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

Proteomics and the search for welfare and stress biomarkers in animal production in the one-health context

Stress and welfare are important factors in animal production in the context of growing production optimization and scrutiny by the general public. In a context in which animal and human health are intertwined aspects of the one-health concept it is of utmost importance to define the markers of stress and welfare. These are important tools for producers, retailers, regulatory agents and ultimately consumers to effectively monitor and assess the welfare state of production animals. Proteomics is the science that studies the proteins existing in a given tissue or fluid. In this review we address this topic by showing clear examples where proteomics has been used to study stress-induced changes at various levels. We adopt a multi-species (cattle, swine, small ruminants, poultry, fish and shellfish) approach under the effect of various stress inducers (handling, transport, management, nutritional, thermal and exposure to pollutants) clearly demonstrating how proteomics and systems biology are key elements to the study of stress and welfare in farm animals and powerful tools for animal welfare, health and productivity

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

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

The phylogenetic relationship between serine proteinase domains from <i>Pm</i>MasSPH1 and <i>Pm</i>MasSPH2 and other serine proteinases.

<p>The deduced amino acid sequences of SP-domains from various clip-SPH species were used to generate a phylogenetic tree by the neighbor-joining method. Percent bootstrap values (1000 replicates) are shown at each branch point. <i>Pm</i>MasSPH1 (ABE03741); <i>Pm</i>MasSPH2 (ACP19560); <i>Pm</i>SP, <i>P</i>. <i>monodon</i> serine proteinase (ABW87872); <i>Pm</i>SPL, <i>P</i>. <i>monodon</i> serine proteinase-like protein (ABD62888); <i>Pm</i>SPL3 (ABO33174); <i>Pm</i>Mas, <i>P</i>. <i>monodon</i> mas-like protein (AAT42131); <i>Fc</i>PPAF, <i>Fenneropenaeus chinensis</i> prophenoloxidase activating factor (AFW98986); <i>Fc</i>Mas (AFW98983); <i>Lv</i>PPAF, <i>Litopenaeus vannamei</i> PPAF (AFW98993); <i>Lv</i>SP (AY368151); <i>Lv</i>Mas (AFW98990); <i>Pl</i>SPH1, <i>P</i>. <i>leniusculus</i> SPH1 (AAX55746); <i>Pl</i>SPH2 (ACB41379); <i>Ms</i>SPH3, <i>Manduca sexta</i> SPH3 (AF413067); <i>Hd</i>PPAFII, <i>Holotrichia diomphalia</i> PPAFII (CAC12665); <i>Mj</i>SPH, <i>Marsupenaeus japonicus</i> SPH (AB161692); <i>Nv</i>SPH21, <i>Nasonia vitripennis</i> SPH21 (NP_001155060); <i>Cq</i>SP, <i>Culexquin quefasciatus</i> SP (XP_001868413); <i>Aa</i>SP, <i>Aedes aegypti</i> SP (XP_001655705); <i>Pt</i>PPAF, <i>Portunus trituberculatus</i> PPAF (ACN87221); <i>Cs</i>PAF, <i>Callinectes sapidus</i> PAF (AAS60227); <i>Es</i>PPAF, <i>Eriocheir sinensis</i> PPAF (ACU65942); <i>Es</i>Trypsin-likeSP (ACT78700); <i>Ls</i>ST, <i>Lucilia sericata</i> salivary trypsin (AEX33291); <i>Cp</i>PPAF, <i>Cancer pagurus</i> PPAF (CCE46009); <i>Pp</i>SP, <i>Papilio polytes</i> SP(BAM19108); <i>Ha</i>SPL1, <i>Helicoverpa armigera</i> SPL1 (ACI32835); <i>Tm</i>PPAF, <i>Tenebrio molitor</i> PPAF (CAC12696); <i>St</i>SPL, <i>Scylla tranquebarica</i> SPL (ADN44616); <i>Lo</i>PPAF1, <i>Lonomia obliqua</i> PPAF1 (AAV91458); <i>Px</i>SP, <i>Papilio xuthus</i> SP (BAM17901); <i>Pr</i>MSPH, <i>Pieris rapae</i> MSPH (ACZ68116); <i>Tc</i>SP4, <i>Tribolium castaneum</i> SP4 (EEZ99183); <i>Dm</i>Mas, <i>Drosophila melanogaster</i> Mas (AAC46512); <i>Sp</i>-SPH, <i>S</i>. <i>paramamosain</i> SPH (ADG83846).</p

Enhancement of PGN-triggered hemolymph PO activity by r<i>Pm</i>MasSPH1 and r<i>Pm</i>PPAE2 <i>in vitro</i>.

<p>The total PO activity of shrimp hemolymph (HL) alone or mixed with <i>B</i>. <i>subtilis</i> PGN, r<i>Pm</i>MasSPH1 and r<i>Pm</i>PPAE2 was determined. BSA served as a protein control. Each bar represents the PO activity as the mean ± standard deviation from three replicates. The percentages of each treatment are relative to the PO activity of HL alone.</p

Increase in bacterial load in hemolymph from <i>Pm</i>MasSPHs silenced shrimp.

<p>Shrimp were injected twice with dsRNA specific to <i>Pm</i>MasSPH1 (A) or <i>Pm</i>MasSPH2 (B). At the second dsRNA injection, shrimp were also injected with <i>V</i>. <i>harveyi</i> (2×10⁵ CFU/shrimp). Control groups given injections of GFP dsRNA or NaCl. The number of viable bacteria in the knockdown shrimp hemolymph is shown as the bacterial CFUs 6 h after challenge. The data are shown as the mean ± standard deviation derived from three independent experiments. Means with lower case letters above each bar indicate significant differences (<i>p</i> < 0.05).</p

Expression of <i>Pm</i>MasSPH1 and <i>Pm</i>MasSPH2 transcripts at various stages of shrimp larval development.

<p>Expression profiles of <i>Pm</i>MasSPH1 and <i>Pm</i>MasSPH2 were examined at four larval stages including nauplius 3 (N3), protozoea 2 (Z2), mysis 2 (M2) and post-larvae 15 (PL15) by semi-quantitative RT-PCR. The elongation factor 1-α (EF1-α) served as an internal control. Each lane represents the result of individual shrimp (n = 3).</p

A multiple amino acid sequence alignment of <i>Pm</i>MasSPH1 and <i>Pm</i>MasSPH2 with other arthropod SPHs.

<p>The amino acid sequence of <i>Penaeus monodon Pm</i>MasSPH1 (ABE03741), <i>Pm</i>MasSPH2 (ACP19560), <i>Pacifastacus leniusculus Pl</i>SPH1 (AAX55746), <i>Pl</i>SPH2 (ACB41379), <i>Scylla paramamosain Sp</i>-SPH (ADG83846) and <i>Holotrichia diomphalia Hd</i>PPAFII (CAC12665) were collectively compared. The predicted signal peptides are in bold and underlined. The dash line indicates the glycine-rich domain of <i>Pm</i>MasSPH1. The conserved clip-domains are indicated below the line. The light-grey highlight indicates the cysteine residues in the clip-domains. The black box shows the conserved serine proteinase-like domain with the grey highlight indicating the catalytic triad (His, Asp, and Gly residues) in the domain.</p