria.
Insights
This study identifies PcToll2 and PcATF4 in Procambarus clarkii, revealing PcATF4
Area of Science:
- Immunology
- Molecular Biology
- Crustacean Research
Background:
- Toll-like receptors (TLRs) are critical immune receptors in pathogen defense.
- Activating transcription factor 4 (ATF4) is involved in TLR signaling, but its role in crustaceans is unknown.
- This study investigates a novel ATF4 homolog in Procambarus clarkii.
Purpose of the Study:
- To identify and characterize a Toll homolog (PcToll2) and an ATF4 homolog (PcATF4) in Procambarus clarkii.
- To analyze the regulatory role of PcATF4 in PcToll2 signaling pathway.
- To understand the immune defense mechanisms against Gram-negative bacteria in crustaceans.
Main Methods:
- Identification and sequencing of PcToll2 and PcATF4 cDNA.
- Gene expression analysis using quantitative PCR.
- Gene silencing (RNA interference) and knockdown experiments.
- Subcellular localization studies of PcATF4.
Main Results:
- PcToll2 and PcATF4 were identified and characterized in Procambarus clarkii.
- PcToll2 and PcATF4 expression was upregulated upon Vibrio parahemolyticus challenge.
- PcToll2 signaling promotes PcATF4 nuclear translocation, upregulating antimicrobial peptides ALF1 and ALF2.
- PcATF4, not PcDorsal, is the key transcription factor in this pathway.
Conclusions:
- PcToll2 signaling pathway regulates antimicrobial peptide expression via PcATF4 nuclear import.
- This pathway is crucial for immune defense against Gram-negative bacteria in Procambarus clarkii.
- Identifies a novel mechanism of innate immunity in crustaceans.
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