Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages

A penaeidin family, mo-penaeidin was cloned from the haemocytes of tiger shrimp Penaeus monodon using genomic polymerase chain reaction (PCR) by gene specific primers. Analysis of nucleotide sequence revealed that this mo-penaeidin consists of 1348 bp containing one intron (680 bp) and two exons (210 and 458 bp). It has an open reading frame (ORF) of 222 p, which encodes a protein of 74 amino acids including a signal peptide of 19 amino acids. The calculated molecular mass of the mature protein (55 amino acids) is 6.059 kDa with an estimated pI of 9.3. The deduced amino acid sequence of mo-penaeidin has similarity to that of penaeidin from Fenneropenaeus chinensis (73%), Farfantepenaeus paulensis (66%), Litopenaeus schmitti (53-67%), L. stylirostris (50-67%), L. setiferus (50-62%), L. vannamei (44-66%), and Marsupenaeus japonicus (33%), respectively. Phylogenetic tree analysis indicated that penaeidin (including mo-penaeidin, penaeidin, and penaeidin 5, 2, 3k, 3c1) of P. monodon is distinct from penaeidin 1, penaeidin 2, penaeidin 3 and penaeidin 4 of other penaeid shrimps. The mo-penaeidin mRNA was detected in various tissues including ovary and mandibular organ. The mo-penaeidin mRNA was present in one cell to postlarva stage with higher level at nauplius I (9h post hatching) and higher expression during the intermoult stage indicating an early innate immunity and different immunity at moulting stage.     

PenBase, the shrimp antimicrobial peptide penaeidin database: sequence-based classification and recommended nomenclature

Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at , has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature.     

Characterization and expression of a new subfamily member of penaeidin antimicrobial peptides (penaeidin 5) from Fenneropenaeus chinensis

Penaeidins are members of a special family of antimicrobial peptides existing in penaeid shrimp and play an important role in the immunological defence of shrimp. Here, we report one penaeidin with a putative isotype newly cloned from fleshy prawn Fenneropenaeus chinensis. The penaeidin open reading frame encodes a 79 amino acid peptide while two exons and an intron were identified within the 1126bp genomic sequence of Fenchi-penaeidin 5. Phylogenetic analysis and sequence comparison with other known penaeidins suggest the new gene belongs to a novel subfamily of penaeidins and the two isoforms were named Fenchi-penaeidin 5-1 and 5-2, respectively. Fenchi-penaeidin 5 mRNA was examined in normal and microbial challenged shrimp and was found to be constitutively expressed in heamocytes, heart, gill, intestine and ovary. Bacterial challenge resulted in mRNA up-regulation, inducing expression in hepatopancreas and stomach. Fenchi-penaeidin 5-1 was also expressed in Pichia pastoris, and recombinant Fenchi-penaeidin 5-1 exhibited activities against Gram-positive and -negative bacteria and fungi.     

Multiplication of Taura syndrome virus in primary hemocyte culture of shrimp (Penaeus vannamei)

The propagation of Taura syndrome virus (TSV) in primary hemocyte culture of Pacific white shrimp (Penaeus vannamei) was investigated. Purified TSV was inoculated into a 24 h old primary hemocyte culture and the development of cytopathic effects was monitored. The cell morphology started changing within 6 h post-inoculation; TSV-infected hemocytes started shrinking and granular structures began to form on the cell surface. There was a gradual loss of cell viability and, by 48 h post-inoculation, most cells detached from the bottom of the 96 well microplate. The propagation of TSV during the 48 h time course studied was measured by real-time RT-PCR. TSV copy number reached the highest level by 12 h post-inoculation and then started to decrease. Using an anti-TSV polyclonal antibody, the 55 kDa VP1 capsid protein was detected by Western blot analysis. The data suggest that shrimp primary hemocyte culture supports TSV replication and could be used as a tool for the study of host-virus interactions in TSV pathogenesis.     

Diversity in penaeidin antimicrobial peptide form and function

Penaeidins are a diverse family of two-domain antimicrobial peptides expressed in shrimp. Variation in penaeidin sequence results in functional diversity, which was discovered using synthetic reproductions of native penaeidins. An isoform of penaeidin class 3 from Litopenaeus setiferus (Litset Pen3-4) was synthesized using native ligation and compared directly with the synthetic penaeidin class 4 known to be expressed in the same organism. New antimicrobial activity data are included in this review that emphasize differences in effectiveness that are apparent from a direct comparison of two classes. A novel approach to intact penaeidin analysis is presented in the form of Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry, which has implications for the identification of individual penaeidin isoforms without chemical modification or enzymatic cleavage. The new information included in this review helps gather the perspective on relevance of penaeidin diversity to antimicrobial function, the use of synthetic peptides as tools to evaluate specific immune functions and the application of high mass resolution, top-down sequencing methods to the intact analysis of individual penaeidin isoforms.     

Expression and regulation of antimicrobial peptides in the gastrointestinal tract

The gastrointestinal (GI) tract is exposed to a wide range of microorganisms. The expression of antimicrobial peptides has been demonstrated in different regions of the GI tract, predominantly in epithelial cells, which represent the first host cells with which the microorganisms have to interact for invasion. The intestinal epithelial monolayer is complex, consisting of different cell types, and most have a limited lifespan. Of the GI antimicrobial peptides, alpha- and beta-defensins have been studied the most and are expressed by distinct types of epithelial cells. Enteric alpha-defensin expression is normally restricted to Paneth and intermediate cells in the small intestine. However, there are important differences between mice and humans in the processing of the precursor forms of enteric alpha-defensins. Parasite infection induces an increase in the number of enteric alpha-defensin-expressing Paneth and intermediate cells in the murine small intestine. In the chronically inflamed colonic mucosa, metaplastic Paneth cells (which are absent in the normal colon) also express enteric alpha-defensins. Epithelial expression of beta-defensins may be constitutive or inducible by infectious and inflammatory stimuli. The production of some members of the beta-defensin family appears to be restricted to distinct parts of the GI tract. Recent studies using genetically manipulated rodents have demonstrated the likely in vivo importance of enteric antimicrobial peptides in innate host defense against microorganisms. The ability of these peptides to act as chemoattractants for cells of the innate- and adaptive-immune system may also play an important role in perpetuating chronic inflammation in the GI tract.     

Expression and regulation of antimicrobial peptides in articular joints

Articular joint infection is a surprisingly rare event considering the frequency of joint arthrocentesis and other invasive procedures applied to limb joints. This observation led us to the hypothesis that a local “chemical shield” in the form of antimicrobial proteins provides synovial membrane and articular cartilage with resistance to infection. We subsequently began a systematic analysis of in vitro and in vivo antimicrobially active proteins in healthy articular joints and in disease states such as pyogenic arthritis, rheumatoid arthritis, and osteoarthritis. An anatomical approach with systematic characterization combined with antimicrobial testing revealed expression and production of human antibiotic peptides and proteins. In this review, we focus on the most prominent antimicrobial proteins in articular joints, which we have identified as lysozyme, lactoferrin, secretory phospholipase A₂, RNase 7, CAP37, the cathelicidin LL37, and especially the human beta-defensin-2 and -3 (HBD-2/-3). Activation pathways and possible antimicrobial functions are discussed and the involvement in non-antimicrobial processes such as tissue remodelling is also considered.     

A new subfamily of penaeidin with an additional serine-rich region from kuruma shrimp (Marsupenaeus japonicus) contributes to antimicrobial and phagocytic activities

Penaeidins are an important family of antimicrobial peptides (AMPs) in penaeid shrimp. To date, five groups of penaeidins have been identified in penaeid shrimp. All are composed of a proline-rich N-terminus and a C-terminus containing six cysteine residues engaged in three disulfide bridges. In this study, a new type of penaeidin from Marsupenaeus japonicus was identified. The full-length penaeidin contains a unique serine-rich region and a penaeidin domain, which consists of a proline-rich region and a cysteine-rich region. Here, we classify all penaeidins into two subfamilies. All reported penaeidins are in subfamily I, and the new penaeidin identified in M. japonicus is designated as Penaeidin subfamily II (MjPen-II). MjPen-II was expressed in hemocytes, heart, hepatopancreas, gills, stomach and intestine, and was upregulated after bacterial challenge. A liquid bacteriostatic assay showed that MjPen-II had antibacterial activity to some Gram-positive and Gram-negative bacteria. MjPen-II could bind to bacteria by binding to polysaccharides on the surface of bacteria, thus promoting bacterial agglutination. The serine-rich region enhanced the agglutination activity of MjPen-II. The proline-rich domain had a stronger bacterial-binding activity and polysaccharide-binding activity than the cysteine-rich domain. MjPen-II was also found to be involved in the phagocytosis of bacteria and efficiently improved the phagocytosis rate. Therefore, MjPen-II eliminates bacteria through direct bacterial inhibition as well as by promoting phagocytosis in shrimp.     

A comparative study of antimicrobial properties of crustinPm1 and crustinPm7 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 crustinPm1 and crustinPm7, the two most abundant crustin isoforms found in the haemocyte of P. monodon. Previously, crustinPm1 showed strong inhibition against Gram-positive bacteria, whilst crustinPm7 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 crustinPm7. Meanwhile, crustinPm1 can cause a visible change on the cell surface of S. aureus and E. coli only. This is in agreement with the fact that crustinPm1 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.     

Evidences of abundant hemocyanin variants in shrimp Litopenaeus vannamei

Hemocyanin (HMC) is a multifunctional immune molecule present in mollusks and arthropods and functions as an important antigen non-specific immune protein. Our previous evidences demonstrated that Litopenaeus vannamei HMC might display extensive molecular diversities. In this study, bioinformatics analysis showed dozens of variant sequences of the HMC subunit with higher molecular weight from L. vannamei (LvHMC). Three variant fragments, named as LvHMCV1-3, which shared 85–99% nucleotide identity with that of the classical form of LvHMC (AJ250830.1), were cloned and characterized. Spatial expression profiles showed that LvHMCV1-3 had different tissue-specific distribution, which were affected by stimulation with six pathogenic bacteria, including Escherichia coli K12, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio fluvialis, Streptococcus pyogenes and Staphylococcus aureus, with each variant fragment showing a specific stress pattern to different bacterial pathogens. Full length cDNA of LvHMCV3 was further cloned and characterized. The deduced amino acid sequence shared 92% identity with that of LvHMC, possessed a conserved structure characteristic of the HMC family and could be clustered into one branch along with other arthropod HMC in a phylogenetic tree. In addition, the recombinant protein of LvHMCV3 (rLvHMCV3) showed obvious agglutination activities against three aquaculture pathogenic bacteria including E. coli K12, V. parahaemolyticus and S.aureus at concentrations ranging from 31.25–62.5 g/mL. It also showed obvious antibacterial activity against V. parahaemolyticus at concentrations 0.02–0.5 mg/mL, and possessed the best inhibitive effects compared with those of rLvHMCV4 and rLvHMC. Co-injection of V. parahaemolyticus and rLvHMCV3 in L. vannamei showed significant decrease of the mortality rate at 24–72 h after injection. Therefore, these studies suggested that L. vannamei had abundant HMC variants, which possessed obvious resistance to pathogenic infection and might specifically target on different pathogens in shrimp.     

皮肤抗菌肽的研究进展

抗菌肽是机体固有免疫的重要组成部分,人类皮肤抗菌肽主要包括cathelicidins家族和β-防御素家族,广泛表达于皮肤角质形成细胞及各种炎症细胞.皮肤抗菌肽除了直接杀菌作用外,还能调节免疫反应,促进伤口愈合和血管新生,其表达受到体内,体外多种因素的影响,与各种皮肤炎症性疾病发病密切相关,也可能在糖尿病创面愈合中发挥着重要作用.     

Litopenaeus vannamei Toll-interacting protein (LvTollip) is a potential negative regulator of the shrimp Toll pathway involved in the regulation of the shrimp antimicrobial peptide gene penaeidin-4 (PEN4)

The Toll-like receptor (TLR)-nuclear factor (NF)-κB signaling pathway is evolutionarily conserved from insects to mammals as a regulator of the expression of immune-related genes. In mammals, TLR-NF-κB signaling is tightly controlled because excessive activation of this pathway can result in severe damage to the host. The mammalian Toll-interacting protein (Tollip) has an important function in the negative regulation of this pathway, but no reports about invertebrate Tollip have been published to date. In this study, we cloned Litopenaeus vannamei Tollip (LvTollip) and investigated its function in the regulation of the NF-κB pathway-controlled antimicrobial peptide genes (AMPs). The LvTollip full-length cDNA is 1231 bp long and contains an open reading frame of 813 bp that encodes a 270-amino acid protein. LvTollip shares significant similarities to mammalian Tollips, which contain a centrally localized protein kinase C conserved region 2 (C2) domain and a C-terminal CUE domain. After challenges with the white spot syndrome virus (WSSV) or Vibrio alginolyticus, the expression levels of LvTollip were altered in the gill, hemocyte, hepatopancreatic, intestinal, and muscle tissues. In Drosophila S2 cells, LvTollip localized in the membrane and the cytoplasm and significantly inhibited the promoter activities of the NF-κB pathway-controlled AMP penaeidin-4 (PEN4). In LvTollip-knockdown shrimp, the expression level of AMP PEN4 was increased. However, the mortality rates of LvTollip-knockdown shrimp in response to WSSV or V. alginolyticus infections were not significantly different from those of the control group. Our results suggested that LvTollip might be involved in the negative regulation of PEN4 and that LvTollip expression was responsive to microbial infections.     

Inhibition of Penaeus monodon densovirus replication in shrimp by double-stranded RNA

Stunted shrimp caused by Penaeus monodon densovirus (PmDNV) infection is one of the main problems leading to a significant economic loss in Thailand. To control this pandemic disease, a double-stranded-RNA-mediated virus-specific gene silencing approach was applied to inhibit viral replication. In this study, two dsRNAs corresponding to the non-structural protein (ns1) and the structural protein (vp) genes of PmDNV were synthesized and introduced into shrimp haemolymph prior to viral challenge. After allowing viral replication for two weeks, the suppression effect by each dsRNA was evaluated by semi-quantitative PCR and compared with the control. A reduction of PmDNV in shrimp treated with each dsRNA was observed. In contrast, a high level of viral infection was detected in the control group (NaCl). Based on a limited sample number, we reached the tentative conclusion that virus-specific dsRNA can inhibit PmDNV replication, in which the dsRNA-ns1was more effective than the dsRNA-vp.     

Localization of the excitable membrane in the myelinated giant fiber of shrimp Penaeus japonicus

A significant DC-potential was recorded from the axon of shrimp myelinated giant nerve fiber, using a dye-filled microelectrode. Tracing of the dye assured that the source of the potential was the axonal membrane. All-or-none action potentials were obtained in the same preparation from which the ends of the axon were removed except for the synaptic region. It was concluded that the functionally excitable membrane is localized only in the synaptic region.