Solution structure of synthetic penaeidin-4 with structural and functional comparisons with penaeidin-3

Antimicrobial peptide structure has direct implications for the complexity of functions and mechanisms of action. The penaeidin antimicrobial peptide family from shrimp is divided into multiple class designations based on primary structure. The penaeidin classes are not only characterized by variability in primary sequence but also by variation in target specificity and effectiveness. Whereas class 4 exhibits low isoform diversity within species and is highly conserved between species, the primary sequence of penaeidin class 3 is less conserved between species and exhibits considerable isoform diversity within species. All penaeidins, regardless of class or species, are composed of two dramatically different domains: an unconstrained proline-rich domain and a disulfide bond-stabilized cysteine-rich domain. The proline-rich domain varies in length and is generally less conserved, whereas the spacing and specific residue content of the cysteine-rich domain is more conserved. The structure of the synthetic penaeidin class 4 (PEN4-1) from Litopenaeus setiferus was analyzed using several approaches, including chemical mapping of disulfide bonds, circular dichroism analysis of secondary structural characteristics, and complete characterization of the solution structure of the peptide by proton NMR. L. setiferus PEN4-1 was then compared with the previously characterized structure of penaeidin class 3 from Litopenaeus vannamei. Moreover, the specificity of these antimicrobial peptides was examined through direct comparison of activity against a panel of microbes. The penaeidin classes differ in microbial target specificity, which correlates to variability in specific domain sequence. However, the tertiary structure of the cysteine-rich domain and indeed the overall structure of penaeidins are conserved across classes.     

Molecular Characterization of a Newly Identified Subfamily Member of Penaeidin from two Penaeid Shrimps, <Emphasis Type="Italic">Fenneropenaeus indicus</Emphasis> and <Emphasis Type="Italic">Metapenaeus monoceros</Emphasis>

Penaeidins are a major group of antimicrobial peptides found in penaeid shrimps. This study reports a new isoform of penaeidin from the hemocytes of Indian white shrimp, Fenneropenaeus indicus (Fi-PEN, JX657680), and the pink shrimp, Metapenaeus monoceros (Mm-PEN, KF275674). Mm-PEN is also the first antimicrobial peptide to be identified from M. monoceros. The complete coding sequences of the newly identified Fi-PEN and Mm-PEN consisted of an ORF of 338 bp encoding 71 amino acids with a predicted molecular weight of 5.66 kDa and a pI of 9.38. The penaeidins had its characteristic signal peptide region (19 amino acids), which was followed by a mature peptide with a proline-rich domain (24 amino acids) at the N-terminal region and a cysteine-rich domain (28 amino acids) at the C-terminal region, designating it to penaeidin-3 subgroup. Structural analysis revealed an alpha-helix in its secondary structure and an extended structure at the proline-rich domain. The newly identified penaeidin isoform showed maximum similarity of 63 % to a penaeidin-3 isoform of P. monodon, which further proves it to be a new isoform. Phylogenetic analysis showed that it possessed similar evolutionary status like other penaeidins, which has subsequently diverged at different phases of evolution. The wide distribution of penaeidins in penaeid shrimps indicates the importance of these AMPs in the innate immunity.     

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp.

Penaeidins are 5.5- to 6.6-kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline-rich N-terminus and of a C-terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen-2 and Pen-3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen-2 and -3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen-2 and -3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram-positive bacteria, with a strain-specific inhibition mechanism. Despite a better efficiency of Pen-3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen-2 and -3a. Finally, no synergistic effect could be observed between the two molecules.     

Sampling and evaluation of white spot syndrome virus in commercially important Atlantic penaeid shrimp stocks

In 1997, white spot syndrome virus (WSSV) was discovered in shrimp culture facilities in South Carolina, USA. This disease was known to cause devastating mortalities in cultured populations in Southeast Asia and prompted concern for the health of wild populations in the USA. Our study surveyed wild shrimp populations for the presence of WSSV by utilizing molecular diagnostics and bioassay techniques. A total of 1150 individuals (586 Litopenaeus setiferus, 477 Farfantepenaeus aztecus and 87 F. dourarum) were examined for the presence of WSSV DNA by PCR. A total of 32 individuals tested positive and were used in a bioassay to examine the transmission of disease to healthy individuals of the culture species L. vannamei. DNA sequencing of PCR products from a positive individual confirmed that the positive individuals carried WSSV DNA. Significant mortalities were seen in test shrimp injected with tissue extracts from heavily infected wild shrimp. These data confirm the existence of WSSV in wild shrimp stocks along the Atlantic Coast and that the virus can cause mortalities in cultured stocks.     

Epidemiological parameters of White Spot Syndrome Virus infections in Litopenaeus vannamei and L. setiferus

An experimental protocol based on a mathematical epidemiology model was developed to study the transmission, virulence, and recovery rates of White Spot Syndrome Virus (WSSV). Two modes of transmission were compared for WSSV in Litopenaeus vannamei. We compared transmission by ingestion of infected cadavers to transmission by cohabitation with infected animals. In addition, we compared the ingestion transmission of WSSV in L. vannamei and in L. setiferus. Finally, we compared the virulence and recovery rates of WSSV in L. vannamei and L. setiferus. The transmission rate of WSSV to L. vannamei by cohabitation was 0.01. The transmission rate by ingestion of infected cadavers was over an order of magnitude larger at 0.46, suggesting that cohabitation is a much less important mode of transmission for WSSV. A statistically significant difference was detected between the estimates of ingestion transmission of L. vannamei (0.46) and those of L. setiferus (0.84), yet no differences in the virulence or recovery rates were detected between hosts. The overall estimated virulence rate was 0.34, and the overall estimated recovery rate from a WSSV infection was 0.007 for both species. According to epidemiological theory the threshold density of hosts necessary for an epidemic to occur is directly related to the virulence and recovery rates and inversely related to the transmission rate. Therefore, the epidemic threshold density may be lower for ingestion transmission than cohabitation transmission and lower for L. setiferus than for L. vannamei.     

Increased tolerance of Litopenaeus vannamei to white spot syndrome virus (WSSV) infection after oral application of the viral envelope protein VP28

It has been generally accepted that invertebrates such as shrimp do not have an adaptive immune response system comparable to that of vertebrates. However, in the last few years, several studies have suggested the existence of such a response in invertebrates. In one of these studies, the shrimp Penaeus monodon showed increased protection against white spot syndrome virus (WSSV) using a recombinant VP28 envelope protein of WSSV. In an effort to further investigate whether this increased protection is limited to P. monodon or can be extended to other penaeid shrimp, experiments were performed using the Pacific white shrimp Litopenaeus vannamei. As found with P. monodon, a significantly lower cumulative mortality for VP28-fed shrimp was found compared to the controls. These experiments demonstrate that there is potential to use oral application of specific proteins to protect the 2 most important cultured shrimp species, P. monodon and L. vannamei, against WSSV. Most likely, this increased protection is based on a shared and, therefore, general defence mechanism present in all shrimp species. This makes the design of intervention strategies against pathogens based on defined proteins a viable option for shrimp culture.     

Solution structure of the recombinant penaeidin-3, a shrimp antimicrobial peptide

Penaeidins are a family of antimicrobial peptides of 47-63 residues isolated from several species of shrimp. These peptides display a proline-rich domain (N-terminal part) and a cysteine-rich domain (C-terminal part) stabilized by three conserved disulfide bonds whose arrangement has not yet been characterized. The recombinant penaeidin-3a of Litopenaeus vannamei (63 residues) and its [T8A]-Pen-3a analogue were produced in Saccharomyces cerevisiae and showed similar antimicrobial activity. The solution structure of the [T8A]-Pen-3a analogue was determined by using two-dimensional 1H NMR and simulated annealing calculations. The proline-rich domain, spanning residues 1-28 was found to be unconstrained. In contrast, the cysteine-rich domain, spanning residues 29-58, displays a well defined structure, which consists of an amphipathic helix (41-50) linked to the upstream and the downstream coils by two disulfide bonds (Cys32-Cys47 and Cys48-Cys55). These two coils are in turn linked together by the third disulfide bond (Cys36-Cys54). Such a disulfide bond packing, which is in agreement with the analysis of trypsin digests by ESI-MS, contributes to the highly hydrophobic core. Side chains of Arg45 and Arg50, which belong to the helix, and side chains of Arg37 and Arg53, which belong to the upstream and the downstream coils, are located in two opposite parts of this globular and compact structure. The environment of these positively charged residues, either by hydrophobic clusters at the surface of the cysteine-rich domain or by sequential hydrophobic residues in the unconstrained proline-rich domain, gives rise to the amphipathic character required for antimicrobial peptides. We hypothesize that the antimicrobial activity of penaeidins can be explained by a cooperative effect between the proline-rich and cysteine-rich features simultaneously present in their sequences.     

Tiger shrimp (Penaeus monodon) penaeidin possesses cytokine features to promote integrin-mediated granulocyte and semi-granulocyte adhesion

Recent studies indicated that antimicrobial peptides (AMPs) play multiple roles in both innate and adaptive immune functions. The penaeidin of tiger shrimp Penaeus monodon shows an antimicrobial activity against Gram-positive bacteria and filamentous fungi. To study immunomodulation functions of the penaeidin, we transfected shrimp hemocytes in primary culture with penaeidin-specific small interfering RNA (siRNA-3) and observed a concomitant 20% reduction in adhesive hemocytes compared with mock-transfected cells. The addition of biosynthesized or chemically synthesized penaeidin or penaeidin proline-rich domain (PRD) to the culture medium of penaeidin knock-down hemocytes led to a full recovery in the number of adhesive hemocytes. The effect of penaeidin knock-down on the expression of tiger shrimp cell adhesion-associated molecules was examined using real-time Q-PCR. Results demonstrated 91% and 64% decreases in the expression of integrin-β and collagen, respectively, and a 396% increase in the expression of collagenase. The addition of chemically synthesized penaeidin after penaeidin knock-down hemocytes normalized the expression of these genes. The addition of the integrin-β ligand competitor RGDS to mock-transfected hemocytes decreased the number of adhesive hemocytes similar to penaeidin knock-down. In conclusion, penaeidin possesses an integrin-β-mediated cytokine feature that promotes shrimp granulocyte and semi-granulocyte adhesion. This is the first report about functional shrimp cytokine.     

Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1h after the rLV-CHH injection and then returned to normal after 3h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.