Characterization of a novel Type-I Crustin (carcininPm2) from black tiger shrimp Penaeus monodon.

Carcinins are type-I crustins from crustaceans and play an important role in innate immune system. In this study, type-I crustins, carcininPm1 and carcininPm2, from the hemocytes of Penaeus monodon were identified. Comparison of their amino acid sequences and the phylogenetic tree revealed that they were closely related to the other crustacean carcinin proteins, but were clustered into different groups of the carcinin proteins. The full-length amino acids of carcininPm1 and carcininPm2 were 92 and 111 residues, respectively. CarcininPm1 and carcininPm2 were expressed mainly in hemocytes and intestine compared to the other tissues. The expression of carcininPm1 and carcininPm2 were dramatically increased in early time of bacterial challenged shrimp hemocytes. In contrast, the carcininPm1 and carcininPm2 were expressed in response to late state of YHV-infected shrimp hemocytes where the copy number of virus was high. The recombinant carcininPm2 (rcarcininPm2) but not its WAP domain (rcarcininPm2_WAP) exhibited antimicrobial activity against Vibrio harveyi and Vibrio parahaemolyticus AHPND but not other bacteria tested. The rcarcininPm2 was able to prolong the survival rate of VH-treated post larval shrimp from about 102 h to 156 h. These studies indicated that the carcininPm2 possessed the potential and challenges as antibacterial in innate immunity of shrimp.

Microfluidic paper-based analytical device for determination of sucrose in sugarcane juice using Benedict's reagent.

This work presents a microfluidic paper-based analytical device (µPAD) for the determination of sucrose using the Benedict's test. An asymmetric dumbbell-shaped hydrophobic barrier was produced by rubber stamping the barrier pattern onto a laboratory filter paper. Hydrochloric acid and solution containing sucrose were successively deposited onto the sample reservoir of the µPAD attached to a glass slide. The device was placed in a plastic bag and dipped into boiling water for accelerating the hydrolysis of sucrose into the reducing sugars. Then the Benedict's reagent was added at the narrow straight channel connecting the two circular zones of the µPAD, which was replaced in the plastic bag and heated again for reduction of Cu(II) by the reducing sugars. Precipitate of brick-red copper(I) oxide was formed. The image of the µPAD was recorded by a smartphone. The ratio of the red to blue intensities gave linear correlation with the concentration of sucrose in the range of 0.5-10% w/v. The relative standard deviation of the measurement was less than 5% for 2 and 4% w/v sucrose (n = 10), with limit of determination, calculated using standard deviation of regression divided by slope of calibration, of 0.26% w/v sucrose. The method was successfully validated using the dinitrosalicylic acid method for sucrose measurement. Percent recoveries of sucrose were evaluated using ten sugarcane samples. The recoveries were in the range of 89 to 101%, demonstrating that there were no significant sample matrix effects on the quantification.

Silencing of a Kazal-type serine proteinase inhibitor SPIPm2 from Penaeus monodon affects YHV susceptibility and hemocyte homeostasis.

In shrimp, the Kazal-type serine proteinase inhibitors (KPIs) are involved in host innate immune defense system against pathogenic microorganisms. A five-Kazal-domain SPIPm2 is the most abundant KPIs in the black tiger shrimp Penaeus monodon and up-regulated in response to yellow head virus (YHV) infection. In this study, the role of SPIPm2 in YHV infection was investigated. The expression of SPIPm2 in hemocytes, gill and heart from 48-h YHV-infected shrimp was increased. The expression of SPIPm2 in hemocytes was significantly increased after 12 h of infection and gradually increased higher afterwards. Silencing of SPIPm2 by dsRNA interference resulted in the increased expression of different apoptosis-related genes, the increased expression of transcriptional factors of antimicrobial synthesis pathways, the reduction of circulating hemocytes in the shrimp hemolymph, and the increased susceptibility of the silenced shrimp to YHV infection. The activities of caspase-3 and caspase-7 in the hemocytes of SPIPm2-silenced shrimp was also increased by 5.32-fold as compared with those of the control shrimp. The results suggested that the SPIPm2 was involved in the hemocyte homeostasis.

A single WAP domain-containing protein from Litopenaeus vannamei possesses antiproteinase activity against subtilisin and antimicrobial activity against AHPND-inducing Vibrio parahaemolyticus.

The single WAP domain-containing protein (SWD) is a type III crustin antimicrobial peptide whose function is to defense the host animal against the bacterial infection by means of antimicrobial and antiproteinase activities. A study of SWD from Litopenaeus vannamei (LvSWD) is reported herein about its activities and function against bacteria, particularly the AHPND-inducing Vibrio parahaemolyticus (VPAHPND) that causes acute hepatopancreatic necrosis disease (AHPND). The LvSWD is mainly synthesized in hemocytes and up-regulated in response to VPAHPND infection. Over-expressed mature recombinant LvSWD (rLvSWD) and its WAP domain (rLvSWD-WAP) are able to strongly inhibit subtilisin but not trypsin, chymotrypsin and elastase. The rLvSWD inhibits subtilisin with the inhibition constant (Ki) of 14.3 nM. However, only rLvSWD exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Unlike the rLvSWD, the rLvSWD-WAP does not possess antimicrobial activity. Therefore, the killing effect of rLvSWD on VPAHPND and Bacillus megaterium was studied. The MIC of 30 µM against VPAHPND is bactericidal whereas the MIC against B. megaterium is not. With four times the MIC of rLvSWD, the VPAHPND-treated post larval shrimp are able to survive longer with 50% survival rate as long as 78 h as compared to 36 h of the infected shrimp without rLvSWD. The antimicrobial activity of LvSWD against the VPAHPND infection suggests its potential application for disease control in aquaculture.

Domain 2 of a Kazal serine proteinase inhibitor SPIPm2 from Penaeus monodon possesses antiviral activity against WSSV.

A 5-domain Kazal type serine proteinase inhibitor SPIPm2 from Penaeus monodon is involved in innate immune defense against white spot syndrome virus (WSSV). To test which domains were involved, the 5 domains of SPIPm2 were over-expressed and tested against WSSV infection. By using hemocyte primary cell culture treated with each recombinant SPIPm2 domain along with WSSV, the expression of WSSV early genes ie1, WSV477 and late gene VP28 were substantially reduced as compared to other domains when the recombinant domain 2, rSPIPm2D2, was used. Injecting the WSSV along with rSPIPm2D2 but not with other domains caused delay in mortality rate of the infected shrimp. The results indicate that the SPIPm2D2 possesses strong antiviral activity and, hence, contributes predominantly to the antiviral activity of SPIPm2.

Type I and type II crustins from Penaeus monodon, genetic variation and antimicrobial activity of the most abundant crustinPm4.

An antimicrobial protein, crustin, is involved in the innate immunity of crustacean by defending the host directly against the microbial pathogens. By data mining the Penaeus monodon EST database, two type I crustins, carcininPm1 and 2, and ten type II crustins, crustinPm1-10, were identified. The abundant crustins were crustinPm1, 4 and 7, each with variation in the length of Gly-rich repeat among its members. A few crustinPm1, 4 and 7 with deletion in the Cys-rich region were also observed. Furthermore, the crustinPm4 with the longest N-terminal Gly-rich region was characterized. The crustinPm4 allelic genes were expressed mainly from the hemocytes. Its expression was up-regulated readily by WSSV infection and gradually decreased to normal level afterwards. The recombinant crustinPm4-1 (rcrustinPm4-1) isoform was produced using the Escherichia coli expression system and tested for its antimicrobial activity. The rcrustinPm4-1 was able to inhibit the growth of a Gram-positive bacterium, Bacillus megaterium but not Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus. It also inhibited the growth of two Gram-negative bacteria, E. coli 363 and Vibrio harveyi 639 at lower potency. The rcrustinPm4-1 affected the WSSV infection because the expression of an intermediate early gene ie1 in WSSV-infected hemocyte cell culture was reduced. It was shown further that the rcrustinPm4-1 could delay by about one and a half days the manifestation of disease by WSSV.

The N-terminal glycine-rich and cysteine-rich regions are essential for antimicrobial activity of crustinPm1 from the black tiger shrimp Penaeus monodon.

An antimicrobial protein crustinPm1 from Penaeus monodon is a WAP domain-containing protein with an antimicrobial activity against Gram-positive bacteria but does not have antiproteinase activity. The lack of antiproteinase is speculated to be due to the P(1)' Met and/or the length of spacing between the conserved Cys2 and Cys3 while the antimicrobial activity may be due to the N-terminal Gly-rich and Cys-rich regions. In this study, the P(1)-P(1)' and the N-terminal Gly-rich and Cys-rich regions of crustinPm1 were mutated by amino acid substitution or deletion. Substitutions of P(1)-P(1)' from Pro-Pro to Leu-Leu, Leu-His, Leu-Met, Leu-Ala and P(1)' from Pro to Met did not make the protein inhibitory to subtilisin, trypsin, chymotrypsin and elastase. The mutations at P(1)-P(1)' positions in rcrustinPm1 had no effect on antibacterial activity. The WAP domain mutant with both Gly-rich and Cys-rich regions deleted did not exhibit antibacterial activity against Staphylococcus aureus while the deletion mutants of either Gly-rich or Cys-rich regions exhibited lower antibacterial activity than the wild type crustinPm1. Therefore, both Gly-rich and Cys-rich regions attached to a WAP domain are essential for efficient antibacterial activity of crustinPm1.

Proteinase inhibitory activities of two two-domain Kazal proteinase inhibitors from the freshwater crayfish Pacifastacus leniusculus and the importance of the P(2) position in proteinase inhibitory activity.

Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors (KPIs), KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P. leniusculus, they are found specific to the hemocytes and contain an uncommon P(2) amino acid residue, Gly. To unveil their inhibitory activities, the two KPIs and their domains were over-expressed. By testing against subtilisin, trypsin, chymotrypsin and elastase, the KPI2 was found to inhibit strongly against subtilisin and weakly against trypsin, while the KPI8 was strongly active against only trypsin. With their P(1) Ser and Lys residues, the KPI2_domain2 and KPI8_domain2 were responsible for strong inhibition against subtilisin and trypsin, respectively. Mutagenesis of KPI8_domain1 at P(2) amino acid residue from Gly to Pro, mimicking the P(2) residue of KPI8_domain2, rendered the KPI8_domain1 strongly active against trypsin, indicating the important role of P(2) residue in inhibitory activities of the Kazal-type serine proteinase inhibitors. Only the KPI2 was found to inhibit against the extracellular serine proteinases from the pathogenic oomycete of the freshwater crayfish, Aphanomyces astaci.

A Kazal-type serine proteinase inhibitor SPIPm2 from the black tiger shrimp Penaeus monodon is involved in antiviral responses.

A five-domain Kazal-type serine proteinase inhibitor, SPIPm2, from Penaeus monodon has recently been implicated in antiviral responses for it is up-regulated upon viral infection and needs further studies. The SPIPm2 genomic gene was composed of seven exons and six introns. The genomic DNA segments coding for each Kazal domain were separated by introns of variable lengths supporting the hypothesis of gene duplication in the Kazal-type gene family. RT-PCR and Western blot analysis revealed that the SPIPm2 transcript and its five-domain protein product were expressed mainly in the hemocytes and less in gill, heart and antennal gland. Upon white spot syndrome virus (WSSV) infection, the SPIPm2 was only detected in the hemocytes and plasma. Immunocytochemical study of P. monodon hemocytes showed that the percentage of SPIPm2-producing hemocytes was reduced by about half after WSSV infection. Quantitative RT-PCR revealed further that the SPIPm2 was up-regulated early in the hemocytes of WSSV-infected shrimp and gradually reduced as the infection progressed. Injection of the recombinant SPIPm2 (rSPIPm2) prior to WSSV injection resulted in a significant inhibition of WSSV replication. The rSPIPm2 injection also prolonged the mortality rate of WSSV-infected shrimp. Therefore, the SPIPm2 was involved in the innate immunity against WSSV infection in shrimp.

Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus.

The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 microM and 3.5-8 microM, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.

Kazal-type serine proteinase inhibitors from the black tiger shrimp Penaeus monodon and the inhibitory activities of SPIPm4 and 5.

Serine proteinase inhibitors (SPIs) play important roles in physiological and immunological processes involving proteinases in all multicellular organisms. In black tiger shrimp Penaeus monodon, nine different Kazal-type SPIs, namely SPIPm1-9, were identified from the cDNA libraries of hemocyte, hepatopancreas, hematopoietic tissue, ovary and lymphoid organ. They are multi-domain SPIs containing 2-7 and possibly more Kazal domains. Two interesting cDNA clones, SPIPm4 and SPIPm5 coding for two-domain Kazal-type SPIs, were identified from the heat-treated hemocyte cDNA libraries. The SPIPm4 and SPIPm5 consist of open reading frames of 387 and 399 bp coding for polypeptides of 128 and 132 amino acids with putative signal peptides of 21 and 19 amino acid residues and mature SPIs of 107 and 113 amino acid residues, respectively. Recombinant expression in an Escherichia coli expression system yielded recombinant proteins, rSPIPm4 and rSPIPm5, with molecular masses of 12.862 and 13.433 kDa, respectively. The inhibitory activities of SPIPm4 and SPIPm5 were tested against trypsin, chymotrypsin, subtilisin and elastase. The SPIPm4 exhibited potent inhibitory activity against subtilisin and weakly against chymotrypsin whereas the SPIPm5 strongly inhibited subtilisin and elastase. The inhibition was a competitive type with inhibition constants (K(i)) of 14.95 nM for SPIPm4 against subtilisin, 4.19 and 59.64 nM, respectively, for SPIPm5 against subtilisin and elastase. They had no bacteriostatic effect against Gram-positive bacteria: Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, and Gram-negative bacteria: Vibrio harveyi 639, E. coli JM109. Gene expression study revealed that the SPIPm5 gene was up-regulated in response to heat treatment suggesting the involvement of SPIs in stress responses.

Domain inhibitory and bacteriostatic activities of the five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon.

Serine proteinase inhibitors (SPIs) in multi-cellular organisms are important modulators of proteinase activities in various biological processes. A five-domain Kazal-type SPI SPIPm2 from the black tiger shrimp Penaeus monodon is presumably involved in innate immune response. The SPIPm2 with the domain P1 residues T, A, E, K and E was isolated from the hemocyte cDNA libraries and found to strongly inhibit subtilisin and elastase, and weakly inhibit trypsin. To unravel further the inhibitory activity of each domain, we subcloned, over-expressed and purified each individual SPI domain. Their inhibitory specificities against trypsin, subtilisin and elastase were determined. Domain 1 was found to be inactive. Domains 2, 3 and 5 inhibited subtilisin. Domain 2 inhibited also elastase. Domain 4 weakly inhibited subtilisin and trypsin. The intact SPIPm2 inhibitor was found to possess bacteriostatic activity against the Bacillus subtilis but not the Bacillus megaterium, Staphylococcus aureus, Vibrio harveyi 639 and Escherichia coli JM109. Domains 2, 4 and 5 contributed to this bacteriostatic activity.

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.