Antibacterial activity and modes of action of a novel hepcidin isoform from the shrimp scad, Alepes djedaba (Forsskål, 1775).

Hepcidin, initially identified in human blood ultrafiltrate as cysteine rich Liver Expressed Antimicrobial Peptide (LEAP-1), is a core molecular conduit between iron trafficking and immune response. Though a great share of studies has been focused on the iron regulatory function of hepcidins, investigations on the antimicrobial aspects are relatively less. The present study is aimed at identification of hepcidin from a teleost fish, Alepes djedaba followed by its recombinant expression, testing antibacterial property, stability and evaluation of cytotoxicity. Modes of action on bacterial pathogens were also examined. A novel hepcidin isoform, Ad-Hep belonging to the HAMP1 (Hepcidin antimicrobial peptide 1) group of hepcidins was identified from the shrimp scad, Alepes djedaba. Ad-Hep with 2.9 kDa size was found to be a cysteine rich, cationic peptide (+4) with antiparallel beta sheet conformation, a furin cleavage site (RXXR) and 'ATCUN' motif. It was heterologously expressed in E. coli Rosettagami B(DE3)PLysS cells and the recombinant peptide, rAd-Hep was found to have significant antibacterial activity, especially against Edwardsiella tarda, Vibrio parahaemolyticus and Escherichia coli. Membrane depolarization followed by membrane permeabilization and Reactive Oxygen Species (ROS) production were found to be the modes of action of rAd-Hep on bacterial cells. Ad-Hep was found to be non-haemolytic to hRBC and non-cytotoxic in mammalian cell line. Stability of the peptide at varying temperature, pH and metal salts qualify them for applications in vivo. With significant bactericidal activity coupled with direct killing mechanisms, the rAd-Hep can be a promising drug candidate for therapeutic applications in medicine and fish culture systems.

ß-Defensins from common goby (Pomatoschistus microps) and silver trevally (Pseudocaranx georgianus): Molecular characterization and phylogenetic analysis.

Antimicrobial peptides (AMPs) are biologically active molecules involved in host defense present in a variety of organisms. They are an integral component of innate immunity, forming a front line of defense against potential pathogens, including antibiotic-resistant ones. Fishes are proven to be a prospective source of AMPs as they are constantly being challenged by a variety of pathogens and the AMPs are reported to play an inevitable role in fish immunity. Among them, ß-defensins form one of the most studied multifunctional peptides with early evolutionary history and recently being considered as host defense peptides. The present study highlights the first-ever report on ß-defensin AMP sequences from common goby (Pomatoschistus microps) and silver trevally (Pseudocaranx georgianus). A 192 bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) comprising a 20 aa signal peptide region at the N-terminal was obtained from the mRNA of gill tissue of both P. microps and P. georgianus by RT-PCR. These peptide sequences when characterized in silico at the molecular level revealed a 43 aa cationic mature peptide with the signature intra-molecular disulphide bonded cysteine residue pattern ascertaining its ß-defensin identity, further confirmed by phylogenetic analysis. The data collected will pave the way for further research on varied facets of the peptide-like, tissue level expressions, antimicrobial activities on commonly encountered pathogens, and its feasibility as a therapeutant in the aquaculture scenario.

Antibacterial Efficacy and Mechanisms of Action of a Novel Beta-Defensin from Snakehead Murrel, Channa striata.

Beta-defensins, identified from fishes, constitute a crucial category of antimicrobial peptides important in combating bacterial fish pathogens. The present investigation centers on the molecular and functional characterization of CsDef, a 63-amino acid beta-defensin antimicrobial peptide derived from snakehead murrel (Channa striata). The physicochemical attributes of CsDef align with the distinctive characteristics observed in AMPs. CsDef was recombinantly produced, and the recombinant peptide, rCsDef, exhibited notable antibacterial efficacy against bacterial fish pathogens with an MIC of 16 µM for V. proteolyticus. A. hydrophila exhibited 91% inhibition, E. tarda 92%, and V. harveyi 53% at 32 µM of rCsDef. The rCsDef exhibited a multifaceted mechanism of action against bacteria, i.e., through membrane depolarization, membrane permeabilization, and generation of ROS. The rCsDef was non-hemolytic to hRBCs and non-cytotoxic to normal mammalian cell line CHO-K1. However, it exhibited anticancer properties in MCF-7. rCsDef demonstrated notable stability with respect to pH, temperature, salt, metal ions, and proteases. These findings suggest it is a potential candidate molecule for prospective applications in aquaculture.

Characterisation of a novel crustin isoform from mud crab, Scylla serrata (Forsskål, 1775) and its functional analysis in silico.

A 336-base pair (bp) sized mRNA sequence encoding 111 amino acid size crustin isoform (MC-crustin) was obtained from the gill sample of the green mud crab, Scylla serrata. MC-crustin possessed an N-terminal signal peptide region comprising of 21 amino acid residues, followed by a 90 amino acid mature peptide region having a molecular weight of 10.164 kDa, charge + 4.25 and theoretical pI of 8.27. Sequence alignment and phylogenetic tree analyses revealed the peptide to be a Type I crustin, with four conserved cysteine residues forming the cysteine rich region, followed by WAP domain. MC-crustin was cationic with cysteine/proline rich structure and was predicted with antimicrobial, anti-inflammatory, anti-angiogenic and anti-hypertensive property making it a potential molecule for possible therapeutic applications.

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression.

BACKGROUND: Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. RESULTS: This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four ß sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. CONCLUSION: Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

Functional characterization of a histone H2A derived antimicrobial peptide HARRIOTTIN-1 from sicklefin chimaera, Neoharriotta pinnata.

Antimicrobial peptides (AMPs) are gene encoded short peptides which play an important role in the innate immunity of almost all living organisms ranging from bacteria to mammals. Histones play a very important role in defense as precursors to bioactive peptides. The present study is an attempt to decipher the antimicrobial activity of a histone H2A derived peptide, Harriottin-1 from sicklefin chimaera, Neoharriotta pinnata. Analysis in silico predicted the molecule with potent antibacterial and anticancer property. The Harriottin-1 was recombinantly produced and the recombinant peptide rHar-1 demonstrated potent antibacterial activity at 25 µM besides anticancer activity. The study strongly suggests the importance of histone H2A derived peptides as a model for the design and synthesis of potent peptide drugs.

A hepatic antimicrobial peptide, hepcidin from Indian major carp, Catla catla: molecular identification and functional characterization.

BACKGROUND: Increase of antibiotic resistance in pathogenic microbes necessitated novel molecules for curing infection. Antimicrobial peptides (AMPs) are the gene-encoded evolutionarily conserved small molecules with therapeutic value. AMPs are considered as an alternative drug for conventional antibiotics. Hepcidin, the cysteine-rich antimicrobial peptide, is an important component in innate immune response. In this study, we identified and characterized hepcidin gene from the fish, Catla catla (Indian major carp) and termed it as Cc-Hep. RESULTS: Open reading frame of Cc-Hep consists of 261 base pair that encodes 87 amino acids. Cc-Hep is synthesized as a prepropeptide consisting of 24 amino acid signal peptide, 36 amino acid propeptide, and 26 amino acid mature peptide. Sequence analysis revealed that Cc-Hep shared sequence similarity with hepcidin from Sorsogona tuberculata. Phylogenetic analysis indicated that Cc-Hep was grouped with HAMP2 family. Structure analysis of mature Cc-Hep identified two antiparallel beta sheets stabilized by four disulphide bonds and a random coil. The mature peptide region of Cc-Hep has a charge of + 2, isoelectric value 8.23 and molecular weight 2.73 kDa. CONCLUSION: Functional characterization predicted antibacterial, antioxidant, and anticancer potential of Cc-Hep, which can be explored in aquaculture or human health care.

Molecular characterization of a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger (Ruppel, 1836).

BACKGROUND: The concern regarding a post-antibiotic era with increasing drug resistance by pathogens imposes the need to discover alternatives for existing antibiotics. Antimicrobial peptides (AMPs) with their versatile therapeutic properties are a group of promising molecules with curative potentials. These evolutionarily conserved molecules play important roles in the innate immune system of several organisms. The ß-defensins are a group of cysteine rich cationic antimicrobial peptides that play an important role in the innate immune system by their antimicrobial activity against the invading pathogens. The present study deals with a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger. Total RNA was isolated from the gills, cDNA was synthesized and the ß-defensin isoform obtained by polymerase chain reaction was cloned and subjected to structural and functional characterization in silico. RESULTS: A ß-defensin isoform could be detected from the gill mRNA of red-toothed trigger fish, Odonus niger. The cDNA encoded a 63 amino acid peptide, ß-defensin, with a 20 amino acid signal sequence followed by 43 amino acid cationic mature peptide (On-Def) having a molecular weight of 5.214 kDa and theoretical pI of 8.89. On-Def possessed six highly conserved cysteine residues forming disulfide bonds between C1-C5, C2-C4, and C3-C6, typical of ß-defensins. An anionic pro-region was observed prior to the ß-defensin domain within the mature peptide. Clustal alignment and phylogenetic analyses revealed On-Def as a group 2 ß-defensin. Furthermore, it shared some structural similarities and functional motifs with ß-defensins from other organisms. On-Def was predicted to be non-hemolytic with anti-bacterial, anti-viral, anti-fungal, anti-cancer, and immunomodulatory potential. CONCLUSION: On-Def is the first report of a ß-defensin from the red-toothed trigger fish, Odonus niger. The antimicrobial profile showed the potential for further studies as a suitable candidate for antimicrobial peptide therapeutics.

A histone H2A-derived antimicrobial peptide, Hipposin from mangrove whip ray, Himantura walga: Molecular and functional characterisation.

Antimicrobial peptides (AMPs) are biologically dynamic molecules produced by all type of organisms as a fundamental component of their innate immune system. The present study deals with the identification of a histone H2A-derived antimicrobial peptide, Hipposin from mangrove whip ray, Himantura walga. A 243 base pair fragment encoding 81 amino acid residues amplified from complementary DNA was identified as Hipposin and termed as Hw-Hip. Homologous analysis showed that Hw-Hip belongs to the Histone H2A superfamily and shares sequence identity with other histone-derived AMPs from fishes. Phylogenetic analysis of Hw-Hip displayed clustering with the fish H2A histones. Secondary structure analysis showed the presence of three α-helices and four random coils with a prominent proline hinge. The physicochemical properties of Hw-Hip are in agreement with the properties of antimicrobial peptides. A 39-mer active peptide sequence was released by proteolytic cleavage in silico. Functional characterisation of active peptide in silico revealed antibacterial, anticancer and antibiofilm activities making Hw-Hip a promising candidate for further exploration.