Single Whey Acidic Protein Research Articles

Identification and functional characterization of C-type lectins and crustins provide new insights into the immune response of Portunus trituberculatus

A meticulous understanding of the immune characteristics of aquaculture animals is the basis for developing precise disease prevention and control strategies. In this study, four novel C-type lectins (PtCTL-5, PtCTL-6, PtCTL-7 and PtCTL-8) including a single carbohydrate-recognition domain (CRD), and four novel crustins (Ptcrustin-1, Ptcrustin-2, Ptcrustin-3 and Ptcrustin-4) with a single whey acidic protein (WAP) domain were identified from the swimming crab Portunus trituberculatus. Tissue distribution analysis indicated that most of the target genes were predominantly expressed in the hepatopancreas in all examined tissues, except for Ptcrustin-1 which were mainly expressed in the gills. Our results showed that the eight genes displayed various transcriptional profiles across different tissues. In hemocytes, the PtCTL-7 responded quickly to Vibrio alginolyticus and exhibited much more strongly up-regulation than other three PtCTLs. The Ptcrustin-1 rapidly responded to V. alginolyticus within 3 h in all the three tested tissues. Furthermore, recombinant proteins of PtCTL-5 and PtCTL-8 were successfully obtained, and both of them displayed bacterial binding activities toward V. alginolyticus, V. harveyi and Staphylococcus aureus, and only showed antibacterial activity against V. harveyi. These findings provided new insights into the diverse immune response of P. trituberculatus and laid theoretical foundations for the development of precise disease prevention and control strategies in P. trituberculatus farming. Moreover, the specific anti-V. harveyi activities exhibited by rPtCTL-5 and rPtCTL-8 suggested their promising application prospects for controlling diseases caused by V. harveyi.

A new crustin is involved in the innate immune response of shrimp Litopenaeus vannamei

Crustin is an antimicrobial peptide (AMP) that plays a key role in the innate immunity of crustaceans. This study cloned a new crustin from Pacific white shrimp Litopenaeus vannamei, which we designated as LvCrustinB, using rapid amplification of cDNA ends (RACE). The full-length cDNA of LvCrustinB is 751 bp with an open reading frame (ORF) of 591 bp encoding a peptide of 196 amino acids that includes a putative signal sequence. LvCrustinB is a type II crustin that has a glycine-rich region and a single whey acidic protein domain (WAP) domain. The mRNA transcript of LvCrustinB was detected in all examined tissues and was found to be most abundantly expressed in the epithelium and muscle. The expression of LvCrustinB in hemocytes was significantly upregulated after L. vannamei was challenged with LPS, Vibrio parahaemolyticus, and white spot syndrome virus (WSSV). When LvCrustinB was knocked down with RNAi, the mortality rate of L. vannamei significantly increased after V. parahaemolyticus or WSSV infection. Recombinant LvCrustinB was produced using Pichia pastoris GS115 and was shown to bind to 2 g-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and 2 g-negative bacteria (Escherichia coli and V. parahaemolyticus) via polysaccharides, which included PGN, LTA, and LPS. In vivo, the recombinant LvCrustinB remarkably protected L. vannamei from V. parahaemolyticus infection. These results suggest that LvCrustinB plays an important role in innate immunity and may be potentially utilized as antibacterial agents in shrimp.

Expression of the Shrimp wap gene in Drosophila elicits defense responses and protease inhibitory activity

The wap gene encodes a single whey acidic protein (WAP) domain-containing peptide from Chinese white shrimp (Fenneropenaeus chinensis), which shows broad-spectrum antimicrobial activities and proteinase inhibitory activities in vitro. To explore the medical applications of the WAP peptide, a wap gene transgenic Drosophila melanogaster was constructed. In wap-expressing flies, high expression levels of wap gene (>100 times) were achieved, in contrast to those of control flies, by qRT-PCR analysis. The wap gene expression was associated with increased resistance to microbial infection and decreased bacterial numbers in the flies. In addition, the WAP protein extract from wap-expressing flies, compared with control protein extract from control flies, showed improved antimicrobial activities against broad Gram-positive and Gram-negative bacteria, including the clinical drug resistant bacterium of methicillin-resistant S. aureus (MRSA), improved protease inhibitor activities against crude proteinases and commercial proteinases, including elastase, subtilis proteinase A, and proteinase K in vitro, and improved growth rate and microbial resistance, as well as wound-healing in loach and mouse models. These results suggest that wap-expressing flies could be used as a food additive in aquaculture to prevent infections and a potential antibacterial for fighting drug-resistant bacteria.

A single WAP domain (SWD)-containing protein with antiviral activity from Pacific white shrimp Litopenaeus vannamei

The single whey acidic protein (WAP) domain (SWD)-containing proteins, also called type III crustins, are a group of antimicrobial peptides (AMPs) in crustaceans. At present, a number of SWDs have been identified in shrimp, which showed essential antibacterial activities. However, the roles of SWDs in antiviral immune responses have not been reported up to now. In this study, a novel SWD (LvSWD3) was identified from Pacific white shrimp, Litopenaeus vannamei, which contained a typical single WAP domain homologous to those of other crustacean SWDs. Although lacking the pro and arg-rich region between the signal peptide and the WAP domain, LvSWD3 was closely clustered with other shrimp SWDs in the phylogenetic tree. Similar to many shrimp SWDs, the highest expression of LvSWD3 was detected in hemocytes. The LvSWD3 expression exhibited only limited changes after challenges with Vibrio parahaemolyticus, Poly (I:C) and lipopolysaccharide, but was significantly up-regulated after white spot syndrome virus (WSSV) infection. Silencing of LvSWDs significantly accelerated the death of the WSSV-infected but not the V. parahaemolyticus-infected shrimp. The recombinant LvSWD3 protein did not show proteinase inhibitory and antibacterial activities but could significantly postpone the death of WSSV-infected shrimp and reduce the viral load in tissues. These suggested that LvSWD3 was a novel SWD with antiviral activity.

Molecular cloning, expression, promoter analysis and functional characterization of a new Crustin from Litopenaeus vannamei

Antimicrobial peptides (AMPs) are the most important players in the innate immune system, providing a principal first-line of defense against the invading pathogens. Crustin, a type of whey acidic protein (WAP) domain-containing and cationic cysteine-rich AMP, can function in a protease inhibition or an effector molecule manner. In the present study, a new Crustin was cloned and identified from Pacific white shrimp Litopenaeus vannamei and designated as LvCrustinA. The full-length cDNA of LvCrustinA was 687 bp, with a 519 bp open reading frame (ORF) that encoded a peptide of 172 amino acids. Domain analysis indicated that LvCrustinA contained a Glycine-rich region in the N-terminal and a single WAP domain within eight cysteines in the C-terminal. The 5′ upstream regulatory sequence of 1249 bp (promoter) was obtained using a genome walking method, and it contained several conserved transcription factors binding motifs including NF-κB, AP-1 and STAT (Signal transducers and activators of transcription). Dual-reporter assay showed that NF-κB transcription factors LvDorsal and LvRelish, and AP-1 transcription factor Lvc-Jun could up-regulate the promoter activity of LvCrustinA, suggesting that NF-κB and JNK-c-Jun pathways could be involved in regulating the expression of LvCrustinA. Moreover, LvCrustinA was abundantly expressed in immune related tissues such as gill, hemocyte and epithelium, and its expression was up-regulated in response to Vibrio parahaemolyticus and White spot syndrome virus (WSSV) challenges in gill tissue, suggesting that LvCrustinA could be involved in the host defense against bacterial and viral infection. Additionally, RNAi mediated knockdown of LvCrustinA resulted in shrimps with the higher cumulative mortality during V. parahaemolyticus and WSSV infection. Taken together, these results provided some insight into the expression and transcriptional regulatory role of LvCrustinA, and its defensive role against pathogenic infection.

A novel single WAP domain-containing protein isoform with antibacterial relevance in Litopenaeus vannamei

Single WAP domain (SWD)-containing protein is a small protein containing a whey acidic protein (WAP) domain at the C-terminal region. SWD-containing protein exhibits structural similarity to the family of serine proteinase inhibitors. As of this writing, some SWD domain-containing proteins have been identified in crustaceans, and their functions included antibacterial and anti-proteinase activities. We identified a SWD protein isoform gene in Litopenaeus vanname (Lv-SWDi). Very high sequence similarity was found between Lv-SWDi and Lv-SWD. Results of time-course analysis for the gene expression profile showed that Lv-SWDi could produce a rapid feedback and an obvious upregulation at 12 h after Vibrio injection. Endogenous Lv-SWDi protein was obviously upregulated, and the highest expression level was reached at 24 h after Vibrio injection. The purified rLv-SWDi could directly bind to Gram-positive and Gram-negative bacteria. Results of the proteinase inhibitory assay also showed that rLv-SWDi could inhibit secretory protease activity from Bacillus subtilis. Lv-SWDi is a part of an important immunity-relevant gene and may serve important functions in defense against bacteria.

A novel crustin from Marsupenaeus japonicus promotes hemocyte phagocytosis

Crustins are cationic cysteine-rich antimicrobial peptides (AMPs) that contain multiple domains (glycine-rich, cysteine-rich, or proline-rich) at the N-terminus and whey acidic protein (WAP) domains at the C-terminus. Crustins have multiple functions, including protease inhibition and antimicrobial activity. Other functions of crustins need to be clarified. In this study, a novel crustin with a cysteine-rich region, and a single WAP domain, belonging to type I crustins, was identified in Marsupenaeus japonicus and designated as MjCru I-1. MjCru I-1 was expressed in various tissues. The expression of MjCru I-1 was upregulated in the hemocytes of shrimp challenged with bacteria. MjCru I-1 could bind to bacteria by binding to the cell wall molecules of the bacteria, such as lipopolysaccharide (LPS), peptidoglycan (PGN), and lipoteichoic acid (LTA). The synthesized WAP domain of MjCru I-1 but not synthesized Cys-rich domain has antibacterial and agglutinative activities. Scanning electron microscope assay showed that the bacterial cells treated with sMjCru I-1 appeared to be disrupted and cracked compared with those of the control samples. The knockdown of MjCru I-1 could reduce bacterial clearance and injection of MjCru I-1 could significantly increase the survival rate of shrimp infected with Vibrio anguillarum and Staphylococcus aureus compared with those of the control samples. Further study discovered that MjCru I-1 could increase the hemocyte phagocytosis against V. anguillarum and S. aureus. These results suggest that MjCru I-1 has dual functions, bactericidal and phagocytosis promoting activities, in the antibacterial immunity of shrimp.

A single whey acidic protein domain containing protein (SWD) inhibits bacteria invasion and dissemination in shrimp Marsupenaeus japonicus

The single whey acidic protein (WAP) domain containing proteins (SWDs) in crustacean belong to type III crustins and have antiprotease activities and/or antimicrobial activities. Their functions in vivo in crustacean immunity need to be clarify. In this study, a new single WAP domain containing protein (SWD) was obtained from Marsupenaeus japonicus, designated as MjSWD. The full-length cDNA of MjSWD was 522 bp.The open reading frame of MjSWD encoded a protein of 79 amino acids, with a 24 amino acid signal peptide and a WAP domain. Tissue distribution analysis revealed that MjSWD transcripts were generally expressed in all the tested tissues, including hemocytes, heart, hepatopancreas, gill, stomach and intestine. The time course expression of MjSWD was analyzed by quantitative real time PCR, and the results exhibited that MjSWD was upregulated after bacteria (Vibrio anguillarum, Staphylococcus aureus) and white spot syndrome virus (WSSV) challenge in gills and stomach of the shrimp. The purified recombinant protein of MjSWD could bind to several Gram-negative and Gram-positive bacteria though binding to microbial polysaccharides (peptidoglycan). MjSWD could inhibit the activity of Subtilisin A and Proteinase K and bacteria-secreted proteases. The results of natural infection with MjSWD incubated bacteria showed that the inhibition of MjSWD against bacterial secreted proteases was contributed to inhibiting bacteria invasion and dissemination in the shrimp. The MjSWD is, thus, involved in the shrimp antibacterial innate immunity.

Crustins from eyestalk cDNA library of swimming crab Portunus trituberculatus: Molecular characterization, genomic organization and expression analysis

Crustins are cationic, cysteine-rich antimicrobial proteins, containing a single whey acidic protein (WAP) domain in the C-terminal end. Different from the reported Ptcrustin in the hemocytes, two novel crustin genes (PtCrustin2 and PtCrustin3) were cloned completely from the eyestalk cDNA library of Portunus trituberculatus in this study. All PtCrustins share the consensus cysteine motif and are considered as Type I crustins. Four exons and three introns are identified in genomic DNA sequence of PtCrustin3 while three exons and two introns in PtCrustin2. The mRNA transcripts of PtCrustin2 and PtCrustin3 are mainly detected in eyestalk and gills, but not in hemocytes. Although both PtCrutins are up-regulated after challenge of three microorganisms, PtCrustin3 seems to respond more quickly to microbial challenge than Ptcrustin2. Unlike most crustins, both recombinant PtCrustin2 and PtCrustin3 exhibit antibacterial activity against Gram-positive bacteria Micrococcus luteus and Staphyloccocus aureus and Gram-negative bacterium Pseudomonas aeruginosa. In addition, rPtCrustin2 is moderately active against yeast Pichia pastoris and rPtCrustin3 show significant activity against Gram-negative bacterium Vibrio alginolyticus. These results indicate that PtCrustin2 and PtCrustin3 are two novel crustins and play different roles in immune response of P. trituberculatus against microbial challenge.

Phylogeny of whey acidic protein (WAP) four-disulfide core proteins and their role in lower vertebrates and invertebrates

Proteins containing WAP (whey acidic protein) domains with a characteristic WFDC (WAP four-disulfide core) occur not only in mammals (including marsupials and monotremes) but also in birds, reptiles, amphibians and fish. In addition, they are present in numerous invertebrates, from cnidarians to urochordates. Many of those from non-mammalian groups are poorly understood with respect to function or phylogeny. Those well characterized so far are waprins from snakes, perlwapins from bivalves and crustins from decapod crustaceans. Waprins are venom proteins with a single WAP domain at the C-terminus. They display antimicrobial, rather than proteinase inhibitory, activities. Perlwapins, in contrast, possess three WAP domains at the C-terminus and are expressed in the shell nacre of abalones. They participate in shell formation by inhibiting the growth of calcium crystals in the shell. The crustin group is the largest of all WFDC-containing proteins in invertebrates with the vast majority being highly expressed in the haemocytes. Most have a single WAP domain at the C-terminus. The presence and type of the domains between the signal sequence and the C-terminus WAP domain separate the different crustin types. Most of the TypeI and II crustins are antimicrobial towards Gram-positive bacteria, whereas the TypeIII crustins tend to display protease inhibition. Expression studies show that at least some crustins have other important biological effects, as levels change with physiological stress, wound repair, tissue regeneration or ecdysis. Thus WAP domains are widely distributed and highly conserved, serving in diverse physiological processes (proteinase inhibition, bacterial killing or inhibition of calcium transport).

A comparative study of antimicrobial properties of crustin Pm1 and crustin Pm7 from the black tiger shrimp Penaeus monodon

Several isoforms of crustin have been identified in the black tiger shrimp Penaeus monodon. These cationic cysteine-rich antimicrobial peptides contain a single whey acidic protein (WAP) domain at the C-terminus and exhibit antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigate the binding properties and antimicrobial actions of crustin Pm1 and crustin Pm7, the two most abundant crustin isoforms found in the haemocyte of P. monodon. Previously, crustin Pm1 showed strong inhibition against Gram-positive bacteria, whilst crustin Pm7 acted against both Gram-positive and Gram-negative bacteria. A binding study showed that both crustins can bind to Gram-positive and Gram-negative bacterial cells. Enzyme-linked immunosorbent (ELISA) assay suggested that crustins bind to the cell wall components, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) with positive cooperativity of Hill slope (H) > 2. This indicates that at least two molecules of crustins interact with one LTA or LPS molecule. In addition, both crustins can induce bacterial agglutination and cause inner membrane permeabilization in Escherichia coli. Scanning Electron Microscopy (SEM) revealed the remarkable change on the cell surface of Staphylococcus aureus, Vibrio harveyi and E. coli after the bacteria were treated with the recombinant crustin Pm7. Meanwhile, crustin Pm1 can cause a visible change on the cell surface of S. aureus and E. coli only. This is in agreement with the fact that crustin Pm1 has shown no antimicrobial activity against V. harveyi. It is likely that the antimicrobial activity of crustins mainly relies on their ability to agglutinate bacterial cells and to disrupt the physiochemical properties of bacterial surface.

A single WAP domain (SWD)-containing protein with antipathogenic relevance in red swamp crayfish, Procambarus clarkii

Single WAP domain-containing proteins (SWDs) are small proteins possessing a whey acidic protein (WAP) domain at the C-terminal region. In this study, a complementary deoxyribonucleic acid (cDNA) of SWD-containing protein was isolated from the hemocytes of red swamp crayfish, Procambarus clarkii called Pc-SWD. The full-length cDNA sequence is 998 bp. The deduced amino acid sequence consists of 74 residues with a signal peptide of 20 residues. The mature peptide has a single WAP domain which contains eight conserved cysteine residues forming a “four-disulphide core” (4-DSC). The predicted molecular mass of the mature protein is 5.97 kDa, with an estimated pI of 7.71. Tissue distribution analysis by reverse-transcribed polymerase chain reaction (RT-PCR) revealed that Pc-SWD transcripts were primarily found in the hemocytes, heart, hepatopancreas, gills, and intestine. The results of time course analysis demonstrated that expression of Pc-SWD was decreased at 6 h followed by a significant upregulation from 48 h to 72 h in hemocytes after white spot syndrome virus (WSSV) injection. A similar expression pattern was found in the hepatopancreas after WSSV injection. In addition, Pc-SWD expression was visibly upregulated in the gills from 6 h to 72 h after WSSV injection. The results of Western bolt revealed that Pc-SWD was constitutively expressed in the heart, hepatopancreas, gills, and intestine of unchallenged crayfish. A weak band was detected in the hemocytes and hemolymph of unchallenged P. clarkii. The Pc-SWD expression was upregulated in the hemocytes and gills after challenging with WSSV; however, no obvious change occurred in the heart and intestine. rPc-SWD could bind to both Gram-negative and -positive bacteria strongly. Moreover, rPc-SWD exhibited specific proteinase inhibitory activity against the secretory proteinase(s) from B. subtilis and P. aeruginosa. All these findings suggest that Pc-SWD possibly functions as an immunity effector in defense against the invasion of crayfish pathogens.

Characterization of crustin antimicrobial proteins from Japanese spiny lobster Panulirus japonicus

Crustin antimicrobial proteins (PJC1–4) were identified from a phyllosoma library of Japanese spiny lobster, Panulirus japonicus. The deduced amino acid sequences of PJC1–4 contained open reading frames of 130, 139, 124 and 150 amino acid residues, respectively. These proteins contained a glycine-rich region at the N-terminus and 12 conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A phylogenetic tree and sequences alignment analyses revealed that PJC1–4 are more closely related to shrimp crustins than to other lobster crustins. Transcripts of PJC1, 3 and 4 were detected in heart, nerves, intestine, hemocytes, gills and hepatopancreas, while transcripts of PJC2 were detected only in nerves.

A single whey acidic protein domain (SWD)-containing peptide from fleshy prawn with antimicrobial and proteinase inhibitory activities

With their inherent immune system, shrimps survive in marine environments despite the presence of different stressors, such as bacteria, fungi, and virus. In order to fight against and subsist even in the presence of such pathogens, the shrimps' ability to produce and release antimicrobial peptides are indeed important. In view of this, a new type of genetic material, an antimicrobial peptide containing a single whey acidic protein domain (SWD), was cloned from the haemocytes of fleshy prawns ( Fenneropenaeus chinensis). Hence, this peptide is named Fc-SWD. The full-length cDNA of Fc-SWD was 399 bp, and it contained a single open reading frame (ORF) of 273 bp encoding an Fc-SWD of 90 amino acids, including a putative 24-amino acid signal peptide and a mature 66 residue peptide. Sequence comparison and phylogenetic analysis of SWDs and other whey acidic protein (WAP) domains containing proteins in crustaceans suggested that the crustacean WAP containing proteins can be divided into four classes: crustin I, crustin II, SWD, and carcinin and carcinin-like proteins. Apparently, the Fc-SWD is a new type in the SWD subgroup. Northern blot analysis demonstrated that Fc-SWD transcripts were constitutively expressed in the haemocytes of non-infected shrimps and increased in the haemocytes of infected shrimps. Meanwhile, Fc-SWD mRNA was not detected in the hearts, hepatopancreas, stomachs, gills, intestines, or ovaries of the non-infected shrimps nor the infected ones. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that the Fc-SWD signal was detected in the haemocytes, hearts, and gills of both the unchallenged and the challenged shrimps, and in the stomach of the challenged shrimps. Western blot analysis indicated that the Fc-SWD protein appeared to be synthesized in the haemocytes with a signal peptide and then secreted to the haemolymph as a mature peptide when the F. chinensis was infected by pathogens. Thus, the Fc-SWD (for the full-length peptide including the signal peptide), the mFc-SWD (for the mature peptide), and the Fc-WAPD (for the single WAP domain) were manifested in the Escherichia coli, respectively . Further findings indicated that the purified recombinant Fc-SWD and the mFc-SWD have antimicrobial activities against Gram-positive (G +) and Gram-negative (G −) bacteria and fungi, as well as a strong inhibitory activity against subtilisin A and protein K. The Fc-WAPD has anti-protease activities and low antimicrobial activity. In view of the foregoing findings, it can be concluded that Fc-SWD is one of the factors that affect shrimps' immunity against various pathogens.

Molecular cloning and characterization of crustin from mud crab Scylla paramamosain

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain.

Shrimp single WAP domain (SWD)-containing protein exhibits proteinase inhibitory and antimicrobial activities

Single WAP domain (SWD)-containing proteins are small proteins with a C-terminal region containing a single whey acidic protein (WAP) domain. In the present study, the cDNAs representing three isoforms of SWD proteins (SWDPm1, SWDPm2 and SWDPm3) were identified from hemocytes of the black tiger shrimp, Penaeus monodon. The deduced peptides revealed that they contain a putative signal peptide of 24 amino acids and encode for a mature peptide of 69, 68 and 56 amino acids, respectively, which contain typical characters similar to those of the shrimp SWD proteins (type III crustin) with a Pro-Arg region and a WAP domain towards the C-terminus. Tissue distribution analysis by RT-PCR showed that all three SWDPm transcripts were primarily found in hemocytes. Transcript expression of SWDPm1 was down-regulated upon injection with Staphylococcus aureus whilst there was no change of SWDPm2 and SWDPm3 expression. In contrast, white spot syndrome virus (WSSV) injection resulted in a biphasic response with up-regulation of SWDPm1 and SWDPm2 transcripts at 6h followed by significant down-regulation by 24h after infection. Genomic organization of the SWDPm2 gene revealed the presence of three exons interrupted by two introns. To characterize the biological functions of the SWD protein, the mature SWDPm2 protein encoding cDNA was cloned and expressed in Escherichia coli. Purified recombinant (r)SWDPm2 exhibits antibacterial activity against several Gram-positive, but not Gram-negative, bacteria and is a competitive inhibitor of subtilisin A with an inhibition constant (Ki) of 1.98nM. Thus, rSWDPm2 may contribute to the inhibitory regulation of subtilisin A from bacterial infection and P. monodon SWD protein likely function as immune effectors in defense against invasion of shrimp pathogens.

Cloning, expression and antimicrobial activity of crustin Pm1, a major isoform of crustin, from the black tiger shrimp Penaeus monodon

Crustin antibacterial homologues, containing a whey acidic protein (WAP) domain, have been identified from the haemocyte library of the black tiger shrimp, Penaeus monodon. Sequence analysis of these cDNAs indicates the presence of several isoforms of crustin in P. monodon. Crustin Pm1, the most abundant isoform, contains an open reading frame of 435 bp encoding a precursor of 145 amino acids that comprises 17 amino acid signal peptides and 128 amino acid mature peptides. The peptides contain a Gly–Pro rich region at the amino-terminus and a single whey acidic protein (WAP) domain at the carboxyl-terminus. In order to characterize the properties and biological activities of this peptide, crustin Pm1 was overexpressed in Escherichia coli. The recombinant crustin Pm1 has a molecular mass of 14.7 kDa with a predicted p I of 8.3. Antimicrobial assays demonstrated that recombinant crustin Pm1 exhibited antimicrobial activity against only Gram-positive bacteria with strong inhibition against Staphylococcus aureus and Streptococcus iniae. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of crustin Pm1 was a result of bactericidal effect. In situ hybridization with crustin Pm1 antisense probes showed strong hybridization signals in a certain haemocyte population of unchallenged shrimp, indicating that crustin Pm1 transcript is differentially expressed in different subsets of haemocyte cells.

Antimicrobial activity of omwaprin, a new member of the waprin family of snake venom proteins

We have isolated and characterized omwaprin, a 50-amino-acid cationic protein from the venom of inland taipan (Oxyuranus microlepidotus). It is a new member of the waprin family of snake venom proteins. A synthetic gene was designed and constructed for expressing the recombinant protein in Escherichia coli. Recombinant omwaprin was used for carrying out functional analyses. The protein is non-toxic to Swiss albino mice at doses of up to 10 mg/kg when administered intraperitoneally. However, it shows selective and dose-dependant antibacterial activity against Gram-positive bacteria. The minimum inhibitory doses were in the range 2-10 microg for selected species of bacteria in radial diffusion assays. The antibacterial activity is salt-tolerant up to 350 mM NaCl. However, omwaprin lost its antibacterial activity upon reduction and alkylation of its cysteine residues, or upon deletion of six N-terminal amino acid residues, four of which are positively charged. These observations indicate that the three-dimensional structure constrained by four disulfide bonds and the N-terminal residues are essential for its activity. The mechanism of action is via membrane disruption, as shown by scanning electron microscopy. Importantly, omwaprin lacks haemolytic activity on human erythrocytes. This demonstrates the specificity of omwaprin for bacterial membranes. Unlike other reported WAP (whey acidic protein) domain-containing antibacterial proteins, including elafin, EPPIN (epididymal proteinase inhibitor), SWAM1 and SWAM2 [single WAP (whey acidic protein) motif proteins 1 and 2] and SLPI (secretory leucocyte proteinase inhibitor), omwaprin shows species-specific activity on the Gram-positive bacteria tested.

Organization and promoter analysis of a tiger shrimp Penaeus monodon single WAP domain-containing protein gene

The main function of the single whey acidic protein domain (SWD)-containing protein in shrimp is unknown. To elucidate the function of the SWD-containing protein in vivo, the SWD-containing protein gene was isolated and characterized. A 9.3-kb shrimp SWD-containing protein gene, and a 3.9-kb 3′-flanking region. The shrimp SWD-containing protein gene contained three exons and two introns. Different fragments of the shrimp SWD-containing protein 5′-flanking region were transfected into HeLa cells. The promoter activities were assayed by basal human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone (LRH), and gonadotropin-releasing hormone (GnRH) treatments. The in vitro actions of the SWD-containing protein promoter expression pattern were studied by transfection of an SWD-containing protein promoter (1 kb)-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene into the HeLa cell line, which was then microinjected into zebrafish Danio rerio embryos. These results indicate that the shrimp SWD-containing protein promoter might play an important role in gene regulation of sex hormones in mammalian cell lines and in gene regulation of developmental stages in zebrafish.

The effect of sodium alginate on the immune response of tiger shrimp via dietary administration: Activity and gene transcription

The total haemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (release of superoxide anions), and superoxide dismutase (SOD) activity, as well as expressions of β-1,3-glucan-binding protein (βGBP), prophenoloxidase (proPO), peroxinectin (PE), cytosolic SOD (cyt-SOD), penaeidin-5 (PA-5), and a single whey acidic protein (WAP) domain protein (SWDP) gene were determined in the tiger shrimp Penaeus monodon (15.6–19.5 g) which had individually been fed diets containing sodium alginate at 0, 1.0, or 2.0 g kg −1 for 5 months. Results showed that shrimp fed a diet containing 1.0 and 2.0 g kg −1 sodium alginate had significantly increased SOD activity but decreased respiratory bursts. The expressions of βGBP, PE, cyt-SOD, PA-5, and SWDP were significantly elevated in shrimp fed the ≤2.0 g kg −1 sodium alginate-containing diet for 5 months. However, no significant differences in THC, PO activity, or proPO mRNA transcription in shrimp were observed among the three treatments. It was concluded that sodium alginate can be used as an immunomodulator for shrimp through dietary administration to modify immune genes expression of shrimp.