RNA-seq transcriptome analysis and identification of the theromacin antimicrobial peptide of the copepod Apocyclops royi.

Copepods, including Apocyclops royi, are small aquatic crustaceans and one of the important foods for fish and shellfish larvae. However, studies of the host-pathogen interactions and understanding of infectious disease in copepods are still very limited, yet they are likely to be a significant factor in the sustainable development of copepod aquaculture. In the present study, we performed de novo RNA sequence analysis of A. royi-TH (a Thai isolate of A. royi), which yielded 4.80 Gb bases of clean data and a total of 29,786 unigenes. Annotation was then performed by comparison against seven functional databases, yielding 17,617 (NR: 59.15%), 2,969 (NT: 9.97%), 15,023 (SwissProt: 50.44%), 14,543 (KOG: 48.82%), 15,077 (KEGG: 50.62%), 6,763(GO: 22.71%), and 15,841 (InterPro: 53.18%) unigenes. In comparison to the components of the shrimp Toll pathway, LGBP, Spätzle, Toll receptors, MyD88, Pelle, TRAF6, Dorsal, and Cactus homologs were successfully identified in A. royi-TH. Additionally, a novel antimicrobial peptide (Theromacin-like) was characterized in A. royi (ArTM-like). The ArTM-like ORF was 279 bp and predicted to encode for 92 amino acid residues, with a mature peptide of 75 amino acids and a molecular mass of 8.56 kDa. The genomic organization of the ArTM-like gene consisted of three exons and two introns. Expression analysis indicated that ArTM-like mRNA was abundantly expressed in copepodid and adult stages as an immune responsive gene after infection with the pathogenic Vibrio parahaemolyticus-(AHPND)-causing strain. Altogether, the knowledge obtained in this study will provide a basis for future functional studies of the molecular mechanisms in copepod immunity that may eventually be applied for disease prevention in copepod aquaculture.

Characterization, Stress Response and Functional Analyses of Giant River Prawn (Macrobrachium rosenbergii) Glucose-Regulated Protein 78 (Mr-grp78) under Temperature Stress and during Aeromonas hydrophila Infection.

The endoplasmic reticulum (ER) is an organelle important for several functions of cellular physiology. This study identified the giant river prawn's glucose-regulated protein 78 (Mr-grp78), which is important for ER stress mechanisms. Nucleotide and amino acid analyses of Mr-grp78, as compared with other species, revealed the highest similarity scores with the grp78 genes of crustaceans. An expression analysis by quantitative RT-PCR indicated that Mr-grp78 was expressed in all tissues and presented its highest expression in the ovary (57.64 ± 2.39-fold), followed by the gills (42.25 ± 1.12), hindgut (37.15 ± 2.47), thoracic ganglia (28.55 ± 2.45) and hemocytes (28.45 ± 2.26). Expression analysis of Mr-grp78 mRNA levels under Aeromonas hydrophila induction and heat/cold-shock exposure was conducted in the gills, hepatopancreas and hemocytes. The expression levels of Mr-grp78 in these tissues were highly upregulated 12 h after bacterial infection. In contrast, under heat- and cold-shock conditions, the expression of Mr-grp78 was significantly suppressed in the gills at 24-96 h and in the hepatopancreas at 12 h (p < 0.05). A functional analysis via Mr-grp78 gene knockdown showed that Mr-grp78 transcription in the gills, hepatopancreas and muscle strongly decreased from 6 to 96 h. Furthermore, the silencing of this gene effectively increased the sensitivity of the tested prawns to heat- and pathogenic-bacterium-induced stress. The results of this study clearly demonstrate the significant functional roles of Mr-grp78 in response to both temperature and pathogen treatments.

Molecular Characterization and Defense Functions of the Nile Tilapia (Oreochromis niloticus) DnaJ B9b and DnaJ C3a Genes in Response to Pathogenic Bacteria under High-Temperature Stress Conditions.

DnaJ proteins or heat shock protein 40s (HSP40s) form one of the largest heat shock protein families. In this study, 2 cDNAs encoding Nile tilapia (Oreochromis niloticus) DnaJ proteins (On-DnaJ B9b and On-DnaJ C3a) were successfully cloned and characterized. The structures and organizations of these two genes are first reported in the present study. On-DnaJ B9b is approximately 2.1 kb long and contains 2 exons and 1 intron, while On-DnaJ C3a is approximately 12 kb long and contains 12 exons and 11 introns. Under normal conditions, On-DnaJ B9b mRNA is highly expressed in gonad and trunk kidney tissues, while On-DnaJ C3a transcripts are abundantly expressed in gills, intestine, liver, and trunk kidney tissues. Following pathogenic infections, the expression of both genes is induced in the liver, spleen and head kidney tissues of Nile tilapia that were infected with two virulent pathogenic bacteria, Streptococcus agalactiae and Flavobacterium columnare. Silencing of these two genes was first carried out, and the results clearly indicated their crucial roles under both heat and bacterial stress conditions. The fundamental knowledge obtained from this study indicates the characteristic basic biofunctions of heat shock proteins in the regulation of intracellular proteins during infection, which involve preventing protein aggregation, promoting protein refolding, and activating unfolded protein degradation.

Stimulator of interferon gene (STING) and interferon regulatory factor (IRF) are crucial for shrimp antiviral defense against WSSV infection.

The cytosolic DNA-sensing immune response is essential for recognizing and establishing an effective host immune response to pathogens. However, the importance of the cytosolic signalling molecules responsible for facilitating an appropriate immune response following infection with a DNA virus in shrimps remains unknown. Here, we report the discovery of the Penaeus monodon stimulator of interferon gene (PmSTING) and interferon regulatory factor (PmIRF) genes and their important roles in the host defense against viral infection. High expression levels of PmSTING transcripts were detected in the midgut, hepatopancreas, and hindgut, with lower levels in foregut, while PmIRF was highly expressed in the hindgut, foregut, and hepatopancreas of P. monodon. The mRNA expression level of both PmSTING and PmIRF was up-regulated in the foregut in response to white spot syndrome virus (WSSV; dsDNA virus) infection. RNA-interference-mediated gene silencing of PmSTING and PmIRF rendered shrimps to be more susceptible to WSSV infection; suppression of PmIRF decreased the mRNA transcript level of PmSTING; and silencing of the cytosolic sensor PmDDX41 suppressed both PmSTING and PmIRF gene transcript levels. Thus, PmSTING and PmIRF are likely to be important for the antiviral innate response against the dsDNA WSSV pathogen and may mediate the antiviral immune defenses via PmDDX41/PmSTING/PmIRF signaling cascade in P. monodon.

Functional and Stress Response Analysis of Heat Shock Proteins 40 and 90 of Giant River Prawn (Macrobrachium rosenbergii) under Temperature and Pathogenic Bacterial Exposure Stimuli.

The aims of this research were to perform molecular characterization and biofunctional analyses of giant river prawn Hsp40 and Hsp90 genes (Mr-hsp40 and Mr-hsp90) under various stress conditions. Comparisons of the nucleotide and amino acid sequences of Mr-hsp40 and Mr-hsp90 with those of other species showed the highest similarity scores with crustaceans. Under normal conditions, expression analysis using quantitative real-time RT-PCR (qRT-PCR) indicated that Mr-hsp40 was highly expressed in the gills and testis, and Mr-hsp90 expression was observed in all tissues, with the highest expression in the ovary. The expression patterns of Mr-hsp40 and Mr-hsp90 transcripts under Aeromonas hydrophila challenge and heat-cold shock conditions were examined in gills, the hepatopancreas and hemocytes, at 0, 3, 6, 12, 24, 48 and 96 h by qRT-PCR. Under bacterial challenge, Mr-hsp40 displayed variable expression patterns in all tissues examined during the tested periods. In contrast, upregulated expression of Mr-hsp90 was quickly observed from 3 to 12 h in the gills and hepatopancreas, whereas obviously significant upregulation of Mr-hsp90 was observed in hemocytes at 12-96 h. Under temperature shock conditions, upregulation of Mr-hsp40 expression was detected in all tested tissues, while Mr-hsp90 expression was quickly upregulated at 3-48 h in all tissues in response to 35 °C conditions, and conditions of 35 and 25 °C stimulated its expression in gills and the hepatopancreas at 12 and 48 h, respectively. Silencing analyses of these two genes were successfully conducted under normal, high-temperature (35 °C) and A. hydrophila infection conditions. Overall, knockdown of Mr-hsp40 and Mr-hsp90 effectively induced more rapid and higher mortality than in the PBS control and GFP induction groups in temperature and infectious treatments. Evidence from this study clearly demonstrated the significant functional roles of Mr-hsp40 and Mr-hsp90, which are crucially involved in cellular stress responses to both temperature and pathogenic bacterial stimuli.

Characterization and functional analysis of fibrinogen-related protein (FreP) in the black tiger shrimp, Penaeus monodon.

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca2+) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity.

Antibiofilm and immunological properties of lectin purified from shrimp Penaeus semisulcatus.

Penaeid prawns are considered as most demanding fishery resources. The current study aims to purify and characterize lectin from the haemolymph of Penaeus semisulcatus. The semisulcatus-lectin was purified by affinity chromatography using mannose coupled Sepharose CL-4B column and purified lectin exhibited a single band of 66 kDa in SDS-PAGE. The purity and crystalline structure of purified lectin was confirmed by HPLC and X-ray diffraction analysis. Semisulcatus-lectin exhibited yeast agglutination activity against Saccharomyces cerevisiae and agglutinated human erythrocytes. Semisulcatus-lectin was evaluated for phenol oxidase activation and phagocytic activities. It was observed that semisulcatus-lectin had antibacterial activity against Gram-negative Vibrio parahaemolyticus and Aeromonas hydrophila, suggesting a potential therapeutic strategy in aquaculture industry for disease management.

Yield improvement and enzymatic dissection of Plasmodium falciparum plasmepsin V.

To survive within a red blood cell (RBC), malaria parasites establish striking modifications to the permeability, rigidity and cytoadherence properties of the host cell. This is mediated by the export of hundreds of proteins from the parasite into the erythrocyte. Plasmodium falciparum plasmepsin V (PfPMV), is an ER resident aspartic protease that processes proteins for export into the host erythrocyte, plays a crucial role in parasite virulence and survival and is considered a potential malaria drug target. Most attempts at its heterologous expression in Escherichia coli have resulted in mainly the production of insoluble proteins. In this study, we employed a multipurpose fusion tag to improve the production of PfPMV in E. coli. Recombinant PfPMVm, comprising residues 84-521, was substantially obtained in soluble form and could be purified in a single step, yielding a 3.7-fold increase in purified PfPMVm compared to previous reports. Additionally, we have mutated the catalytic residues (D118N and D365N), individually and together, and the unpaired cysteine residue C178 to evaluate the effects on catalytic efficiency. Mutation of D365 had more pronounced effects on the catalytic efficiency than that of D118, suggesting that the D365 may act as a catalytic nucleophile to activate the water molecule. The importance of C178 was also confirmed by the inhibition by metal ions, indicating that C178 is partially involved in the substrate recognition. Collectively, our results describe an improved system to produce recombinant PfPMVm in E. coli and dissect the amino acids involved in catalysis and substrate recognition.

Molecular and functional analyses of novel anti-lipopolysaccharide factors in giant river prawn (Macrobrachium rosenbergii, De Man) and their expression responses under pathogen and temperature exposure.

Anti-lipopolysaccharide factor (ALF) is an immune-related protein that is crucially involved in immune defense mechanisms against invading pathogens in crustaceans. In the current study, three different ALFs of giant river prawn (Mr-ALF3, Mr-ALF8 and Mr-ALF9) were discovered. Based on sequence analysis, Mr-ALF3 and Mr-ALF9 were identified as new members of ALFs in crustaceans (groups F and G, respectively). Structurally, each newly identified Mr-ALF contained three α-helices packed against a four-stranded ß-sheet bearing the LPS-binding motif, which usually binds to the cell wall components of bacteria. Tissue expression analysis using quantitative real-time RT-PCR (qRT-PCR) demonstrated that Mr-ALF3 was expressed in most tissues, and the highest expression was in the heart and hemocytes. The Mr-ALF8 gene was highly expressed in the heart, hemocytes, midgut, hepatopacreas and hindgut, respectively, while the Mr-ALF9 gene was modestly expressed in the heart and hemocytes, respectively. The transcriptional responses of the Mr-ALFs to Aeromonas hydrophila and hot/cold temperatures were investigated by qRT-PCR in the gills, hepatopancreas and hemocytes. We found that all Mr-ALFs were clearly suppressed in all tested tissues when the experimental prawns were exposed to extreme temperatures (25 and 35 °C). Moreover, the expression levels of these genes were significantly induced in all examined tissues by 2 different concentrations of A. hydrophila (1 × 106 and 1 × 109 CFU/ml), particularly 12 and 96 h after the injection. Finally, binding activity analysis of LPS-motif peptides of each Mr-ALF revealed that the LPS peptide of Mr-ALF3 exhibited the strongest adhesion to two pathogenic Gram-negative bacteria, A. hydrophila and Vibrio harveyi, and the non-pathogenic Gram-positive Bacillus megaterium. The results also showed that the Mr-ALF8 and Mr-ALF9 peptides had mild antimicrobial effects against similar tested bacteria. Based on information obtained in this study, novel ALF genes were clearly identified. Analyses of their responses under pathogenic and temperature stresses demonstrated the binding and antimicrobial activities of these ALFs and the consequent physiological effects, indicating their crucial functional roles in the prawn immune system.

A novel C-type lectin in the black tiger shrimp Penaeus monodon functions as a pattern recognition receptor by binding and causing bacterial agglutination.

C-type lectins are pattern recognition proteins that play important roles in innate immunity in invertebrates by mediating the recognition of pathogens. In this study, a novel C-type lectin gene, PmCLec, was cloned and characterized from the black tiger shrimp Penaeus monodon. The open reading frame of PmCLec is 657 bp in length. It encodes a predicted protein of 218 amino acids with a calculated molecular mass and an isoelectric point of 24086 Da and 4.67, respectively. Sequence analysis of PmCLec showed similarity to members of the C-type lectin gene superfamily. The deduced protein contains a single carbohydrate recognition domain (CRD) and four conserved cysteine residues (Cys58, Cys126, Cys141, Cys149) that are involved in the formation of disulfide bridges. PmCLec transcripts are expressed in various tiger shrimp tissues, with the highest expression in the lymphoid organ. RNAi-mediated silencing of PmCLec resulted in higher cumulative mortality of knockdown shrimp after Vibrio harveyi infection compared to the control groups. Recombinant PmCLec was successfully expressed in the E. coli system. In the presence of Ca2+, purified rPmCLec protein binds and agglutinates Gram-positive bacteria (Staphylococcus aureus, S. hemolyticus), but only slightly binds and agglutinates E. coli and could not bind to the Gram-negative bacteria Bacillus megaterium and Vibrio harveyi. These results suggest that PmCLec functions as a pattern recognition receptor that is implicated in shrimp innate immunity.

Characterization and identification of calmodulin and calmodulin binding proteins in hemocyte of the black tiger shrimp (Penaeus monodon).

Calmodulin (CaM), a ubiquitous intracellular calcium (Ca(2+)) sensor in all eukaryotic cells, is one of the well-known signaling proteins. Previously, CaM gene has shown a high transcriptional level in hemocyte of the pathogen infected shrimp, suggesting that shrimp CaM does not only regulate Ca(2+) metabolism, but is also involved in immune response cascade. In the present study, the CaM gene of shrimp Penaeus monodon was identified and the recombinant P.monodon CaM (rPmCaM) was produced and biochemically characterized. The identification of CaM-binding proteins was also performed. The PmCaM cDNA consisted of an open reading frame of 447 bp encoding for 149 amino acid residues with a calculated mass of 16,810 Da and an isoelectric point of 4.09. Tissue distribution showed that the PmCaM transcript was expressed in all examined tissues. The results of gel mobility shift assay, circular dichroism spectroscopy and fluorescence spectroscopy all confirmed that the conformational changes of the rPmCaM were observed after the calcium binding. According to the gene silencing of PmCaM transcript levels, the shrimp's susceptibility to pathogenic Vibrio harveyi infection increased in comparison with that of the control groups. Protein pull-down assay and LC-MS/MS analysis were performed to identify rPmCaM-binding proteins involved in shrimp immune responses and transglutaminase, elongation factor 1-alpha, elongation factor 2 and actin were found. However, by computational analysis, only the first three proteins contained CaM-binding domain. These findings suggested that PmCaM may play an important role in regulation of shrimp immune system.

Purification, crystallization and preliminary X-ray analysis of recombinant betaine aldehyde dehydrogenase 2 (OsBADH2), a protein involved in jasmine aroma, from Thai fragrant rice (Oryza sativa L.).

Fragrant rice (Oryza sativa L.) betaine aldehyde dehydrogenase 2 (OsBADH2) is a key enzyme in the synthesis of fragrance aroma compounds. The extremely low activity of OsBADH2 in catalyzing the oxidation of acetaldehyde is believed to be crucial for the accumulation of the volatile compound 2-acetyl-1-pyrroline (2AP) in many scented plants, including fragrant rice. Recombinant fragrant rice OsBADH2 was expressed in Escherichia coli as an N-terminal hexahistidine fusion protein, purified using Ni Sepharose affinity chromatography and crystallized using the microbatch method. Initial crystals were obtained within 24 h using 0.1 M Tris pH 8.5 with 30%(w/v) PEG 4000 and 0.2 M magnesium chloride as the precipitating agent at 291 K. Crystal quality was improved when the enzyme was cocrystallized with NAD(+). Improved crystals were grown in 0.1 M HEPES pH 7.4, 24%(w/v) PEG 4000 and 0.2 M ammonium chloride and diffracted to beyond 2.95 Å resolution after being cooled in a stream of N(2) immediately prior to X-ray diffraction experiments. The crystals belonged to space group C222(1), with unit-cell parameters a = 66.03, b = 183.94, c = 172.28 Å. An initial molecular-replacement solution has been obtained and refinement is in progress.

Biochemical and enzymatic study of rice BADH wild-type and mutants: an insight into fragrance in rice.

Betaine aldehyde dehydrogenase 2 (BADH2) is believed to be involved in the accumulation of 2-acetyl-1-pyrroline (2AP), one of the major aromatic compounds in fragrant rice. The enzyme can oxidize ω-aminoaldehydes to the corresponding ω-amino acids. This study was carried out to investigate the function of wild-type BADHs and four BADH2 mutants: BADH2_Y420, containing a Y420 insertion similar to BADH2.8 in Myanmar fragrance rice, BADH2_C294A, BADH2_E260A and BADH2_N162A, consisting of a single catalytic-residue mutation. Our results showed that the BADH2_Y420 mutant exhibited less catalytic efficiency towards γ-aminobutyraldehyde but greater efficiency towards betaine aldehyde than wild-type. We hypothesized that this point mutation may account for the accumulation of γ-aminobutyraldehyde/Δ(1)-pyrroline prior to conversion to 2AP, generating fragrance in Myanmar rice. In addition, the three catalytic-residue mutants confirmed that residues C294, E260 and N162 were involved in the catalytic activity of BADH2 similar to those of other BADHs.

Abundantly expressed transcripts in the lymphoid organ of the black tiger shrimp, Penaeus monodon, and their implication in immune function.

The lymphoid organ of penaeid shrimps is proposed to play an important role in the innate immune system. To investigate the potential immune function of the lymphoid organ, we analyzed the expressed genes from the lymphoid organ of normal and Vibrio harveyi-infected Penaeus monodon using an expressed sequence tag (EST) approach. Sequence analysis of the EST clones derived from the two lymphoid organ cDNA libraries (408 clones from the normal and 625 clones from the infected libraries), revealed a high redundancy of specific transcripts. Transcripts of the lysosomal cysteine proteinases, cathepsins B and L, were abundantly expressed in the lymphoid organ of both libraries, whilst the transcripts of the related genes peritrophin and thrombospondin predominated and were found only in the V. harveyi-infected library, making them interesting candidate functional genes. Moreover, immune-related genes were found at a significant proportion (approximately 15%) in both normal and infected libraries, but different expressed genes were observed between the two libraries. The expression levels of P. monodon cathepsins B and L in the lymphoid organ following injection with either V. harveyi or white spot syndrome virus (WSSV) showed only a slight change in the transcript abundance compared to that seen in the mock-infection (control). Immunohistochemistry confirmed that cathepsin L protein was localized in the lymphoid organ with intense cathepsin L staining observed in the lymphoid organ spheroids of WSSV-infected shrimps. The results suggest that cathepsins L and B likely play a major role in the lymphoid organ function and are probably implicated in degradation of foreign material that is sequestrated in the lymphoid organ spheroids, although any additional role in control of viral or cellular mediated apoptosis remains to be evaluated.