Co-Interactive DNA-Binding between a Novel, Immunophilin-Like Shrimp Protein and VP15 Nucleocapsid Protein of White Spot Syndrome Virus

White spot syndrome virus (WSSV) is one of the most serious pathogens of penaeid shrimp. Although its genome has been completely characterized, the functions of most of its putative proteins are not yet known. It has been suggested that the major nucleocapsid protein VP15 is involved in packaging of the WSSV genome during virion formation. However, little is known in its relationship with shrimp host cells. Using the yeast two-hybrid approach to screen a shrimp lymphoid organ (LO) cDNA library for proteins that might interact with VP15, a protein named PmFKBP46 was identified. It had high sequence similarity to a 46 kDa-immunophilin called FKBP46 from the lepidopteran Spodoptera frugiperda (the fall armyworm). The full length PmFKBP46 consisted of a 1,257-nucleotide open reading frame with a deduced amino acid sequence of 418 residues containing a putative FKBP-PPIase domain in the C-terminal region. Results from a GST pull-down assay and histological co-localization revealed that VP15 physically interacted with PmFKBP46 and that both proteins shared the same subcellular location in the nucleus. An electrophoretic mobility shift assay indicated that PmFKBP46 possessed DNA-binding activity and functionally co-interacted with VP15 in DNA binding. The overall results suggested that host PmFKBP46 might be involved in genome packaging by viral VP15 during virion assembly

Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp

BACKGROUND: RNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called “multi-WSSV dsRNA.” A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners. RESULTS: For a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (μg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge. CONCLUSION: The present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV

In Vitro Antimicrobial Activity of Piper retrofractum Fruit Extracts against Microbial Pathogens Causing Infections in Human and Animals

Long pepper (Piper retrofractum Vahl) is a Thai medicinal herb which has been used as one of the common ingredients in variety of Thai foods. Here, we investigated antimicrobial activities of crude bioactive metabolites extracted from fruits of P. retrofractum against 10 pathogenic organisms (bacteria and yeast) causing opportunistic infections in human or animals including Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC2921, Escherichia coli ATCC25922, Klebsiella pneumonia TISTR1843, Pseudomonas aeruginosa ATCC741, Salmonella typhi (clinical isolate), Vibrio parahaemolyticus (XN98 and 5HP), and Candida albicans ATCC90020. The results of disk diffusion test showed that the extract from methanol solvent exhibited greater antibacterial activity than other solvents with inhibition zones ranging from 0.5 to 8.0 mm, respectively. Subsequently, minimal inhibition concentration (MIC) determined by the colorimetric assay confirmed that methanol extracts showed consistent results with disk diffusion method. In summary, in vitro assays suggest that methanol is the best solvent for extraction of bioactive metabolites from P. retrofractum fruits. This crude extract can inhibit the majority of human and animal pathogens. This opens up a potential use of pepper fruits in prevention of food-contaminating microorganisms

In Vitro Antimicrobial Activity of <i>Piper retrofractum</i> Fruit Extracts against Microbial Pathogens Causing Infections in Human and Animals

Long pepper (Piper retrofractum Vahl) is a Thai medicinal herb which has been used as one of the common ingredients in variety of Thai foods. Here, we investigated antimicrobial activities of crude bioactive metabolites extracted from fruits of P. retrofractum against 10 pathogenic organisms (bacteria and yeast) causing opportunistic infections in human or animals including Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC2921, Escherichia coli ATCC25922, Klebsiella pneumonia TISTR1843, Pseudomonas aeruginosa ATCC741, Salmonella typhi (clinical isolate), Vibrio parahaemolyticus (XN98 and 5HP), and Candida albicans ATCC90020. The results of disk diffusion test showed that the extract from methanol solvent exhibited greater antibacterial activity than other solvents with inhibition zones ranging from 0.5 to 8.0 mm, respectively. Subsequently, minimal inhibition concentration (MIC) determined by the colorimetric assay confirmed that methanol extracts showed consistent results with disk diffusion method. In summary, in vitro assays suggest that methanol is the best solvent for extraction of bioactive metabolites from P. retrofractum fruits. This crude extract can inhibit the majority of human and animal pathogens. This opens up a potential use of pepper fruits in prevention of food-contaminating microorganisms.</jats:p

Shrimp serine proteinase homologues PmMasSPH-1 and -2 play a role in the activation of the prophenoloxidase system.

Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system