Anti-lipopolysaccharide Factor Isoform Research Articles

Integration of metabolomics and transcriptomics reveals the toxicological mechanism of deltamethrin exposure in Chinese mitten crab Eriocheir sinensis

This study investigated the toxicological mechanism of deltamethrin on Chinese mitten crab Eriocheir sinensis juveniles in fresh water. We first conducted an acute toxicity test, followed by laboratory methods to detect changes in immune-related indices in terms of antioxidant enzyme markers, lipid metabolism-related genes, and autophagy-related and apoptosis genes. The acute toxicity (96-h LC50) of deltamethrin to E. sinensis was 7.195 μg/L. After 48 h of exposure, serum showed elevated immune-related indices (P < 0.05) for alkaline phosphatase (AKP), acid phosphatase (ACP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), complement components C3 and C4, and the key pro-inflammatory cytokines interleukin-6, interleukin-1β, and tumor necrosis factor alpha (TNF-α). In hepatopancreas at 48 h, indicators related to the antioxidant system, namely superoxide dismutase (SOD) and glutathione (GSH), were significantly elevated, whereas nitric oxide and total antioxidant capacity (T-AOC) were decreased (P < 0.05). In contrast, lipid metabolism indices for triglyceride (TG), total cholesterol (TC), and malondialdehyde (MDA) were increased (P < 0.05). Transcriptomics and metabolomics revealed that exposure to deltamethrin disrupted the lipid metabolic process in the hepatopancreas mainly by altering fatty acid synthesis, amino acid metabolism, immune signaling, and autophagy activation, while the exposure increased the content of phospholipids and cholesterol but decreased the levels of amino acids and palmitoleic acid. Quantitative genetics revealed significantly aberrantly expressed (P < 0.05) lipid metabolism-related genes, including acc1, fasn, scd1, and pnpla2, all key genes involved in lipid accumulation. Deltamethrin exposure also significantly altered (P < 0.05) gene expression levels for Toll-like receptor (tlr), myeloid differentiation factor 88 (myd88), crustin1, anti-lipopolysaccharide factor isoform 3 (alf3), tumor necrosis factor alpha (tnf-α), and NF-κB transcription factor relish. Furthermore, deltamethrin activated the toll-like receptor/major myeloid differentiation response gene 88/nuclear factor kappa-light-chain-enhancer of activated B cells (TLR/MyD88/NF-kB) signaling pathway, which activates a nonspecific immune response in E. sinensis. Additionally, carnitine palmitoyltransferase 1 A (cpt1a), cytochrome c (cyt-c), adenosine 5′-monophosphate (amp)-activated protein kinase (ampk), the autophagosomal protein microtubule-associated protein 1 light chain 3c (lc3c), and the autophagy-related proteins beclin1, atg5, atg12 were significantly induced (P < 0.05) in the adenosine monophosphate-activated protein kinase/rapamycin (AMPK/mTOR) signaling pathway. These changes resulted in excess free radicals, causing oxidative stress in the mitochondrial membrane, promoting mitochondrial autophagy. The results confirm that deltamethrin exposure can induce hepatopancreatic injury by promoting mitochondrial autophagy, activating an immune response, and inhibiting lipid metabolism. Overall, this study provides multi-level information to reveal the toxic effects of deltamethrin on E. sinensis.

Oral administration of anti-lipopolysaccharide factor isoform 3 via the bait microalga for controlling bacterial infection of Atlantic Bay scallop (Argopecten irradians)

In order to reduce antibiotic application in aquaculture, an antimicrobial peptide anti-lipopolysaccharide factor isoform 3 (ALFPm3) with highly effective inhibitory activity against pathogens was introduced to bay scallop (Argopecten irradians) using bait microalga Tetraselmis subcordiformis as a delivery system. To improve its expression and stability, a fusion of two alfpm3 genes (alfpm3–2) was inserted into an expression vector and transformed into T. subcordiformis chloroplast, followed by in vitro and in vivo functional evaluations of the antimicrobial activities of ALFPm3–2 by minimum inhibitory concentration method and bay scallop breeding experiment, respectively. The results showed that the transformants of T. subcordiformis and the purified ALFPm3–2 had significant inhibitory effects on Vibrio parahaemolyticus and Staphylococcus aureus in vitro, while feeding the transformants reduced the colonization of the two pathogens in bay scallops, which further reduced bay scallops’ mortality by 28.67 % as revealed in infection test. Furthermore, daily oral administration of ALFPm3–2 could improve the gut microbial community and reduce the inflammatory response. Combined with changes in immune factors and endotoxin, it indicated that exogenous ALFPm3–2 played antimicrobial and anti-inflammatory functions in the guts of bay scallops. Overall, this study provided strong evidence to support the oral administration of antimicrobial peptides via microalgae in aquaculture.

Roles of qseC mutation in bacterial resistance against anti-lipopolysaccharide factor isoform 3 (ALFPm3).

Propelled by global climate changes, the shrimp industry has been facing tremendous losses in production due to various disease outbreaks, particularly early mortality syndrome (EMS), a disease caused by Vibrio parahaemolyticus AHPND. Not only is the use of antibiotics as EMS control agents not yet been proven successful, but the overuse and misuse of antibiotics could also worsen one of the most challenging global health issues-antimicrobial resistance. To circumvent antibiotic usage, anti-lipopolysaccharide factor isoform 3 (ALFPm3), an antimicrobial peptide (AMP) derived from the shrimp innate immune system, was proposed as an antibiotic alternative for EMS control. However, prolonged use of AMPs could also lead to bacterial cross resistance with life-saving antibiotics used in human diseases. Here, we showed that ALFPm3-resistant strains of E. coli could be induced in vitro. Genome analysis of the resistant mutants revealed multiple mutations, with the most interesting being a qseC(L299R). A study of antibiotic susceptibility profile showed that the resistant strains harboring the qseC(L299R) not only exhibited higher degree of resistance towards polymyxin antibiotics, but also produced higher biofilm under ALFPm3 stress. Lastly, a single cell death analysis revealed that, at early-log phase when biofilm is scarce, the resistant strains were less affected by ALFPm3 treatment, suggesting additional mechanisms by which qseC orchestrates to protect the bacteria from ALFPm3. Altogether, this study uncovers involvement of qseC mutation in mechanism of resistance of the bacteria against ALFPm3 paving a way for future studies on sustainable use of ALFPm3 as an EMS control agent.

PmAP2-\u03b2 depletion enhanced activation of the Toll signaling pathway during yellow head virus infection in the black tiger shrimp Penaeus monodon

Yellow head virus (YHV) is a pathogen which causes high mortality in penaeid shrimp. Previous studies suggested that YHV enters shrimp cells via clathrin-mediated endocytosis. This research investigated the roles of clathrin adaptor protein 2 subunit β (AP-2β) from Penaeus monodon during YHV infection. PmAP2-β was continuously up-regulated more than twofold during 6–36 hpi. Suppression of PmAP2-β significantly reduced YHV copy numbers and delayed shrimp mortality. Quantitative RT-PCR revealed that knockdown of PmAP2-β significantly enhanced the expression level of PmSpätzle, a signaling ligand in the Toll pathway, by 30-fold at 6 and 12 hpi. Moreover, the expression levels of gene components in the Imd and JAK/STAT signaling pathways under the suppression of PmAP2-β during YHV infection were also investigated. Interestingly, anti-lipopolysaccharide factor isoform 3 (ALFPm3) was up-regulated by 40-fold in PmAP2-β knockdown shrimp upon YHV infection. In addition, silencing of PmAP2-β dramatically enhanced crustinPm1 expression in YHV-infected shrimp. Knockdown of ALFPm3 and crustinPm1 significantly reduced shrimp survival rate. Taken together, this work suggested that PmAP2-β-deficiency promoted the Toll pathway signalings, resulting in elevated levels of ALFPm3 and crustinPm1, the crucial antimicrobial peptides in defence against YHV.

Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity.

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.

Anti-lipopolysaccharide Factor from Crucifix Crab Charybdis feriatus, Cf-ALF2: Molecular Cloning and Functional Characterization of the Recombinant Peptide

Antilipopolysaccharide factors (ALFs) are important effectors of innate immunity in crustaceans with broad spectrum antimicrobial activity. Present study deals with the molecular and functional characterization of a 98-amino acid ALF isoform from, crucifix crab, Charybdis feriatus termed as Cf-ALF2. The ALF isoform Cf-ALF2 exhibits characteristic features of an AMP including a cationic net charge of +9 and a total hydrophobic ratio of 34%. Recombinant peptide rCf-ALF2 showed remarkable antimicrobial activity against Gram-negative and Gram-positive bacteria especially against Staphylococcus aureus (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 5µM) and Escherichia coli (MIC 10µM and MBC 20µM). Using scanning electron microscopy, bacterial membrane blebbing, disruption, and cell content leakage were observed in peptide treated E. coli. The recombinant peptide was found to be non-hemolytic and non-cytotoxic in NCI-H460 cell line at the highest tested concentration (20µM). Thus, this study identified a novel isoform of ALF from C. feriatus and revealed the potent antimicrobial property of the recombinant peptide Cf-ALF2 and the future prospects of using the peptide for therapeutic applications in the future.

Recombinant expression of an oriental river prawn anti-lipopolysaccharide factor gene in Pichia pastoris and its characteristic analysis

Anti-lipopolysaccharide factors (ALFs), as main host-defense molecules of crustaceans, form a unique family of antimicrobial peptides (AMPs). MnALF4 is one isoform of ALFs isolated from the freshwater prawn Macrobrachium nipponense. In the present study, MnALF4 gene was successfully expressed in the yeast Pichia pastoris and the recombinant MnALF4 protein exhibited efficient and broad-spectrum antimicrobial activities against both Gram-positive bacteria and Gram-negative bacteria in vitro. When prawns were injected with rMnALF4 before bacterial challenge with E. coli, the recombinant protein effectively promote the elimination of bacteria by the host. It manifested that rMnALF4 could effectively kill the invading bacteria in vivo. Treatment with rMnALF4 led to remarkable changes in bacterial morphology, such as spheroidization, oversized bacteria, and cell lysis. In addition, rMnALF4 showed weak hemolysis activity to the rabbit red blood cells. Our work suggests that MnALF4 plays an important role in Macrobrachium immunity and is worthy of further investigation as a potential antibacterial agent with high efficacy against bacterial infection and low toxicity to host cells.

A Proteomic Study of Hemocyte Proteins from Mud Crab (Scylla paramamosain) Infected with White Spot Syndrome Virus or Vibrio alginolyticus.

In this study, we investigated the hemocytes’ immune response to white spot syndrome virus (WSSV) or Vibrio alginolyticus infection at the protein level. The differential proteomes from crab hemocytes infected with WSSV or V. alginolyticus were analyzed using the isobaric tags for relative and absolute quantitation approach immediately after infection. Using this approach, we identified 1,799 proteins by their by LC–MS/MS spectra and sequencing data. These included 157 upregulated proteins and 164 downregulated proteins after WSSV infection. Similarly, 243 proteins were determined to be differentially expressed during V. alginolyticus infection, of these, 121 were upregulated and 122 were downregulated after infection. Interestingly, among these differentially expressed proteins, 106 were up- or downregulated significantly in both WSSV and V. alginolyticus infection. Six genes, β-actin, myosin-9, anti-lipopolysaccharide factor isoform 4, anti-lipopolysaccharide factor 4, transketolase-like protein 2-like isoform 1, and sarcoplasmic calcium-binding protein 1 were chosen for further study. The expression of these genes all showed a trend of upregulation at 24 h post-WSSV or V. alginolyticus infection except for myosin-9 in response to WSSV. To confirm the protective effects of the six genes, crabs were injected with specific dsRNAs before WSSV or V. alginolyticus challenge. The results showed that the knockdown of these genes led to an increase in the morbidity and mortality (P < 0.01) rate, and a decrease in infection time in WSSV-infected crabs. During the first 84 h, knockdown of these genes also led to an increase in the morbidity rates in V. alginolyticus -infected crabs, and results of four genes showed a higher mortality rate than that of the control after they were knocked down. This is the first report of the proteome response in crab hemocytes during WSSV or V. alginolyticus infection. These findings will contribute to our understanding of the immune response to WSSV and V. alginolyticus infection in crabs.

Antiviral action of the antimicrobial peptide ALFPm3 from Penaeus monodon against white spot syndrome virus

The anti-lipopolysaccharide factor isoform 3 (ALFPm3), the antimicrobial peptide from Penaeus monodon, possesses antibacterial and antiviral activities. Although the mechanism of action of ALFPm3 against bacteria has been revealed but its antiviral mechanism is still unclear. To further study how the ALFPm3 exhibits antiviral activity against the enveloped virus, white spot syndrome virus (WSSV), the ALFPm3-interacting proteins from WSSV were sought and identified five ALFPm3-interacting proteins, WSSV186, WSSV189, WSSV395, WSSV458, and WSSV471. Only the interaction between ALFPm3 and WSSV189, however, has been confirmed to be involved in anti-WSSV activity of ALFPm3. Herein, the interactions between ALFPm3 and rWSSV186, rWSSV395, rWSSV458, or rWSSV471 were further analyzed and confirmed by in vitro pull-down assay. Western blot analysis and immunoelectron microscopy showed that the uncharacterized proteins, WSSV186 and WSSV471, were nucleocapsid and envelope proteins, respectively. The decrease of shrimp survival after injection the shrimp with mixtures of each rWSSV protein, rALFPm3 and WSSV as compared to those injected with rALFPm3-neutralizing WSSV was clearly observed indicating that all rWSSV proteins could interfere with the neutralization effect of rALFPm3 on WSSV similar to that reported previously for WSSV189. Morphological change on WSSV after incubation with rALFPm3 was observed by TEM. The lysed WSSV virions were clearly observed where both viral envelope and nucleocapsid were dismantled. The results lead to the conclusion that the ALFPm3 displays direct effect on the viral structural proteins resulting in destabilization and breaking up of WSSV virions.

Recombinant Expression of a Modified Shrimp Anti-Lipopolysaccharide Factor Gene in Pichia pastoris GS115 and Its Characteristic Analysis.

Anti-lipopolysaccharide factors (ALFs) with a LPS-binding domain (LBD) are considered to have broad spectrum antimicrobial activities and certain antiviral properties in crustaceans. FcALF2 was one isoform of ALFs isolated from the Chinese shrimp Fenneropenaeus chinensis. Our previous study showed that a modified LBD domain (named LBDv) of FcALF2 exhibited a highly enhanced antimicrobial activity. In the present study, a modified FcALF2 gene (mFcALF2), in which the LBD was substituted by LBDv, was designed and synthesized. This gene was successfully expressed in yeast Pichia pastoris GS115 eukaryotic expression system, and the characteristics of the recombinant protein mFcALF2 were analyzed. mFcALF2 exhibited apparent antibacterial activities against Gram-negative bacteria, including Escherichia coli, Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus, and Gram-positive bacteria, including Bacillus licheniformis and Staphylococcus epidermidis. In addition, mFcALF2 could reduce the propagation of white spot syndrome virus (WSSV) in vivo by pre-incubation with virus. The present study paves the way for developing antimicrobial drugs in aquaculture.

Differentially expressed transcripts in stomach of Penaeus monodon in response to AHPND infection

Acute Hepatopancreatic Necrosis Disease (AHPND) is an emerging disease in aquacultured shrimp caused by a pathogenic strain of Vibrio parahaemolyticus. As with several pathogenic bacteria, colonization of the stomach appeared to be the initial step of the infection for AHPND-causing Vibrio. To understand the immune responses in the stomach of black tiger shrimp (Penaeus monodon), differentially expressed transcripts (DETs) in the stomach during V. parahaemolyticus strain 3HP (VP3HP) infection was examined using Ion Torrent sequencing. From the total 42,998 contigs obtained, 1585 contigs representing 1513 unigenes were significantly differentially expressed with 1122 and 391 unigenes up- and down-regulated, respectively. Among the DETs, there were 141 immune-related unigenes in 10 functional categories: antimicrobial peptide, signal transduction pathway, proPO system, oxidative stress, proteinases/proteinase inhibitors, apoptotic tumor-related protein, pathogen recognition immune regulator, blood clotting system, adhesive protein and heat shock protein. Expression profiles of 20 of 22 genes inferred from RNA sequencing were confirmed with the results from qRT-PCR. Additionally, a novel isoform of anti-lipopolysaccharide factor, PmALF7 whose transcript was induced in the stomach after challenge with VP3HP was discovered. This study provided a fundamental information on the molecular response in the shrimp stomach during the AHPND infection that would be beneficial for future research.

Molecular cloning, recombinant expression and functional characterization of a novel isoform of anti-lipopolysaccharide factor from the crucifix crab, Charybdis feriatus

Anti-lipopolysaccharide factor (ALF), one of the key effector molecules in innate immune system of crustaceans is a cationic Antimicrobial Peptide (AMP) having broad spectrum antibacterial and antiviral property. In the present study we identified and characterized one isoform of ALF homolog (Cf-ALF2) from the haemocytes of crucifix crab, Charybdis feriatus. The partial cDNA of 294bp encoded for a mature peptide with 98 amino acid constituting a molecular mass of 10.923 kDa with a net charge of +9 and pI of 10.09. Furthermore, different from the previously identified ALF isoforms, Cf-ALF2 has a LPS binding domain with 23 amino acids including two cysteines instead of 22 amino acids and also exhibited less sequence similarity. Secondary structure of Cf-ALF2 predicted using PSIPRED constitutes two alpha helices packed against three beta strands. Multiple alignment performed for Cf-ALF2 with representatives of ALFs from limulids and decapod crustaceans using MEGA 6.0 revealed the existence of conserved regions within the sequence. The recombinant form of Cf-ALF2 (rCf-ALF2) was expressed as a fusion protein in E. coli, Rosettagami B DE3 pLysS using the pET-32a+ vector. The purified and refolded rCf-ALF2 protein revealed antimicrobial activity even at 2.5 μM against Gram-negative bacteria viz., Pseudomonas aeruginosa, Aeromonas hydrophila and Edwardsiella tarda and Gram-positive bacteria viz., Bacillus cereus and Staphylococcus aureus. Recombinant Cf-ALF2 was found to be non-haemolytic and non-cytotoxic even at a concentration of 8 μM (250μg/mL). These preliminary functional studies strongly suggest that Cf-ALF2 is a potent AMP against bacterial infection and might function as a promising therapeutic candidate in aquaculture and medicine.

A potential application of shrimp antilipopolysaccharide factor in disease control in aquaculture

Antilipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) exhibits broad antimicrobial activities against various microbes, including bacteria, fungi and viruses, suggesting its potential use in disease control in shrimp aquaculture. Herein, the recombinant ALFPm3 protein (rALFPm3) exhibited effective antibacterial action against Vibrio parahaemolyticus, the causative agent of the new severe emerging disease, early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) at the minimum inhibitory concentration value of 1.25–5.00 μM. The morphological changes such as bleb formation and cell distortion of V. parahaemoluticus were introduced after rALFPm3 treatment. Furthermore, to control V. parahaemoluticus infection, shrimp were treated with rALFPm3 by immersion and reduction in the cumulative mortality of V. parahaemoluticus-infected Penaeus vannamei was observed. As a potential immunostimulant, the administration of dietary rALFPm3 enhanced the transcript level of five immune-related genes; ALFPm3, haemocyte homoeostasis-associated protein, phagocytosis-activating protein, prophenoloxidase-activating enzyme 1 and cytosolic manganese superoxide dismutase. In addition, dietary rALFPm3 increased the shrimp survival after white spot syndrome virus (WSSV) injection by >35% compared to the 0% survival for the control group. Taken together, these results indicate the potential use of dietary rALFPm3 as immunostimulant in controlling AHPND and WSSV infections in shrimp aquaculture.

Molecular characterization and phylogenetic analysis of an anti-lipopolysaccharide factor from the crucifix crab, Charybdis feriatus

m Abstract: Antimicrobial peptides are small cationic, gene-encoded, amphipathic, host defense peptides with a ubiquitous distribution in all living kingdoms. They are ,10 kDa in size, with 15-100 amino acids having a net positive charge of +2 to +9. Anti-lipopolysaccharide factor (ALF) is a cationic antimicrobial peptide which constitutes one of the key effector molecules in the innate immune system of crustaceans, and is capable of binding and neutralizing lipopoly- saccharides. In the present study, an ALF homolog (Charybdis feriatus (Cf)-ALF1)-encoding cDNA sequence from the hemocytes of the crucifix crab, C. feriatus, was cloned, identified, and characterized. The deduced peptide of Cf-ALF1 encoded for a 123 amino acid peptide with a 97 residue mature peptide (11.16 kDa) that had a net charge of +10. Two conserved cysteine residues and a putative lipopolysaccharide binding domain were observed in the Cf-ALF1 mature peptide. BLAST analysis of Cf-ALF1 nucleotides showed a 99% similarity to Scylla serrata. The spatial structure of Cf-ALF1 was composed of three α-helices packed against a four-strand β-sheet. Two of these helices were linked by a disulfide bond to form an amphipathic loop similar to the structure of anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALF-Pm3). All these features suggest that Cf-ALF1 could play a significant role in the innate immune defense mechanism of C. feriatus.

Molecular Characterization and Phylogenetic Analysis of Novel Isoform of Anti-lipopolysaccharide Factor from the Mantis Shrimp, Miyakea nepa.

Anti-lipopolysaccharide factor (ALF) is a cationic anti-microbial peptide representing humoral defence system exhibiting a diverse spectrum of activity against microbial pathogens, including gram-negative and gram-positive bacteria, fungi, parasites and viruses. In this study, we identified and characterized a novel ALF homologue (MnALF) encoding cDNA sequence from the haemocytes of stomatopod mantis shrimp Miyakea nepa. The deduced peptide of MnALF encoded for a 123-amino acid peptide with a 25-residue signal peptide containing selenocysteine followed by a highly cationic mature peptide comprised of a putative LPS-binding domain flanked by two cysteine residues. BLAST analysis of MnALF showed that it exhibits identity to crustacean and limulid ALFs. The mature peptide of MnALF has a net charge of +7 and predicted molecular weight of 10.998kDa with a theoretical isoelectric point (pI) of 9.93. Spatial structure of MnALF comprises three α-helices packed against a four-stranded β-sheet of which two were linked by a disulphide bond to form an amphipathic loop similar to the structure of Penaeus monodon, ALF-Pm3. All these features suggest that MnALF could play an imperative role in the innate defence mechanism of M. nepa. To our knowledge, this study accounts for the first report of an anti-microbial peptide from the order stomatopoda.

Recombinant expression and functional analysis of an isoform of anti-lipopolysaccharide factors (FcALF5) from Chinese shrimp Fenneropenaeus chinensis

Antimicrobial peptides (AMPs) have a great potential to be used as a substitute for antibiotics since AMPs don't lead to bacteria's drug resistance. Anti-lipopolysaccharide factors (ALFs) are one type of AMPs and exist in crustaceans. In the present study, we produced a recombinant protein (rFcALF5) of an ALF isoform (FcALF5) from Chinese shrimp Fenneropenaeus chinensis through a prokaryotic expression system. The rFcALF5 exhibited varied antibacterial activities against different bacteria. Besides its antibacterial activities, it could also inhibit the infection of white spot syndrome virus (WSSV) to shrimp after pre-incubation with this virus. In order to learn the antiviral mechanism on how rFcALF5 influences WSSV infection, the interaction between the total proteins of WSSV and rFcALF5 was analyzed and the data showed that rFcALF5 had direct interaction with the envelope protein VP24 of WSSV. The LPS binding domain (LBD) of FcALF5 also showed direct interaction with VP24 of WSSV. Therefore we inferred that the antiviral activity of FcALF5 might be achieved through the binding of its LBD to VP24 of WSSV. These findings provided more information to develop new strategies for the control of shrimp disease in aquaculture.

Characterization of a novel anti-lipopolysaccharide factor isoform (SpALF5) in mud crab, Scylla paramamosain

Anti-lipopolysaccharide factors (ALFs), the potential antimicrobial peptides that bind and neutralize lipopolysaccharide (LPS), are common effectors of innate immunity in crustaceans. In this study, a novel isoform of ALFs (SpALF5) was isolated from the hemocytes of mud crab Scylla paramamosain. The full-length 975bp SpALF5 contains a 375bp open reading frame (ORF) encoding 125 amino acids. Although SpALF5 exhibits a low degree of nucleotide homology with other reported ALFs, it contains the conserved amino acid sequence with a signal peptide and a LPS-binding domain including two conservative cysteine residues. The genomic organization of SpALF5 consists of four exons and three introns, with each intron containing one or more tandem repeats. Unlike most of ALFs mainly distributed in crab hemocytes, SpALF5 transcript was predominantly observed in the brain, muscle and skin, while barely detected in the hemocytes in our study. In situ hybridization assay also showed that SpALF5 mRNA was localized in brain, muscle and skin tissues of mud crab. Further, SpALF5 transcript was significantly up-regulated after challenge with LPS, polyinosinic polycytidylic acid (PolyI:C) (with the except of that in brain), Vibrio parahemolyticus or white spot syndrome virus (WSSV). The recombinant SpALF5 protein showed a varying degree of binding activity towards bacteria and fungus. Moreover, in vitro, the recombinant SpALF5 revealed a strong antimicrobial activity against Gram-negative bacteria (V. parahemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) and fungus (Sacchromyces cerevisiae), but could only inhibited the growth of some Gram-positive bacteria like Staphylococcus aureus. The results suggest that SpALF5 is a potent immune protector and plays an important role in immune defense against invading pathogens in S. paramamosain.

Anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) exhibits antiviral activity by interacting with WSSV structural proteins

In innate immunity, antimicrobial peptides (AMPs) play a vital role in combating microbial pathogens. Among the AMPs identified in Penaeus monodon, only anti-lipopolysaccharide factor isoform 3 (ALFPm3) has been reported to exhibit activity against white spot syndrome virus (WSSV). However, the mechanism(s) involved are still not clear. In the present study, ALFPm3-interacting proteins were screened for from a WSSV library using the yeast two-hybrid screening system, revealing the five potential ALFPm3-interacting proteins of WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471. Temporal transcriptional analysis in WSSV-infected P. monodon revealed that all five of these WSSV gene transcripts were expressed in the late phase of infection (24h and 48h post-infection). Of these, WSSV189 that was previously identified as a structural protein, was selected for further analysis and was shown to be an enveloped protein by Western blot and immunoelectron microscopy analyses. The in vitro pull-down assay using recombinant WSSV189 (rWSSV189) protein as bait confirmed the interaction between ALFPm3 and WSSV189 proteins. Moreover, pre-incubation of rWSSV189 protein with rALFPm3 protein interfered with the latter’s neutralization effect on WSSV in vivo, as shown by the increased cumulative mortality of shrimp injected with WSSV following prior treatment with pre-incubated rWSSV189 and rALFPm3 proteins compared to that in shrimp pre-treated with rALFPm3 protein. Thus, ALFPm3 likely performs its anti-WSSV action by binding to the envelope protein WSSV189 and possibly other WSSV structural proteins.

Characterization and function analysis of an anti-lipopolysaccharide factor (ALF) from the Chinese shrimp Fenneropenaeus chinensis

Anti-lipopolysaccharide factor (ALF) is one of the widely-studied antimicrobial peptides (AMPs) with broad-spectrum antibacterial activity and antiviral property. Previous studies show the existence of multiform of ALFs in crustacean which are important for immunity of the animals. In the present study, we characterized one isoform of ALF from the Chinese shrimp Fenneropenaeus chinensis (FcALF2). Tissue distribution analysis revealed that FcALF2 showed the highest expression level in the lymphoid organ (Oka) of the shrimp. The expression level of FcALF2 in shrimp was significantly up-regulated when they were injected with Micrococcus lysodeikticus and Vibrio anguillarum. A peptide corresponding to the LPS-binding domain of FcALF2 (FcALF2-LBD) was synthesized to analyze its antimicrobial activities. Data demonstrated that FcALF2-LBD possessed strong antibacterial activity against Gram-positive bacteria Micrococcus luteus and M.lysodeikticus with MIC ranges of 2–4μM and 1–2μM respectively and significant inhibition activity against white spot syndrome virus (WSSV). The antibacterial activities of the sequence modified peptides (FcALF2-LBDb, FcALF2-LBDv) were apparently enhanced and broadened after the amount of basic amino acids was increased in the synthetic LPS-binding domain. These data provide more insights into understanding the function of LPS-binding domain of ALF and the role of ALF in shrimp immunity.

A new anti-lipopolysaccharide factor (ALF) gene with its SNP polymorphisms related to WSSV-resistance of Litopenaeus vannamei

Anti-lipopolysaccharide factors (ALFs) of crustacean play an important role against bacteria or virus infection. In this study, the cDNA sequence and genomic sequence of one new isoform of ALF designated as nLvALF1 were reported. The open reading frame (ORF) of nLvALF1 consisted of 369 bp encoding 123 amino acids and the genomic structure of nLvALF1 comprised four introns and three exons. The predicted pI of the deduced protein was 8.82 and the molecular weight (MW) was 13.72 KDa. The deduced amino acid sequence of nLvALF1 contained a typical functional domain of ALF: LPS-binding domain. Phylogenetic analysis showed that nLvALF1 had the closest relationship with FcALF1 from Fenneropenaeus chinensis. The nLvALF1 was specifically expressed in lymphoid organ (Oka) of shrimp. Its transcriptional level was significantly up-regulated after white spot syndrome virus (WSSV) challenge, suggesting that nLvALF1 might participate in defense against WSSV in Litopenaeus vannamei. In order to search potential genetic markers associated with WSSV-resistance, we scanned the polymorphisms of the genomic fragment with 397 bp where the LPS-binding domain encoding sequence located and 18 SNPs were found. The distribution frequency of these SNPs was analyzed in WSSV susceptible shrimp and resistant shrimp separately. Significant differences existed in allelic frequencies at loci g.1361-T > C, g.1370-T > C, g.1419-T > A between the WSSV-resistant group and the WSSV-susceptible/susceptible group (P < 0.05). The specific haplotype CT consisted of g.1415-C > A and g.1419-T > A was associated with susceptibility to WSSV (P < 0.05). These findings provide theoretical support for selection of WSSV-resistant varieties of L. vannamei.