The flagellin of Vibrio parahaemolyticus induces the inflammatory response of Tetraodon nigroviridis through TLR5M.

Vibrio parahaemolyticus is an important marine pathogen that cause inflammation even death in teleost. It has brought huge economic losses to aquaculture and serious threats to the sustainable development of marine fisheries. Here, we isolated the DNA, RNA, and total flagellin from V. parahaemolyticus, and obtained the primary spleen and head kidney cells (including leukocytes) from Tetraodon nigroviridis. V. parahaemolyticus DNA, RNA, and total flagellin were used to treat the T. nigroviridis primary cells described above. The results show that the nitric oxide (NO) production and respiratory burst response were significantly induced after stimulation with V. parahaemolyticus total flagellin in T. nigroviridis head kidney and spleen cells. And total flagellin could promote the gene expression and protein production of IL-1ß in T. nigroviridis leukocytes. T. nigroviridis TLR5M (TnTLR5M) and TLR5S (TnTLR5S) ORF sequences were obtained as the main recognition receptor for flagellin. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression of pattern recognition receptor TnTLR5M and TnTLR5S, the important signal molecule of inflammatory system TnMyD88 and TnTRAF6, and inflammatory cytokines TnIL-1ß and TnIFN-γ2. The results show that there were a significant upregulation after challenge with V. parahaemolyticus total flagellin. We further demonstrated that the total flagellin of V. parahaemolyticus could activate the luciferase activity of the NF-κB reporter gene mediated by TnTLR5M. Overall, our results suggest that V. parahaemolyticus total flagellin activated the NO production, respiratory burst response, and inflammatory cytokines expressions, such as TnIL-1ß and TnIFN-γ2, through the TnTLR5M-NF-κB signaling pathway in T. nigroviridis.

Efficiency of monovalent and polyvalent Vibrio alginolyticus and Vibrio Parahaemolyticus vaccines on the immune response and protection in gilthead sea bream, Sparus aurata (L.) against vibriosis.

This experimental studies investigated the protective efficiencies and the potential immune mechanisms of vibrio monovalent and polyvalent autogenous formalin-inactivated whole-cell bacterins (FKC) in Gilthead sea bream (Sparus aurata) cultured in Egypt. Two months post-vaccination, the relative percentage survival (RPS) was estimated after challenge with the vaccine's homologues pathogenic strains. The survival values were 100% and 83.3% in groups immunized with monovalent V. alginolyticus or V. parahaemolyticus FKC bacterins, respectively. On the other hand, survival values were 91.75% and 75% in fish groups subjected to polyvalent (V. parahaemolyticus O11: K40 & V. alginolyticus) and (V. parahaemolyticus O3: K6 & V. alginolyticus) FKC bacterins, respectively. Overall, the tested vaccine preparations were significantly increased (P < 0.05) the agglutination antibody titer, phagocytic activity, respiratory burst activity, when compared to the non-immunized control group. The current results conclude that, autogenous Vibrio vaccines provoked a promising protection against vibriosis in Gilthead sea bream cultured in Egypt, it was superior in monovalent FKC V. alginolyticus vaccine and polyvalent FKC of V. parahaemolyticus O11: K40 with V. alginolyticus vaccine that could be useful means of prevention and control of vibriosis.

The role of caspase 3 in the mud crab (Scylla paramamosain) after Vibrio parahaemolyticus infection.

Apoptosis plays essential roles in the immune defense mechanism against pathogen infection. Caspase 3 is a family of cysteine proteases involved in apoptosis and the immune response. In this study, the full-length of mud crab (Scylla paramamosain) caspase 3 (designated as Sp-caspase 3) was cloned and characterized. The open reading frame of Sp-caspase 3 was comprised a 1035 bp, which encoded a putative protein of 344 amino acids. Sp-caspase 3 was ubiquitously expressed in various tissues with a high-level expression in hemocytes. Cellular localization analysis revealed that Sp-caspase 3 was located in the cytoplasm and nucleus. Over-expression of Sp-caspase 3 could induce cell apoptosis. In addition, V. Parahaemolyticus infection induced the relative expression of caspase-3 mRNA and increased caspase-3 activity. Knocking down Sp-caspase 3 in vivo significantly reduced cell apoptosis and increased mortality of mud crab after V. parahaemolyticus infection. These results indicated that Sp-caspase 3 played important roles in the immune response and apoptosis against bacterial infection.

Evaluation of the ß-barrel outer membrane protein VP1243 as a candidate antigen for a cross-protective vaccine against Vibrio infections.

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that causes acute gastroenteritis after the consumption of contaminated food, wound infection, and seizures. Antibiotic therapy is the main method for controlling Vibrio infections, which inevitably leads to drug resistance. Therefore, a vaccine is urgently needed to avoid this problem. Outer membrane proteins (OMPs) play a pivotal role in the interaction between the host immune system and bacteria. VP1243 is an OMP of V. parahaemolyticus, and it possessed immunogenicity in our previous study. The present study found that VP1243 was widely distributed, highly conserved and possessed similar surface epitopes among the major Vibrio species. The protein stimulated a strong antibody response and induced cross-reactive immune responses in V. parahaemolyticus, V. alginolyticus and V. vulnificus. Notably, it provided 100% immune protection against lethal challenges by the three Vibrio species in mice immunized with VP1243. Efficient clearance of cells of the three Vibrio bacterial species was observed in immunized mice. These findings provide solid evidence for VP1243 as a promising candidate for the development of a versatile vaccine to protect against Vibrio infections.

Selection and characterization of a Vibrio parahaemolyticus OmpU antibody by phage display.

Vibrio parahaemolyticus (V. parahaemolyticus) is a well-known food-borne human pathogen that can cause a variety of clinical manifestations after the consumption of raw or undercooked seafoods. The crucial roles of Vibrio OmpU in bacterial pathogenesis have been found in recent studies. In the present study, we screened for single domain antibody fragment (sdAb) candidates that bind to V. parahaemolyticus OmpU by using a sdAb phage display library and isolated several positive phage clones. The UAb28, which was one of the positive clones, was shown high enrichment and affinity. The CDRs of UAb28 are speculated to perform the OmpU binding function by molecular docking. The capable of recognizing OmpU was verified by binding and inhibition assays. The UAb28 might be useful in future studies to develop the potential sdAb-based immunotherapeutics against V. parahaemolyticus infection.

Immunomagnetic separation-based nanogold enhanced surface plasmon resonance and colloidal gold test strips for rapid detection of Vibrio parahaemolyticus.

Nanogold enhanced surface plasmon resonance (SPR), colloidal gold immunochromatographic test strips (ICTS), and polymerase chain reaction (PCR), combined with immunomagnetic separation (IMS) were established in this study for the rapid detection of Vibrio parahaemolyticus (VP). The sensitivities of SPR, ICTS, and PCR was determined to be 101, 103, and 103 CFU/mL for VP, respectively. After separation and enrichment by IMS, the sensitivities of SPR, ICTS, and PCR were 100, 101, and 102 CFU/mL for VP, respectively, which were improved by 10-, 100-, and 10-fold compared to the direct detection by SPR, ICTS, and PCR, respectively. When the VP-polluted water samples were directly assessed by SPR, ICTS, and PCR, the results were negative. By contrast, after separation and enrichment for 45 min by IMS, the results were all positive. The IMS-SPR, IMS-ICTS, and IMS-PCR detection methods were able to yield results in approximately 1.5 h, 55 min, and 3.5 h, respectively. These combined detection methods have advantages in being high-throughput and easy to operate without the need for sophisticated equipment or specialized skills. These methods might aid in the development of SPR, ICTS, and PCR technologies for simultaneously examining multiple food-borne pathogens in food products.

Recombinant Vibrio parahaemolyticus ghosts protect zebrafish against infection by Vibrio species.

Aquatic animals are frequently threated by bacterial pathogens. The most economic and efficient protection against bacterial infection are through vaccine immunization. The various serotypes of the pathogens, such as Vibrios, hurdle the development of the vaccines, especially polyvalent vaccines. Here, we demonstrate that recombinant bacterial ghost is a good candidate for multivalent vaccine. By expressing PhiX174 gene E alone or co-expressing the gene E with two genes encoding outer membrane proteins (VP1667 and VP2369) in V. parahaemolyticus, we generated the recombinant V. parahaemolyticus ghosts VPG and rVPGs respectively. Fish immunized with either VPG or rVPG showed increased survival against the infection by either V. parahaemolyticus or V. alginolyticus, with a better protective effect by immunization with rVPG. Our furthermore studies show that rVPG stimulates stronger innate immune responses by increasing the expression of tnfα, il1ß, il6, il8 and il10 as well as that of c3b, lyz, and tlr5, the key players linking the innate and adaptive immune responses upon microbial stimulation. In summary, VPG and rVPG can protect zebrafish against the infection from at least two Vibrio species, suggesting its potential value for further aquaculture vaccines development.

Identification, functional characterization and the potential role of variable lymphocyte receptor EsVLRA from Eriocheir sinensis in response to secondary challenge after Vibrio parahaemolyticus vaccine.

Variable lymphocyte receptors (VLRs) play an important role via their antigen-special reorganization in jawless vertebrates (agnathans) adaptive immune response. In the present study, the open reading frame (ORF) of Eriocheir sinensis VLRA (designated as EsVLRA) was identified. EsVLRA comprised a 799-amino-acid polypeptide with one LRR_NT domain, thirteen LRR domains and one LRR_CT domain, which showed a high domain consistency of the VLR genes in lamprey (Petromyzon marinus). The transcript of EsVLRA was detected in all examined tissues with the highest level detected in hepatopancreas. Notably, the expression of EsVLRA in hepatopancreas, gonads, gill and intestine of male crabs was significantly higher than that in females. The recombinant EsVLRA exhibited strong bacteria-binding activity rather than antibacterial activity, suggesting its crucial role in immune recognition. Furthermore, 6 h earlier response and a significantly higher peak of EsVLRA mRNA expression was observed after challenge with live Vibrio parahaemolyticus (240.6-fold, P < 0.01, crabs receive secondary challenge after V. parahaemolyticus vaccine to the carbs only receive twice PBS injection, N = 6), compared with those only received first injection with formalin-inactivated V. parahaemolyticus (39.7-fold, P < 0.01, challenge 6 h to vaccination 12 h). The findings of this study together demonstrated that EsVLRA plays an important role in the immune system of E. sinensis, serving as a pattern recognition receptor and involving in the immune priming.

Effects of Chitosan-Gentamicin Conjugate Supplement on Non-Specific Immunity, Aquaculture Water, Intestinal Histology and Microbiota of Pacific White Shrimp (Litopenaeus vannamei).

When the aquaculture water environment deteriorates or the temperature rises, shrimp are susceptible to viral or bacterial infections, causing a large number of deaths. This study comprehensively evaluated the effects of the oral administration of a chitosan-gentamicin conjugate (CS-GT) after Litopenaeus vannamei were infected with Vibrio parahaemolyticus, through nonspecific immunity parameter detection, intestinal morphology observation, and the assessment of microbial flora diversification by 16S rRNA gene sequencing. The results showed that the oral administration of CS-GT significantly increased total hemocyte counts and reduced hemocyte apoptosis in shrimp (p < 0.05). The parameters (including superoxide dismutase, glutathione peroxidase, glutathione, lysozyme, acid phosphatase, alkaline phosphatase, and phenoloxidase) were significantly increased (p < 0.05). The integrity of the intestinal epithelial cells and basement membrane were enhanced, which correspondingly alleviated intestinal injury. In terms of the microbiome, the abundances of Vibrio (Gram-negative bacteria and food-borne pathogens) in the water and gut were significantly reduced. The canonical correspondence analysis (CCA) showed that the abundances of Vibrio both in the water and gut were negatively correlated with CS-GT dosage. In conclusion, the oral administration of CS-GT can improve the immunity of shrimp against pathogenic bacteria and significantly reduce the relative abundances of Vibrio in aquaculture water and the gut of Litopenaeus vannamei.

Differential Recognition of Vibrio parahaemolyticus OmpU by Toll-Like Receptors in Monocytes and Macrophages for the Induction of Proinflammatory Responses.

Vibrio parahaemolyticus is a human pathogen, and it is a major cause of severe gastroenteritis in coastal areas. OmpU is one of the major outer membrane porins of V. parahaemolyticus Host-immunomodulatory effects of V. parahaemolyticus OmpU (VpOmpU) have not been elucidated yet. In this study, in an effort towards characterizing the effect of VpOmpU on innate immune responses of the host, we observed that VpOmpU is recognized by the Toll-like receptor 1/2 (TLR1/2) heterodimer in THP-1 monocytes but by both TLR1/2 and TLR2/6 heterodimers in RAW 264.7 macrophages. To the best of our knowledge, this is the first report of a natural pathogen-associated molecular pattern (PAMP) recognized by both TLR1/2 and TLR2/6 heterodimers; so far, mainly the synthetic ligand Pam2CSK4 has been known to be recognized by both the TLR1/2 and TLR2/6 heterodimers. We also have shown that VpOmpU can activate monocytes and macrophages, leading to the generation of proinflammatory responses as indicated by tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO production in macrophages and TNF-α and IL-6 production in monocytes. VpOmpU-mediated proinflammatory responses involve MyD88-IRAK-1 leading to the activation of mitogen-activated protein (MAP) kinases (p38 and Jun N-terminal protein kinase [JNK]) and transcription factors NF-κB and AP-1. Further, we have shown that for the activation of macrophages leading to the proinflammatory responses, the TLR2/6 heterodimer is preferred over the TLR1/2 heterodimer. We have also shown that MAP kinase activation is TLR2 mediated.

Investigation of direct repeats, spacers and proteins associated with clustered regularly interspaced short palindromic repeat (CRISPR) system of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a ubiquitous bacterium of the marine environment is an important food-borne pathogen responsible for gastroenteritis worldwide. In this study, we aimed to investigate the occurrence and diversity of the CRISPR-Cas system in V. parahaemolyticus genomes using a bioinformatics approach. The CRISPR-Cas system functions as an adaptive immune system in prokaryotes that provides immunity against foreign genetic elements. In total, 570 genomes V. parahaemolyticus genomes were analyzed of which 200 confirmed for the presence of CRISPR-Cas system. The CRISPR-Cas loci were further analyzed for their repeats, spacers and associated Cas proteins. Among the 200 V. parahaemolyticus strains analyzed, 16 (8%) strains possessed the CRISPR-Cas system of complete subtype I-F, while the remaining 184 (92%) harbored the minimalistic type, a subtype I-F variant. Orphan CRISPR repeats and Cas genes were found in one strain each. The CRISPR-associated direct repeat had an unit length of 28 bases. The number of repeat units in each array ranged from 3 to 5 or 5-41 depending on whether they belonged to the minimalistic or complete subtype-IF CRISPR-Cas system, respectively. Of the 768 spacers analyzed in this study, 295 were found to be unique to V. parahaemolyticus. Homology analysis of the conserved spacers revealed matches to plasmids, phages and gut viruses and self chromosomes. Among the CRISPR-associated proteins, Cas5 and Cas7 proteins were found to be conserved. However, variations were seen in the Cas6 protein, which could be grouped into four different types based on their protein length as well as amino acid composition. We present here the diversity and main features of the CRISPR-Cas system in V. parahaemolyticus, which could provide valuable insights in elucidating the role and mechanism of CRISPR/Cas elements in this pathogen.

Attenuated Listeria monocytogenes protecting zebrafish (Danio rerio) against Vibrio species challenge.

Live attenuated bacteria is a promising candidate vector for the delivery of vaccines in clinic trials. In the field of aquaculture industry, live vector vaccine also could provide long-term and effective protection against fish bacterial diseases. In our previous work, we demonstrated attenuated Listeria monocytogenes (Lm) had the potential to be an aquaculture vaccine vector in cellular level and zebrafish model. To further investigate the potential application of attenuated Lm in aquaculture vaccines, the outer membrane protein K (OmpK) from Vibrio parahaemolyticus (V. parahaemolyticus), as a conservative protective antigen, was fused to a new antigen-delivery system, and introduced into double-gene attenuated Lm strain (EGDe-ΔactA/inlB, Lmdd) to get live-vector vaccine strain Lmdd-OmpK. The strain Lmdd-OmpK showed the stable secrete efficacy of OmpK and was tested the cross-protective immunity against Vibrio species. After intraperitoneal administration in zebrafish, Lmdd and Lmdd-OmpK strain both improved the survival rates of zebrafish infected by V. parahaemolyticus, Vibrio alginolyticus (V. alginolyticus) and Vibrio anguillarum (V. anguillarum), respectively. In summary, attenuated Lm is able to protect zebrafish against Vibrio species challenge, illustrating its potential value for further aquaculture vaccines development.

Effects of a co-culture of marine algae and shrimp (Litopenaeus vannamei) on the growth, survival and immune response of shrimp infected with Vibrio parahaemolyticus and white spot virus (WSSV).

In aquaculture, fighting infectious diseases is a necessity. This study measured the immuno-stimulating effect of live macroalgae consumption on Litopenaeus vannamei against Vibrio parahaemolyticus and WSSV infection in two independent bioassays. Shrimps and macroalgae were cultivated in a co-culture with two species of macroalgae separately (Gracilaria vermiculophylla and Dictyota dichotoma), and later, shrimp were infected with V. parahaemolyticus. In another bioassay, shrimp and macroalgae (G. vermiculophylla, D. dichotoma and Ulva lactuca) were grown and subsequently infected with WSSV. For both bioassays, survival after 120 h was determined, the total hemocyte count (TCH) was measured and the activity of superoxide dismutase (SOD) and catalase (CAT) in tissue were measured. The results indicate that the use of macroalgae in co-culture with L. vannamei provides a nutritional benefit that achieves higher growth than the control organisms, as well as improvements of the ammonium concentration and immune response after infection with V. parahaemolyticus and WSSV. A better immune response was obtained in organisms cultured with macroalgae in both bioassays at a ratio of 1.6-1.9 for organisms infected with bacteria and 1.4 to 1.6 times for organisms infected with the virus. In turn, the enzymatic activity of SOD and CAT were higher in the treated organisms relative to the controls in both experiments.

Identification of two LGBPs (isoform1 and isoform2) and their function in AMP expression and PO activation in male hepatopancreas.

Two lipopolysaccharides (LPS) and ß-1, 3-glucan binding protein (LGBP), designated as PcLGBP isoform1 and PcLGBP isoform2, respectively, were identified from Procambarus clarkii in this study. The full-length cDNA of PcLGBP isoform1 was 1308 bp containing an open reading frame (ORF) of 1113 bp encoding a protein of 370 amino acids. The full-length cDNA of PcLGBP isoform2 was 1440 bp containing an ORF of 1245 bp encoding a protein of 414 amino acids. Predicted PcLGBP isoform1 and PcLGBP isoform 2 proteins contained a signal peptide, a glycoside hydrolase domain, and a low-complexity region. The difference between the two LGBP isoforms was that PcLGBP isoform2 had 44 more amino acids behind the signal peptide than the PcLGBP isoform1. The PcLGBP isoform1 and PcLGBP isoform2 transcripts mainly expressed in the hepatopancreas in female and male crayfish. Moreover, the expression levels of the two genes in the hepatopancreas were higher in male than that in female crayfish. Upon being challenged with Vibrio parahaemolyticus or LPS, the expression levels of PcLGBP isoform1 and PcLGBP isoform2 in the hepatopancreas of female and male crayfish were most significantly up-regulated at different time points. The transcripts of anti-lipopolysaccharide factors (ALF5, ALF6, ALF8, and ALF9) and crustins (CRU1, CRU2, CRU3, and CRU4) were evidently down-regulated in the hepatopancreas of V. parahaemolyticus-challenged total PcLGBP (including PcLGBP isoform1 and PcLGBP isoform2)-silenced male crayfish. In addition, the phenoloxidase (PO) activity in the hepatopancreas of male crayfish was evidently higher than that of female crayfish. PcLGBP knock down could significantly decrease the PO activity in the hepatopancreas lysate (HLS) in male crayfish. The PO activity of male crayfish HLS was significantly increased when incubated with a mixture of recombinant LGBP protein and LPS or ß-1, 3 glucan. We conclude that LGBP isoforms from P. clarkii function as a pattern recognition protein for recognizing and binding LPS and ß-1, 3 glucan, and thus regulate the synthesis of antimicrobial peptides and activate the prophenoloxidase system.

Innate immune responses and metabolic alterations of mud crab (Scylla paramamosain) in response to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is one of the major pathogens caused diseases in cultured mud crab (Scylla paramamosain). Mud crabs lack an adaptive immune system, their defenses depend almost on innate immunity. Evaluation of the molecular responses of mud crabs to pathogens is essential for control of disease occurrence in farmed animals. In this study, the impacts of V. parahaemolyticus on immunity-related genes and metabolites in mud crabs of different groups (PG, SG and MG refer to controlled, survival and moribund groups, respectively) were investigated. Our results revealed that V. parahaemolyticus infection stimulated significant expressions of immune-related genes (prophenoloxidase, alpha 2-macroglobulin, lysosomal-associated membrane protein, Rab5, C-type lectin B and anti-lipopolysaccharide factor 5) in the MG within 72 h post-infection. The ATP content was significantly reduced in all tissues except muscle of moribund mud crabs. A total of 668 metabolites (including 190 down-regulated and 145 up-regulated) were identified and assigned to 77 pathways in both SG and MG. Metabolites involved in the saturated fatty acid are up-regulated, whereas unsaturated fatty acid and amino acid metabolisms are down-regulated in the immune system of mud crabs during the bacterial infection in MG. Furthermore, a reduction of hemocyte number and an increase of microbial abundance was found in MG. Our results demonstrated that V. parahaemolyticus induced death of mud crabs through reducing the metabolites associate with energy biosynthesis and innate immune system (i.e. proliferation of hemocyte and melanization), resulting in decrease of ATP in different tissues and failed to clearance of pathogens, respectively. The findings of this study provide a basic information of the responses of mud crab on bacterial infection, which is essential for prevention and control of diseases in mud crab aquaculture.

Antimicrobial properties and phenoloxidase activation of the lectin isolated from kadal shrimp (Metapenaeus dobsoni).

The present study reveals purification and characterization of the lectin from the haemolymph of Metapenaeus dobsoni. The Md-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 68 kDa in SDS-PAGE. Furthermore, the molecular mass was confirmed by MALDI-TOF and functional groups present were analysed by FTIR. The surface morphology of purified Md-Lec displays the homogeneous nature of protein. The X-ray diffraction (XRD) analysis expresses three peaks at 10.7716̊, 21.6258̊ and 31.7523̊which indicate the crystalline nature of the protein and the retention time of 3.068 min evident from HPLC reveals the purity of the sample. Functional analysis of purified Md-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy. It also exhibited phenoloxidase activation, encapsulation and phagocytic activities. In addition, purified Md-Lec showed the broad spectrum of bacterial agglutination activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila, important fish pathogens. Antiviral potential and anticancer activity of purified Md-Lec against CyHV-2 virus and MDA-MB-231 breast cancer cell lines were also evaluated in this study.

Effects of dietary Bacillus subtilis and Shewanella algae in expression profile of immune-related genes from hemolymph of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

B. subtilis and S. algae effects in growth, survival and innate immunity were assessed on L. vannamei juveniles. During 60 days, shrimp were reared in three treatments: Bs, fed with 106 CFU of B. subtilis per gram of commercial feed, Sa, fed with 106 CFU of S. algae per gram of commercial feed and Control (without bacterial addition). Then, the animals were subjected to a V. parahaemolyticus challenge. For this purpose, four treatments were established: Control (shrimp not submitted to probiotic treatments), Vibrio (Vibrio challenged shrimp), Vibrio + Bs (Bs challenged shrimp) and Vibrio + Sa (Sa challenged shrimp). Shrimp hemolymph was sampled 45-days after rearing and 24 h post-challenge for quantification of prophenoloxidase (proPO), lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) and hemocyanin (HEM) transcripts by qPCR. Moreover, shrimp final weight and survival were also verified. B. subtilis administration enhanced shrimp growth and improved proPO, LGBP and HEM expression levels before and after challenge. After 60-days of feeding, Sa final weight was higher than the Control, whereas Vibrio + Sa cumulative mortality after 48 h of Vibrio challenge was lower than Vibrio group. These results could be correlated with the proPO and LGBP up regulation in Vibrio + Sa compared to Vibrio group, protecting L. vannamei from the bacterial infection. Together, these results suggest the probiotic potential of B. subtilis e S. algae in the modulation of immune-related genes as a tool to control V. parahaemolyticus infection inside shrimp.

A Kruppel-like factor from Macrobrachium rosenbergii (MrKLF) involved in innate immunity against pathogen infection.

Kruppel-like factors (KLFs) belong to a family of zinc finger-containing transcription factors that are widely present in eukaryotes. In the present study, a novel KLF from the giant river prawn Macrobrachium rosenbergii (designated as MrKLF) was successfully cloned and characterized. The full-length cDNA of MrKLF was 1799 bp with an open reading frame of 1332 bp that encodes a putative protein of 444 amino acids, including three conserved ZnF_C2H2 domains at the C-terminus. Multiple alignment analysis showed that MrKLF and other crustacean KLFs shared high similarity. Quantitative real-time PCR analysis revealed that MrKLF mRNA was found in different tissues of prawns and detected in the gills, hepatopancreas, and intestines. After the challenge with Vibrio parahaemolyticus and Aeromonas hydrophila, different expression patterns of MrKLF in the gills, intestines, and hepatopancreas were observed. RNA interference analysis indicated that MrKLF was involved in regulating the expression of four antimicrobial peptides, namely, Crustin (Crus) 2, Crus8, anti-lipopolysaccharide factor (ALF) 1, and ALF3. These results help promote research on M. rosenbergii innate immunity.

Identification of two ferritin genes and their expression profiles in response to bacterial challenge in noble scallop Chlamys nobilis with different carotenoids content.

As a major intracellular iron storage protein, ferritin plays important roles in iron homeostasis and innate immunity. In this study, two novel ferritin subunits from noble scallop Chlamys nobilis (CnFer1 and CnFer2) were identified and analyzed. The open reading frame of CnFer1 and CnFer2 was 522 and 519bp long, encoding 173 and 172 amino acids, respectively. Both ferritins contained a putative iron-binding region signature (IBRS). Analysis of putative conserved domains showed the two CnFer genes contained three key domains of ferritin subunits, a ferroxidase diiron center (E25, Y32, E59, E60, H63, E105, and Q139), an iron ion channel (H116, D129, E132) and a ferrihydrite nucleation center (D58, E59, and E62) that present in M type subunits. A putative iron response element (IRE) was observed at both CnFer genes in the 5' UTR. Phylogenetic analysis result suggested that the two genes are cytoplasmic ferritins and have the closest evolution relationship with ferritins from Mizuhopecten yessoensis. The two ferritin genes were wildly expressed in examined tissues and the highest level was found in gill. After V. parahaemolyticus challenged, both CnFer genes were significantly up-regulated suggesting that they are important proteins involved in host immune defense. Moreover, under bacterial challenge, the expression levels of both two genes in Golden scallops (rich in carotenoids) were significantly higher than that in Brown scallops (less in carotenoids) which suggesting that carotenoids enhance the immunity in scallops to defense against the bacterial stress.

Exploration of the influence of surface proteins on the probiotic activity of Lactobacillus pentosus HC-2 in the Litopenaeus vannamei midgut via label-free quantitative proteomic analysis.

Our previous work showed that using Lactobacillus pentosus HC-2 as a probiotic could improve the growth performance, immune response, gut bacterial diversity and disease resistance of Litopenaeus vannamei. However, the probiotic mechanism had not been fully characterized. In the present study, histology and proteomic analysis were performed to explore the influence of HC-2 surface protein on its probiotic effects on L. vannamei after feeding either the intact surface proteins, the probiotic treated with lithium chloride (LiCl) to remove noncovalently bound surface proteins or no probiotic for four weeks. Histological observation found that feeding with normal HC-2 obviously improved the intestinal histology and enhanced the protective effect against pathogen damage, but feeding with LiCl-treated HC-2 did not improve the intestinal environment. A total of over 2764 peptides and 1118 uniproteins were identified from the L. vannamei midgut; 211 proteins were significantly differentially expressed in the normal HC-2 group compared with the control group; 510 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the control group, and 458 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the normal HC-2 group. GO/KEGG enrichment analysis of the significantly different proteins demonstrated that feeding normal HC-2 mainly induced immune response, metabolic, cell adhesion and cell-cell signaling-related protein upregulation, which contributed to bacterial adhesion and colonization in the midgut to improve the shrimp immune system and growth, but these proteins were suppressed after the shrimp were fed bacteria deprived of surface proteins. Taken together, these results indicate that the surface proteins were indispensable for HC-2 to execute probiotic effects in the shrimp midgut.