Identification of a crustacean ß-1,3-glucanase related protein as a pattern recognition protein in antibacterial response.

Prophenoloxidase (proPO) activating system is an important immune response for arthropods. ß-1, 3-glucanase related protein (previously named as lipopolysaccharide and ß-1, 3-glucan binding protein (LGBP) in crustaceans) is a typical pattern recognition receptor family involved in the proPO activation by recognizing the invading microbes. In this study, we pay special attention to a bacteria-induced ß-1,3-glucanase related protein from red swamp crayfish Procambarus clarkii, an important aquaculture specie in China. This protein, designated PcBGRP, was found a typical member of crustacean BGRP family with the glucanase-related domain and the characteristic motifs. PcBGRP was expressed in hemcoyes and hepatopancreas, and its expression could be induced by the carbohydrate and bacteria stimulants. The induction by lipopolysaccharide (LPS) and ß-1,3-glucan (ßG) was more significant than by peptidoglycan (PG). The response of PcBGRP to the native Gram-negative bacterial pathogen Aeromonas hydrophila was more obvious than to Gram-positive bacteria. Using RNA interference and recombinant protein, PcBGRP was found to protect crayfish from A. hydrophila infection revealed by the survival test and morphological analysis. A mechanism study found PcBGRP could bind LPS and ßG in a dose-dependent manner, and the LPS recognizing ability determined the Gram-negative bacterium binding activity of PcBGRP. PcBGRP was found to enhance the PO activation both in vitro and in vivo, and the protective role was related to the PO activating ability of PcBGRP. This study emphasized the role of BGRP family in crustacean immune response, and provided new insight to the immunity of red swamp crayfish which suffered serious disease during the aquaculture in China.

Identification and characterization of two arasin-like peptides in red swamp crayfish Procambarus clarkii.

Antimicrobial peptides (AMPs) are small effectors in host defense by directly targeting microorganisms or by indirectly modulating immune responses. In the present study, two arasin like AMPs, named as Pc-arasin1 and Pc-arasin2, were identified in red swamp crayfish Procambarus clarkii with sequence similarity to the arasins found in Hyas araneus. Both Pc-arasins consisted of signal peptide, N-terminal proline-rich region and C-terminal region containing four conserved cysteine residues. The similarity of two Pc-arasins was 44.44%, and Pc-arasin2 contained several additional residues in the N-terminus. Multiple alignment of arasin family suggested the conservation of the C-terminus and the variation of the N-terminus of Pc-arasins. Both AMPs were found hemocytes-specific, and the expression could be induced the challenge of bacteria, espeacially by the pathogenic bacterium Aeromonas hydrophila. Knockdown of each Pc-arasin expression by double strand RNA would suppress the host immunity against A. hydrophila, and the commercially synthetic Pc-arasins could rescue the knockdown consequence. Both synthetic peptide showed broad antimicrobial activity towards 3 Gram-positive bacterium and 3 Gram-negative bacterium, and the minimal inhibitory concentrations varied from 6.25 µM to 50 µM. These results presented new data about the sequence, expression and function of arasin family, and emphasized the role of this family in host immune response against bacterial pathogens. The characterization of Pc-arasins also provided potential of therapeutic agent development for disease control in aquaculture based on these two newly identified AMPs.

Three STATs are involved in the regulation of the expression of antimicrobial peptides in the triangle sail mussel, Hyriopsis cumingii.

Janus kinase (Jak) and signal transducers and activators of transcription (STAT) signaling pathway is associated in antiviral and antibacterial immune response. Previous studies primarily investigated the function of STATs in mammals. For most invertebrates, only one STAT was found in each species, such as STAT92E was found in Drosophila melanogaster. The studies, which focus on the functional difference between various STATs in the same species of invertebrate, are limited. In the present study, three STATs (HcSTAT1, HcSTAT2 and HcSTAT3) were identified in triangle shell pearl mussel, Hyriopsis cumingii. Phylogenetic analysis showed that HcSTAT1 and HcSTAT3 were clustered with Homo sapiens STAT5, and HcSTAT2 was clustered with Pinctada fucata STAT and Crassostea gigas STAT6. All three STATs could be detected in all tested tissues (hemocytes, hepatopancreas, gill, mantle and foot), and were induced expression when challenged with Staphylococcus aureus or Aeromonas hydrophilia in hemocytes and hepatopancreas. HcSTAT1 regulated the expression of HcDef, HcWAP, HcThe and HcTNF. The expression of HcWAP and HcTNF was down-regulated in HcSTAT2-RNAi mussel. And HcSTAT3 affected the expression of HcTNF. The study is the first report of different functions in antibacterial immune responses between STATs in mollusks.

The Function and Modification of Human Defensin 5.

The antibacterial and antiviral functions of human defensin 5 lay the foundation for its role as a core host protective component. In addition, HD5 also has the function of inhibiting tumor proliferation and immune regulation. However, everything has two sides; cytotoxic and proinflammatory properties may exist, while HD5 performs physiological functions. Accordingly, the modification and engineering of HD5 are particularly important. Therefore, this review summarizes the role of HD5 in various aspects of host defense, as well as modification of HD5 to ameliorate the biological activity, with a view to promoting the clinical use of HD5.

Steroid hormone 20-hydroxyecdysone promotes CTL1-mediated cellular immunity in Helicoverpa armigera.

Mermithid nematodes, such as Ovomermis sinensis, are used as biological control agents against many insect pests, including cotton bollworm (Helicoverpa armigera). However, given the host's robust immune system, the infection rate of O. sinensis is low, thus restricting its widespread use. To understand the host defense mechanisms against mermithid nematodes, we identified and characterized a protein involved in the recognition of O. sinensis, the potential O. sinensis-binding protein C-type lectin 1 (HaCTL1a and/or HaCTL1b), which was eluted from the surface of O. sinensis after incubation with H. armigera plasma. HaCTL1b is homologous to the previously reported HaCTL1a protein. HaCTL1 was predominantly expressed in hemocytes and was induced by the steroid hormone 20-hydroxyecdysone through ecdysone receptor (HaEcR) or ultraspiracle (HaUSP), or both. Binding assays confirmed the interactions of the HaCTL1 proteins with O. sinensis but not with Romanomermis wuchangensis, a parasitic nematode of mosquito. Moreover, the HaCTL1 proteins were secreted into the hemocoel and promoted hemocyte-mediated encapsulation and phagocytosis. A knockdown of HaEcR and/or HaUSP resulted in compromised encapsulation and phagocytosis. Thus, HaCTL1 appears to modulate cellular immunity in the defense against parasitic nematodes, and the 20-hydroxyecdysone-HaEcR-HaUSP complex is involved in regulating the process.

Molecular cloning and characterization of a C-type lectin from the cotton bollworm, Helicoverpa armigera.

C-type lectins participate in pathogen recognition and other defense responses in innate immunity as well as in cell-cell interactions. A new cDNA encoding a 335-residue polypeptide containing two tandem C-type lectin domains was cloned from the haemocytes of Helicoverpa armigera (Ha-lectin). Northern hybridizations revealed that the mRNA of Ha-lectin was expressed constitutively in haemocytes, and was up-regulated following injections of bacteria, yeast, or virus. Ha-lectin expression was also induced in the fat body when larvae were injected with bacteria, yeast or 20-hydroxyecdysone and a non-steroidal ecdysone agonist, RH-2485. The Ha-lectin was detected in granular haemocytes. The recombinant protein (rHa-lectin) expressed in Escherichia coli had hemagglutinating and sugar-binding activities. The native Ha-lectin protein was identified in haemocytes and plasma using a polyclonal antiserum raised against rHa-lectin by immunoblotting techniques. All together, our results suggest that the Ha-lectin gene is involved in innate immunity, and may also participate in the molting process.

Identification of genes differentially expressed during larval molting and metamorphosis of Helicoverpa armigera.

BACKGROUND: Larval molting and metamorphosis are important physiological processes in the life cycle of the holometabolous insect. We used suppression subtractive hybridization (SSH) to identify genes differentially expressed during larval molting and metamorphosis. RESULTS: We performed SSH between tissues from a variety of developmental stages, including molting 5th and feeding 6th instar larvae, metamorphically committed and feeding 5th instar larvae, and feeding 5th instar and metamorphically committed larvae. One hundred expressed sequence tags (ESTs) were identified and included 73 putative genes with similarity to known genes, and 27 unknown ESTs. SSH results were further characterized by dot blot, Northern blot, and RT-PCR. The expression levels of eleven genes were found to change during larval molting or metamorphosis, suggesting a functional role during these processes. CONCLUSION: These results provide a new set of genes expressed specifically during larval molt or metamorphosis that are candidates for further studies into the regulatory mechanisms of those stage-specific genes during larval molt and metamorphosis.

Higher methylation in genomic DNA indicates incomplete reprogramming in induced pluripotent stem cells.

Pluripotent stem cells can be created successfully through the inner cell mass (ICM), nuclear transfer, and defined-factor induction. Unfortunately, the epigenetic characteristics of the cells produced are poorly understood. In this article, we compared expression levels of enzymes involved in epigenetic modifications across six pluripotent stem cell lines. Six of the 11 genes evaluated here (Dnmt3a, Dnmt3b, Tet1, Ezh2, Mll1, and Lsd1) showed abnormally low levels of expression in the two germ-line chimeric induced pluripotent stem cell (iPSC) lines. We also conducted locus-specific analysis of DNA methylation at 9 loci. Although iPSCs did express Oct4, the Oct4 promoter region was shown to have a higher level of DNA methylation. The Xist and Line-1 repeating sequences differed relatively little in methylation level across the cell lines, but Peg3, Peg10, and H19 exhibited high degrees of variation in the pattern of DNA methylation. Meg3 in the Dlk1-Dio3 imprinting cluster was incompletely methylated in embryonic stem cells (ESCs) and nuclear transfer (nt) ESCs. However, in germ-line chimeric iPSCs, Meg3 was almost entirely methylated. ESC and ntESC lines showed twice as much Meg3 expression than in the iPSC lines. The genomic 5mC contents detected by reverse-phase high-performance liquid chromatography (HPLC) indicated that, despite their germ-line chimeric abilities, iPSCs remained incompletely reprogrammed, even though no direct evidence is shown here.

Molecular cloning and characterization of Hearm caspase-1 from Helicoverpa armigera.

Members of the caspase family play a central and evolutionary role in programmed cell death (PCD), which removes unwanted, damaged and dangerous cells during development to maintain homeostasis. In this paper, we describe the cloning and characterization of a caspase from the cotton bollworm, Helicoverpa armigera, named Hearm caspase-1. The 1,350 bp full-length cDNA contains an 885 bp open reading frame (ORF) that encodes a Hearm caspase-1 proenzyme of 294 amino acids. The deduced protein is highly homologous to Spodoptera frugiperda Sf caspase-1 and Drosophila melanogaster ICE and has the highly conserved pentapeptide QACQG, the recognized catalytic site of caspases, suggesting that it is an effector caspase of the cotton bollworm. Northern blot and RT-PCR analyses demonstrate that Hearm caspase-1 is expressed in embryos and the fat body, midgut and haemocytes of feeding and wandering larvae. Expression of Hearm caspase-1 in the haemocytes appears to be correlated with the pulse of ecdysone, and it is up-regulated by ecdysone agonist RH-2485, implying that Hearm caspase-1 activation is regulated by ecdysone.