Swapna P. Antony

Molecular characterization of a crustin­like antimicrobial peptide in the giant tiger shrimp, Penaeus monodon, and its expression profile in response to various immunostimulants and challenge with WSSV

A crustin-like antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustin-like AMP from other shrimp crustins. The differential expression of the crustin-like AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two β-glucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two gram-positive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded pre- and post-challenge white spot syndrome virus (WSSV) by semi-quantitative RT-PCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustin-like AMP was found to be constitutively expressed in the animal and a significant down-regulation could be noted post-challenge WSSV. Remarkable down-regulation of the gene was observed in the immunostimulant fed animals pre-challenge followed by a significant up-regulation post-challenge WSSV. Tissue-wise expression of crustin-like AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustin-like AMP and confer significant protection to P. monodon against WSSV infection.

Molecular characterization of a crustin-like, putative antimicrobial peptide, Fi-crustin, from the Indian white shrimp, Fenneropenaeus indicus

Antimicrobial peptides are important innate immune defense, especially in those animals which lack adaptive immunity [1e8]. Due to their small size, amphipathic structure and cationic character they can rapidly diffuse to the point of infection [9], a mechanism that presumably makes it easier to circumvent microbial resistance against the peptides [10]. Besides providing an immediate and broad-spectrum microbicidal activity, AMPs can kill bacteria in micromolar range, are promptly synthesized at low metabolic cost, and are easily stored in large amounts and readily available shortly after an infection [11e13]. Many AMPs show a remarkable specificity for prokaryotes with low toxicity for eukaryotic cells; a phenomenon which has favored their investigation and exploitation as potential new antibiotics [14]. AMP gene expression and distribution are regulated through haemocyte reactions [15]. Transcripts of crustin-encoding genes have also been observed in gills, heart and intestine [16e18] but as these tissues are highly vascularised, it is assumed the transcripts from these organs are due primarily to the haemocytes. In penaeid shrimps, four main families of AMPs have been currently described and characterized from the haemocytes: penaeidins, crustins, anti-lipopolysaccharide factors (ALFs) and lysozymes. Penaeidins are mainly active against Gram-positive bacteria, filamentous fungi [19], viruses and protozoans [20] whereas ALFs have a broader antimicrobial spectrum including Gram-negative bacteria [21,22]. Conversely, crustins are reported to have a more-restricted activity spectrum, affecting mainly marine Gram-positive bacteria [17,23,24] Crustins, a widely distributed family of AMPs was first isolated from the shore crab, Carcinus maenas as an 11.5 kDa peptide [23]. Crustins are cationic, cysteinerich antimicrobial AMPs having molecular weight of 7e14 kDa, with an isoelectric point in the range of 7.0e8.7, and contain one whey-acidic protein (WAP) domain at the carboxy terminus [25]. Crustins have been proved to be an important antimicrobial protein in the plasma and haemocyte granules of crustaceans and described as a component of the innate immune system [8]. These AMPs are dominantly synthesized and stored in haemocytes [4,8,16,18,23,24,26e30] and their release from haemocytes is induced by bacterial infection [15,27,31]. Crustin mechanisms of action and function are still largely unknown, although they contain a whey-acidic protein (WAP) domain common to proteinase inhibitory activities as well as antimicrobial activities [8]. Many full-length cDNA and several ESTs of crustins have been described in a wide range of penaeid prawns including Litopenaeus vannamei [8,24,30,32], Litopenaeus setiferus [24,32,33], Penaeus monodon [16,17,29,30,34e37], Marsupenaeus japonicus [17,38], Litopenaeus schmitti [33], Fenneropenaeus chinensis [17,29], Farfantepenaeus brasiliensis [33], Farfantepenaeus paulensis [33] and Farfantepenaeus subtilis [33]. However, no antimicrobial peptide sequences have been reported from Fenneropenaeus indicus. In the current study a crustin cDNA has been characterized from the Indian White Shrimp, F. indicus. Healthy adult F. indicus (8e10 g body weight) were purchased from a local shrimp farm in Vypeen, Kochi. Theywere transferred to aquaria of 500 l capacity and acclimatized for one week under laboratory conditions. Prawns were fed with a standard feed (Higashimaru, India). Aeration was provided in all tanks during the experiment and bioreactorwas set in all the aquaria for the removal of ammonia and nitrate. Only shrimps in the intermoult stage were sampled during the study. Haemolymph was collected from the rostral sinus using specially designed capillary tubes (RNase-free) rinsed using precooled anticoagulant solution (RNase-free, 10% sodium citrate, pH 7.0). Total RNA was extracted from the haemocytes using TRI * Corresponding author. Tel.: þ91 484 2368120; fax: þ91 484 2381120. E-mail addresses: rose@cusat.ac.in, rosammap@gmail.com (R. Philip).

Marine yeast Candida aquaetextoris S527 as a potential immunostimulant in black tiger shrimp Penaeus monodon.

A marine yeast Candida aquaetextoris S527 as a source of immunostimulant in Penaeus monodon was studied. Yeast diet was prepared by incorporating 10% C. aquaetextoris S527 biomass into a standard shrimp diet and administered in P. monodon at different frequencies (daily, once in three days, once in seven days and once in ten days) followed by challenge with white spot syndrome virus (WSSV). Immune parameters such as total protein, total hemocyte count, pro-phenoloxidase, nitroblue tetrazolium reduction, alkaline phosphatase activity and acid phosphatase activity were tested. Expression profile of antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5; immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, peroxinectin, prophenol oxidase (proPO) and transglutaminase, and WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, ribonucleotide reductase, thymidine kinase and VP28 were analyzed. The study demonstrated that marine yeast diet administered once every seven days conferred better protection to P. monodon against WSSV infection, supported by the hematological and immune gene expression profiles analyzed.

Immune gene expression profile of Penaeus monodon in response to marine yeast glucan application and white spot syndrome virus challenge.

Immunostimulant potential of eight marine yeast glucans (YG) from Candida parapsilosis R20, Hortaea werneckii R23, Candida spencermartinsiae R28, Candida haemulonii R63, Candida oceani R89, Debaryomyces fabryi R100, Debaryomyces nepalensis R305 and Meyerozyma guilliermondii R340 were tested against WSSV challenge in Penaeus monodon post larvae (PL). Structural characterization of these marine yeast glucans by proton nuclear magnetic resonance (NMR) indicated structures containing (1-6)-branched (1-3)-β-D-glucan. PL were fed 0.2% glucan incorporated diet once in seven days for a period of 45 days and the animals were challenged with white spot syndrome virus (WSSV). The immunostimulatory activity of yeast glucans were assessed pre- and post-challenge WSSV by analysing the expression profile of six antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5 and 13 immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, caspase, catalase, glutathione peroxidase, glutathione-s-transferase, haemocyanin, peroxinectin, pmCathepsinC, prophenol oxidase (proPO), Rab-7, superoxide dismutase and transglutaminase. Expression of seven WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, thymidine kinase and VP28 were also analysed to detect the presence and intensity of viral infection in the experimental animals post-challenge. The study revealed that yeast glucans (YG) do possess immunostimulatory activity against WSSV and also supported higher survival (40-70 %) post-challenge WSSV. Among the various glucans tested, YG23 showed maximum survival (70.27%), followed by YG20 (66.66%), YG28 (60.97%), YG89 (58.53%), YG100 (54.05%), YG63 (48.64%), YG305 (45.7%) and YG340 (43.24%).

Molecular identification and characterization of Type I crustin isoforms from the hemocytes of portunid crabs, Scylla tranquebarica and Portunus pelagicus

Crustins are cationic antimicrobial peptides of ca. 7-14kDa with a characteristic four-disulphide core containing WAP domain, present in the hemocytes of crustaceans. The present study reports the first crustin sequences from two portunid crabs, viz. the mud crab Scylla tranquebarica (St-Crustin, JQ965930) and the blue swimmer crab Portunus pelagicus (Pp-Crustin, JQ753312). St-Crustin and Pp-Crustin represented the complete cDNA sequence of Type I crustin, with an ORF of 336bp encoding 111aa with a predicted molecular weight of 10kDa and a pI of 8. The signal sequence contained 21aa residues, which was followed by a mature peptide with a WAP domain at the C-terminus. Peptide model of St-Crustin and Pp-Crustin indicated a randomly coiled structure enclosing two β-sheets but no helices. St-Crustin and Pp-Crustin shared significant similarities with crustins of portunid crabs (68-95%) and other crabs (60-73%). Phylogenetic analysis showed that St-Crustin and Pp-Crustin possess the same ancestral origin and have a similar evolutionary status like other crustins, which has subsequently diverged at different phases of evolution. St-Crustin and Pp-Crustin were closely related to crab crustins rather than to the crustins of other crustacean groups. The wide distribution of crustins in crustaceans indicates the importance of these AMPs in the innate immunity. Discovery of novel crustins might pave way to the discovery of promising therapeutic/prophylactic agents in health management and disease control in crustacean aquaculture.

Two isoforms of anti-lipopolysaccharide factors identified and characterized from the hemocytes of portunid crabs, Portunus pelagicus and Scylla tranquebarica

Anti-lipopolysaccharide factors (ALFs), a type of cationic antimicrobial peptides (AMPs), and their derivatives are becoming predominant candidates for potential drugs in viral and bacterial diseases. This study reports the first ALF from the mud crab Scylla tranquebarica (StALF, JQ899453) and the second ALF isoform from the blue swimmer crab Portunus pelagicus (PpALF2, JQ899452). Both sequences encoded for precursor molecules, starting with a signal peptide containing 26 amino acid residues, followed by a highly cationic mature peptide, containing two conserved cysteine residues flanking a putative lipopolysaccharide (LPS)-binding domain. BLAST analysis revealed that both PpALF2 and StALF exhibited significant similarity with crustacean ALF sequences. The predicted molecular mass of the mature ALFs was 11.2 kDa with an estimated pI of 10.0. PpALF2 and StALF also showed the typical pattern of alternating hydrophobic and hydrophilic residues in their putative disulphide loop, suggesting that they comprise the same functional domain. Phylogenetic analysis showed that PpALF2 and StALF have similar evolutionary status and they were phylogenetically ancient immune effector molecules which may play an essential role in the host defense mechanism. The spatial structures of PpALF2 and StALF possessed four beta-strands and two alpha-helices. The results indicated that there were more than one ALF involved in crab immunity against various pathogens. ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control in crustacean aquaculture.

Molecular cloning, recombinant expression and functional characterization of an antimicrobial peptide, Crustin from the Indian white shrimp, Fenneropenaeus indicus

Abstract Antimicrobial peptides (AMPs) comprise molecules that involve in the defense mechanism of various organisms towards pathogens such as bacteria, fungi, parasites and viruses. Crustins are generally defined as multi‐domain cationic antimicrobial peptides containing one whey acidic protein (WAP) domain at the C‐terminus as the functional unit. In this study, we identified and characterized a novel crustin homolog (Fi‐Crustin2) with 354 bp fragment cDNA encoding 117 amino acids and an ORF of 100 amino acids with a net charge of +1 from the mRNA of F. indicus haemocytes. This study forms the second report of a crustin isoform from F. indicus. Blast analysis revealed that Fi‐crustin2 exhibits similarity to shrimp crustins already reported. The active mature peptide has a molecular weight of 10.61 kDa and pI of 7.59 with a beta sheeted structure. The mature peptide was cloned into pET‐32a(+) with a N‐terminal hexa‐histidine tag fused in‐frame, and expressed in Escherichia coli, and the recombinant crustin, Fi‐crustin2 inhibited the growth of Gram‐negative bacteria with low MIC. All these features suggest that Fi‐crustin2 is a potent antibacterial protein against Gram‐negative bacteria and could play an important role in the innate immune mechanism of F. indicus. HighlightsA novel type‐II crustin was identified from Fenneropenaeus indicus.Recombinant production of Fi‐crustin2 done in E. coli Rosettagami™B(DE3)pLysS.Significant inhibition against E. tarda and A. hydrophila with MIC of 5 &10 &mgr;M.Membrane blebbing observed in rFi‐crustin2 treated E. tarda by SEM analysis.Peptide found to be non‐haemolytic & non‐cytotoxic up to 20 &mgr;M & thus biocompatible.