Identification of novel antimicrobial peptides using high-throughput transcriptome data of black tiger shrimp, Penaeus monodon (Fabricius, 1798)

Bahaaeldin, Hassan Saad Abdella (2022) Identification of novel antimicrobial peptides using high-throughput transcriptome data of black tiger shrimp, Penaeus monodon (Fabricius, 1798). Doctoral thesis, Sunway University.

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Black tiger shrimp, Penaeus monodon, is one of the most promising candidates for shrimp aquaculture. The production is unavoidably affected by microbial pathogens in all stages of production including the hatchery, nursery, and grow-out ponds. Shrimps, like all invertebrates, depend primarily on their non-specific innate immune system to defend against microbial infections. The most crucial effector molecules are antimicrobial peptides (AMPs). The AMPs are characterized to be chains of pre-made short polypeptides and have antimicrobial properties against microbial pathogens. Due to the lack of a reference genome/transcriptome of P. monodon, the active investigation of the shrimp’s AMP repertoire becomes more difficult for studying the hostmicrobe interactions at the molecular level. Thus, to identify new gene-encoded and cryptides AMPs from P. Monodon transcriptome, deep transcriptome mining was used in combination with other bioinformatic tools, for instance AntiBP server, Antimicrobial Peptide Database (APD3). The proper identification of AMPs in shrimp will help in better understanding of host-microbe interactions in shrimp aquaculture and might lead to the discovery of new antimicrobial agents to replace the conventional overused antibiotics. New gene-encoded AMPs and AMP fragments were discovered from the P. monodon transcriptome. Based on homology search and annotations, 154 genes were predicted to have a role related to innate immune responses. Nevertheless, only ~14% of these annotated sequences already had hits in P. monodon. This indicates that ~86% of proteins annotated to be AMPs are indeed new in P. monodon, for instance, the ubiquitin & polyubiquitin-like protein, proteases, and C-type lectins. The identified gene-encoded AMPs belong to the AMP families from penaeid shrimp, for example, anti-lipopolysaccharide factor-like and crustin-like AMPs. Additionally, histone and Enhancer of rudimentary homolog (ERH) were also identified to have cryptid AMP fragments. From the analyzed transcriptome, two gene-encoded AMPs have been identified to be members of group F and group E Anti-lipopolysaccharide factors (ALFs). These ALFlike peptides were amplified and characterized in silico, in vitro, and in vivo in response to Vibrio infection. The newly identified ALFs were constitutively expressed in all shrimp tissues with different levels and were induced after bacterial infection in heart tissues. The 3D structural analysis portrayed the structural signature of ALFs consisting of three α-helix and four β-sheets motifs with a central β-hairpin scaffold stabilized with the sulfur bridge formed between conserved cysteine moieties of the lipopolysaccharide-binding domain (LBD). The mature peptide of group F is cationic (net charge +2.01) and has a theoretical pI value in the alkaline range (pI 9.04). However, group E mature peptide is anionic (net charge -1.99) and has a theoretical pI value in the acidic range (pI 6.12). However, both LBD β-hairpins demonstrated cationic charges with values of +2.646 (pI 9.31) and +2.644 (pI 9.24) for group F and group E, respectively. The antimicrobial activity of groups F and E showed activity mainly against Gram-positive bacteria and less activity exerted against Gramnegative bacteria. Additionally, from the transcriptome analysis mentioned above, AMP fragments have been identified from both immune-related and non-immune-related proteins. Among them, two AMPs denoted as AMP08 and AMP13, have been identified from core histone H2A-like protein and enhancer of rudimentary homolog (ERH), respectively. Three AMPs from the ALF-like peptide, designated AMP02, AMP04, and ALF05, have been identified in the second α-helix motif of ALF-like AMP. Moreover, AMP21 was identified from the signal peptide region of an ALF-like AMP. Thus, the ALFs in penaeid shrimp were found to not rely only on the β-hairpin scaffold of the anti-LBD. This finding gives a new insight into the mechanism of action of such ALFs in penaeid shrimps. Furthermore, the activity of an AMP and structural analysis showed that the antimicrobial activities do not only depend on the percentage of hydrophobic residues present but are crucially dependent on how these hydrophobic residues are distributed on the peptide surface. In conclusion, the identified AMPs were divided into two categories, the gene-encoded AMPs which are named ALF9 and ALF10. Both are expressed mainly in the heart tissue and showed increased expression upon challenge with Vibrio parahaemolyticus. Concerning their lipopolysaccharide domains activity, they showed activity mainly toward Gram-positive bacteria. Moreover, the second category was the identified cryptides showed different activities against tested microbes. The cryptides and the ALFs are considered a fruitful base for a new antimicrobial agent development.

Item Type: Thesis (Doctoral)
Subjects: Q Science > QL Zoology
R Medicine > RS Pharmacy and materia medica
Divisions: Sunway University > School of Medical and Life Sciences [formerly School of Healthcare and Medical Sciences until 2020] > Dept. Biological Sciences
Depositing User: Ms Yong Yee Chan
Date Deposited: 27 Sep 2023 04:28
Last Modified: 27 Sep 2023 04:28

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