Novel Lectins from Freshwater Prawn Macrobrachium rosenbergii and their induction in response to V. harveyi and M. rosenbergii Nodavirus Snigdha Baliarsingh1 and Bharat Bhusan Patnaik2 1School
of Biotech Sciences, Trident Academy of Creative Technology (TACT), F2- B, Chandaka Industrial Estates, Chandrasekharpur, Bhubaneswar-751024, Odisha, India 2PG Department of Biosciences and Biotechnology, Fakir Mohan University, Vyasa Vihar, Nuapadhi, Balasore-756089, Odisha, India
E-mail: snigdhasami@gmail.com, drbharatbhusan4@gmail.com ABSTRACT Background: Lectins are non-self recognition factors and bind to glycosylated surface of pathogens. Rationale: The commercial cultivation of M. rosenbergii has been affected due to infection of Vibrio species and M. rosenbergii nodavirus (MrNV). MrNV is responsible for 100% mortality in post-larval prawns within 2-3 days of infection. The white-tail disease (WTD) caused by MrNV accounts for an economic loss of about US$15 million annually. Presently, studies are limited reporting the underlying immune mechanisms of M. rosenbergii against MrNV and V. harveyi infection. Strategy: In this study, we identified novel C- and L-type lectins from M. rosenbergii that could putatively act as pathogen-recognition receptors in the host against the non-self factors associated with the pathogens. We cloned and characterized the full-length cDNA using RACE-PCR, sequencing and bioinformatics approaches. Results: Out of the three, two of them [MrCTL and Mr(T)Lec4] showed C-type lectin domain (CTLD) and one L-type lectin domain, later identified as Endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (MrERGIC-53). While MrCTL and Mr(T)Lec4 encoded polypeptides of 217 and 330 amino acid (aa) residues, MrERGIC-53 encoded a protein of 504 aa. MrCTL mRNA was expressed in stomach and intestine tissue at 48 h and 6 h, respectively post-challenge with MrNV, while Mr(T)Lec4 mRNA was induced at 6 h in hepatopancreas after V. harveyi challenge. MrERGIC-53 was also found to be induced in stomach and intestine tissues of the host post MrNV challenge. Conclusion: These results demonstrate that MrCTL, Mr(T)Lec4, and MrERGIC-53 may putatively act in the innate immune defense response of M. rosenbergii against the pathogens.
MrERGIC-53
MrCTL
5’-UTR (59 bp)
2.888 5.000 1 IC
6 hr
12 hr
6.000
1 0.5 0.06
0.05
0
24 hr 3.149
INTESTINE
1
4.000 2.000
0.601
1
0.497
IC
0 hr
0.000
Temporal expression analysis of Mr(T)Lec-4 mRNA showed an upregulation at 6h post-infection with Vibrio harveyi in the hepatopancreas tissue
6 hr
Relative expression of MrCTL
10.000
0.000
Relative expression of MrERGIC 53
1.5
11.472
24 hr
Expression of MrERGIC mRNA was induced in stomach at 6 h and 12 h and intestine at 24 h after challenge of M. rosenbergii with MrNV
Relative expression of MrCTL
2
9.613
Molecular cloning and in silico characterization of a Ctype lectin domain (CTLD) containing lectin gene represented as MrCTL. The cDNA sequence of MrCTL comprised of 654 nucleotides encoding 217 amino acid residues. 6 5.169 STOMACH
Relative expression of MrERGIC 53
1.83
STOMACH
2.5
15.000
INTESTINE
Relative expression of Mr(T)Lec-4
Molecular cloning and in silico characterization of a dual C-type lectin domain (CTLD) containing lectin gene represented as Mr(T)Lec-4. The cDNA sequence of Mr(T)Lec-4 comprised of 993 nucleotides encoding 330 amino acid residues. Each CTLD contained conserved ligand-binding residues.
3’-UTR (1283 bp)
ORF Sequence (1515 bp) Protein Sequence (504 aa)
Mr(T)Lec-4
4 2
1
0.357
1.102
0.7
0 2
1.564
1.5 1
1 0.443
0.5
0.211
0 IC
0 hr
6 hr
24 hr
Expression of MrCTL mRNA was induced in stomach and intestine at 48 h and 6 h, respectively after challenge of M. rosenbergii with MrNV
This work was supported by grant from Department of Biotechnology, Government of India (BT/PR12710/AAQ/03/713/2015) under Aquaculture and Marine Biotechnology Category.