The involvement of VEGF and VEGFR in bacterial recognition and regulation of antimicrobial peptides in Eriocheir sinensis.

Vascular endothelial growth factor (VEGF) and VEGF reporter (VEGFR) are essential molecules in VEGF signalling pathway. Although the functions of VEGF and VEGFR have been well reported in vertebrates, their functions are still poorly understood in invertebrates. In this study, the open reading frame sequences of EsVEGF1 and EsVEGFR4 were cloned from Eriocheir sinensis, and their corresponding proteins shared typical structure characteristics with their counterparts in other species. EsVEGF1 were predominantly expressed in hepatopancreas and muscle while EsVEGFR4 mainly expressed in hemocytes and intestine. The expression levels of EsVEGF1 in hemocytes were rapidly induced by Staphylococcus aureus and Vibrio parahaemolyticus, and it also increased rapidly in hepatopancreas after being challenged with V. parahaemolyticus. The expression levels of EsVEGFR4 only increased in hepatopancreas of crabs injected with S. aureus. The extracellular immunoglobulin domain of EsVEGFR4 could bind with Gram-negative and Gram-positive bacteria as well as lipopolysaccharide and peptidoglycan. EsVEGF1 could act as the ligand for EsVEGFR4 and Toll-like receptor and regulate the expression of crustins and lysozyme with a tissue-specific manner, while have no regulatory function on that of anti-lipopolysaccharide factors. This study will provide new insights into the immune defense mechanisms mediated by VEGF and VEGFR in crustaceans.

Expression characteristics and inhibitory activity of a leucine-rich repeat (LRR)-only protein in the Chinese mitten crab, Eriocheir sinensis.

The leucine-rich repeat (LRR) domain is a crucial structure in a variety of immune related proteins and displays multiple immune functions. In this study, the open reading frame (ORF) of an LRR-only protein was cloned from the Chinese mitten crab, Eriocheir sinensis (EsLRRop1). The protein sequence of EsLRRop1 contained seven LRR motifs, three LRR-TYP motifs and an LRRCT motif. Tissue distribution exhibited that EsLRRop1 mainly expressed in nervous tissues including thoracic ganglion, eyestalk and brain while showed relatively lower transcriptional level in hemocyte. Based on the above expression characteristics, the responses of EsLRRop1 to the challenge of Vibrio parahaemolyticus and Staphylococcus aureus were tested. The result showed that the transcript of EsLRRop1 in thoracic ganglion and eyestalk up-regulated after being challenged with S. aureus, while it decreased post injection with V. parahaemolyticus. The transcript of EsLRRop1 in hemocytes up-regulated sharply at 3 h and decreased at 12 h and 24 h after being challenged with V. parahaemolyticus, while it decreased at 12 h and 24 h post injection with S. aureus. The recombinant protein of EsLRRop1 (His-EsLRRop1) displayed binding activities to V. alginolyticus, V. harveyi, V. parahaemolyticus, S. aureus, Corynebacterium glutamicum and Micrococcus lysodeikticus as well as lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, the His-EsLRRop1 exhibited inhibitory activity against V. parahaemolyticus and V. harveyi with minimum inhibitory concentration (MIC) of 3.57-7.14 µM and 7.14-14.28 µM, respectively. These results provide theoretical basis for the application of EsLRRop1 in inhibiting bacteria in aquaculture practice.

The antibacterial activity and antibacterial mechanism analyses of an LRR-IG protein in the Chinese mitten crab, Eriocheir sinensis.

Leucine-rich repeat and immunoglobulin domain containing protein (LRR-IG) family is an important class of immune molecules in invertebrates. Herein, a novel LRR-IG, named as EsLRR-IG5, was identified from Eriocheir sinensis. It contained typical structures of LRR-IG including an N-terminal LRR region and three IG domains. EsLRR-IG5 was ubiquitously expressed in all the tested tissues, and its transcriptional levels increased after being challenged with Staphylococcus aureus and Vibrio parahaemolyticus. Recombinant proteins of LRR and IG domains from the EsLRR-IG5 (named as rEsLRR5 and rEsIG5) were successfully obtained. rEsLRR5 and rEsIG5 could bind to both gram-positive bacteria and gram-negative bacteria as well as lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, rEsLRR5 and rEsIG5 exhibited antibacterial activities against V. parahaemolyticus and V. alginolyticus and displayed bacterial agglutination activities against S. aureus, Corynebacterium glutamicum, Micrococcus lysodeikticus, V. parahaemolyticus and V. alginolyticus. The scanning electron microscopy (SEM) observation revealed that the membrane integrity of V. parahaemolyticus and V. alginolyticus was destroyed by rEsLRR5 and rEsIG5, which may lead to the leakage of cell contents and death. This study provided clues for further studies on the immune defense mechanism mediated by LRR-IG in crustaceans and provided candidate antibacterial agents for prevention and control of diseases in aquaculture.

HSP40 mediated TLR-Dorsal-AMPs pathway in Portunus trituberculatus.

Heat shock protein 40 (HSP40) is a kind of molecular chaperone involved in various immune responses. However, the exact roles of HSP40 in immune defense against bacteria remain largely unclear. In this study, the activation function of a type Ⅰ HSP40 from Portunus trituberculatus (PtHSP40-Ⅰ) in the TLR pathway was investigated. The results showed that PtHSP40-Ⅰ can bind to lipopolysaccharide (LPS) and peptidoglycan (PGN). The PtHSP40-Ⅰ also exhibited binding activity toward the extracellular leucine-rich repeat (LRR) domain of Toll-like receptor (TLR). Moreover, the PtHSP40-Ⅰ could promote the transcription factor Dorsal translocated from cytoplasm into the nucleus in hemocytes and participated in regulating the expression of anti-lipopolysaccharide factor (ALF) and crustin. These findings provided insights into the activation mechanisms of TLR pathway mediated by HSP40 and offered basic theory of disease control in P. trituberculatus aquaculture.

Spätzle, a signaling molecule that interacts with pathogen-associated molecules and Toll-like receptor in Portunus trituberculatus.

Spätzle is a crucial ligand for Toll-like receptor (TLR) that triggers the activation of TLR signal pathway in insects. In this study, open reading frames (ORFs) of two spätzles were cloned from Portunus trituberculatus (PtSpz1 and PtSpz2). Both of PtSpzs contained the typical cystine-knot domain of spätzle. Tissue distribution analysis showed that both of PtSpzs were predominantly expressed in the gills. Transcriptional levels of the two PtSpzs in hemocytes and gill rapidly increased at 3 h and 6 h post Vibrio alginolyticus challenge, respectively. The two PtSpzs could bind to several pathogen-associated molecules including lipopolysaccharide (LPS), peptidoglycan (PGN) and envelope proteins of white spot syndrome virus (WSSV). Moreover, the two PtSpzs could directly interact with the extracellular leucine-rich repeats (LRR) domain of TLR. This study revealed that spätzle could interact with pathogen-associated molecules and TLR of host, which may be two important steps for spätzle to deliver signals into host cells.

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.

Full-Length Transcriptome Analysis Provides New Insights Into the Diversity of Immune-Related Genes in Portunus trituberculatus.

Generally, invertebrates were thought to solely rely on their non-specific innate immune system to fight against invading microorganisms. However, increasing studies have implied that the innate immune response of invertebrates displayed diversity and specificity owing to the hyper-variable immune molecules in organisms. In order to get an insight into the diversity of immune-related genes in Portunus trituberculatus, a full-length transcriptome analysis of several immune-related tissues (hemocytes, hepatopancreas and gills) in P. trituberculatus was performed and the diversity of several immune-related genes was analyzed. The full-length transcriptome analysis of P. trituberculatus was conducted using a combination of SMRT long-read sequencing and Illumina short-read sequencing. A total of 17,433 nonredundant full-length transcripts with average length of 2,271 bp and N50 length of 2,841 bp were obtained, among which 13,978 (80.18%) transcripts were annotated. Moreover, numerous transcript variants of various immune-related genes were identified, including pattern recognition receptors, antimicrobial peptides, heat shock proteins (HSPs), antioxidant enzymes and vital molecules in prophenoloxidase (proPO)-activating system. Based on the full-length transcriptome analysis, open reading frames (ORFs) of four C-type lectins (CTLs) were cloned, and tissue distributions showed that the four CTLs were ubiquitously expressed in all the tested tissues, and mainly expressed in hepatopancreas and gills. The transcription of the four CTLs significantly increased in several immune-related tissues (hemocytes, hepatopancreas and gills) of P. trituberculatus challenged with Vibrio alginolyticus and displayed different profiles. Moreover, the four CTLs displayed distinct bacterial binding and antibacterial activities. The recombinant protein PtCTL-1 (rPtCTL-1) and rPtCTL-3 displayed bacterial binding and antibacterial activities against all tested bacteria. rPtCTL-2 only showed bacterial binding and antibacterial activities against V. alginolyticus. No obvious bacterial binding or antibacterial activities for PtCTL-4 was observed against the tested bacteria. This study enriches the transcriptomic information on P. trituberculatus and provides new insights into the innate immune system of crustaceans. Additionally, our study provided candidates of antibiotic agents for the prevention and treatment of bacteriosis.

Antibacterial activity of four recombinant carbohydrate recognition domain proteins identified from the kuruma shrimp Marsupenaeus japonicus.

The carbohydrate recognition domain (CRD) is the key component of C-type lectins (CTLs) with the capacity to recognize and eliminate invading pathogens. Herein, the recombinant proteins of four CRDs identified from the kuruma shrimp, Marsupenaeus japonicus, were produced and purified by an Escherichia coli expression system and affinity chromatography. Bacterial binding and antibacterial assays showed that the four CRDs displayed various bacterial binding and antibacterial activities against different bacteria. Among the four recombinant CRDs, His-CRD2-3 exhibited the broadest spectrum of bacterial binding and antibacterial activities against gram-negative bacteria (Vibrio parahaemolyticus, V. alginolyticus and V. harveyi) and gram-positive bacteria (Staphylococcus aureus and Micrococcus lysodeikticus). Moreover, the four recombinant CRDs showed different capacities to regulate the expression of several immune effector genes (MjCTL3, MjCTL4, MjCTL, Mjily and Mjsty), among which His-CRD2-3 displayed broader and stronger inductive effects on these immune effector genes. This study indicated that the four CRDs participated in immune defense by binding and killing bacteria and regulating the transcription of other immune effector genes. In addition, our results suggested that His-CRD2-3 might be a promising agent for the prevention and treatment of bacteriosis.