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Bacteria contamination in Auricularia auricularculture bags reduces yield and increases the risk of food safety. In this study, 5 species of bacteria, mainly gram-positive bacteria including three species of Bacillusspp., Arthrobacter arilaitensisand Staphylococcus warneri, were isolated and identified from bacteria-contaminated A. auricularculture bags. An in silicopredicted antimicrobial peptide from the β-1,3-glucan-binding protein sequence of Penaeus vannamei, designated PvGBP2 (FLKLGRKSRYGMLKL), was screened and its antibacterial effect and mechanism of action on the isolated Bacillusspp. explored. The minimal inhibitory concentrations (MIC) of PvGBP2 on Bacillusspp. were 15.6–31.25 μg/mL. Peptide PvGBP2 could inhibit Bacillus subtilisin A. auricularculture bags to maintain growth and yield of A. auricular. Transmission electron microscopy (TEM) revealed that PvGBP2 kills bacteria by perforating the cell wall, destroying membrane integrity and resulting in the leakage of intracellular solutes. In addition, PvGBP2 inhibits biofilm formation by B. subtilisby 90.6 % at 1 × MIC. Thus, peptide PvGBP2 could be potentially applied as an antibacterial agent to control bacterial infection of A. auricularcultivation and the spread of foodborne pathogens.

ABSTRACTAcute hepatopancreatic necrosis disease (AHPND) is a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. The Pir toxin proteins secreted by a unique strain of Vibrio parahaemolyticusplay an essential role in the pathogenesis of AHPND. At present, most studies on the effects of Pir toxin proteins in shrimp focus on digestive tissues or organs such as hepatopancreas, stomach, etc., with none on the immune organs. In the present study, two recombinant Pir toxin proteins (rPirA and rPirB) of V. parahaemolyticuswere expressed with rPirB shown to enter shrimp hemocytes. Employing pull-down and LC-MS/MS analysis, GST-rPirB was found to interact with 13 proteins in hemocytes, including histone H3 and histone H4 and among which histone H4 had the highest protein score. Further analysis using GST pull-down and Far-Western blot analysis revealed that rPirB could interact with histone H4. In addition, using the purified nucleosome protein from DrosophilaS2 cells, it was found that PirB protein could specifically bind to histones. When flow cytometry was applied, it was observed that the interaction between PirB and histones in shrimp hemocytes induces apoptosis, which results in the dephosphorylation of Serine 10 in histone H3. Collectively, the current study shows that in addition to its effect on the digestive tract of shrimp, the PirB toxin protein interacts with histones to affect the phosphorylation of histone H3-S10, thereby inducing apoptosis.

•We investigate a novel LHPP homologue in shrimp P. vannamei(PvLHPP).•PvLHPP responsed to various pathogenic infection.•PvLHPP participate in hemocyte apoptosis with Vibrio parahaemolyticuschallenge.

As a common aquaculture pathogen, Citrobacter freundiihas resulted in inconceivable economic losses in aquaculture. In this study, we investigated the spleen immune response of Lithobates catesbeianusto C. freundiiinfection at the protein level. A comparative proteomic analysis of spleens from C. freundii-infected L. catesbeianuswas performed by using isobaric tags for relative and absolute quantitation (iTRAQ) approaches. A total of 2400 proteins were identified by LC-MS/MS spectra and sequencing data. In addition, 316 differentially expressed proteins (DEPs) were identified, of which 154 were significantly upregulated and 162 were significantly downregulated. Seven DEPs that are immune related including Kelch-like protein 22 (KLHL 22), saxiphilin (Sax), integrin beta-3 (ITGB3), retinoic acid-inducible gene I (RIG-I), laboratory of genetics and physiology 2 (LGP2), E3 ubiquitin-protein ligase TRIM39-like (TRIM39L), and phospholipid scramblase 1 (PLSCR1) were selected for further study. The expression profile of these genes in the spleen at 48 h post-C. freundii-infection showed a similar trend as the iTRAQ data. Altogether, the current study is the first to report the spleen proteome of bullfrogs (L. catesbeianus), providing insight into the immune response to C. freundiiinfection.

NACHT, LRR and PYD domains-containing protein 3 (NLRP3) protein is the core actor involved in inflammasome formation, and plays a pivotal role in the innate immune response. However, whether NLRP3 participates in the regulation of innate immunity in invertebrates is still unknown. In the present study, we characterized a NLRP3 ortholog in Penaeus vannamei(designated PvNLRP3-like) with 2514 bp length of open reading frame (ORF) encoding a putative protein of 837 amino acids. Sequence analysis revealed that PvNLRP3-like contained only NACHT domain and shared closely homology with other invertebrates, but the topological structure of NACHT domain of PvNLRP3-like is similar with that in human NLRP3. PvNLRP3-like was ubiquitously expressed in tissues and induced in hemocytes by Vibrio parahaemolyticus, Streptococcus iniaeand White Spot Syndrome Virus (WSSV) challenge, suggesting that PvNLRP3-like participated in the immune responses to pathogens. Furthermore, silencing of PvNLRP3-like followed by V. parahaemolyticusstimulation negatively regulated the transcripts of antimicrobial peptides (AMPs) including Lysozyme (LYZ) 3, Crustin (CRU) 2, Anti-lipopolysaccharide factor (ALF) 2/3 and Penaeidins (PEN) 3/4. This study enriches our current knowledge on shrimp innate immunity, and provides novel perspective to understand the immune regulation role of PvNLRP3-like.