Protease SfpB plays an important role in cell membrane stability and immune system evasion in Streptococcus agalactiae.

Bacteria possess the ability to develop diverse and ingenious strategies to outwit the host immune system, and proteases are one of the many weapons employed by bacteria. This study sought to identify S. agalactiae additional serine protease and determine its role in virulence. The S. agalactiae THN0901 genome features one S8 family serine peptidase B (SfpB), acting as a secreted and externally exposed entity. A S8 family serine peptidase mutant strain (ΔsfpB) and complement strain (CΔsfpB) were generated through homologous recombination. Compared to the wild-type strain THN0901, the absorption of EtBr dyes was significantly reduced (P < 0.01) in ΔsfpB, implying an altered cell membrane permeability. In addition, the ΔsfpB strain had a significantly lower survival rate in macrophages (P < 0.01) and a 61.85 % lower adhesion ability to the EPC cells (P < 0.01) compared to THN0901. In the in vivo colonization experiment using tilapia as a model, 210 fish were selected and injected with different bacterial strains at a concentration of 3 × 106 CFU/tail. At 6, 12, 24, 48, 72 and 96 h post-injection, three fish were randomly selected from each group and their brain, liver, spleen, and kidney tissues were isolated. Subsequently, it was demonstrated that the ΔsfpB strain exhibited a markedly diminished capacity for colonization in tilapia. Additionally, the cumulative mortality of ΔsfpB in fish after intraperitoneal injection was reduced by 19.92-23.85 %. In conclusion, the findings in this study have demonstrated that the SfpB plays a significant role in S. agalactiae cell membrane stability and immune evasion. The immune evasion is fundamental for the development and transmission of invasive diseases, the serine protease SfpB may be a promising candidate for the development of antimicrobial agents to reduce the transmission of S. agalactiae.

Gill lesions are the main cause of death in yellowfin seabream (Acanthopagrus latus) following infection with Amyloodinium ocellatum.

Amyloodiniosis, caused by the ectoparasite Amyloodinium ocellatum, affects the healthy development of mariculture. This study used a local infection method to identify the pathogenic target organ responsible for the death of infected fish. Comparing the relationship between the abundance of trophonts in gills and skin with the mortality of infected fish using local infection showed that severe gill infections cause the mortality of infected fish. At the 40 % survival rate of infected fish, the parasite abundance in the gill was 14,167 ± 4371. The gill filaments of the infected fish were structurally disordered, with pronounced lesions associated with the presence of trophonts, such as epithelial cell degeneration and massive lymphocytic infiltration. However, the skin showed no obvious pathological changes. The TUNEL assay showed a significant presence of apoptotic cells concentrated in the area of A. ocellatum infection. The trophonts on the gills developed faster than those parasitising the skin and fins. Microbiome analysis revealed that at the phylum level, Proteobacteria, Bacteroidota, and Firmicutes are abundant in the skin, while Verrucomicrobiota, Bacteroidota, and Proteobacteria are abundant in the gills of A. latus. Furthermore, A. ocellatum infection significantly reduced (p < 0.05) the richness and diversity of the gill microbial community of A. latus. Infection by A. ocellatum increased the relative abundance of several putative pathogenic bacteria (Flavobacterium and Nocardia) in the gill and skin of A. latus, possibly increasing the likelihood of disease in the host. In conclusion, these results evidenced that severe gill infections by A. ocellatum cause mortality in infected fish, which clarifies the direction for exploring the pathogenesis of amyloodiniosis.

Establishment of a gill cell line from yellowfin seabream (Acanthopagrus latus) for studying Amyloodinium ocellatum infection of fish.

Amyloodinium ocellatum is among the most devastating protozoan parasites, causing huge economic losses in the mariculture industry. However, the pathogenesis of amyloodiniosis remains unknown, hindering the development of targeted anti-parasitic drugs. The A. ocellatum in vitro model is an indispensable tool for investigating the pathogenic mechanism of amyloodiniosis at the cellular and molecular levels. The present work developed a new cell line, ALG, from the gill of yellowfin seabream (Acanthopagrus latus). The cell line was routinely cultured at 28°C in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS). ALG cells were adherent and exhibited an epithelioid morphology; the cells were stably passed over 30 generations and successfully cryopreserved. The cell line derived from A. latus was identified based on partial sequence amplification and sequencing of cytochrome B (Cyt b). The ALG was seeded onto transwell inserts and found to be a platform for in vitro infection of A. ocellatum, with a 37.23 ± 5.75% infection rate. Furthermore, scanning electron microscopy (SEM) revealed that A. ocellatum parasitizes cell monolayers via rhizoids. A. ocellatum infection increased the expression of apoptosis and inflammation-related genes, including caspase 3 (Casp 3), interleukin 1 (IL-1), interleukin 10 (IL-10), tumour necrosis factor-alpha (TNF-α), in vivo or in vitro. These results demonstrated that the in vitro gill cell monolayer successfully recapitulated in vivo A. latus host responses to A. ocellatum infection. The ALG cell line holds great promise as a valuable tool for investigating parasite-host interactions in vitro.

Skin transcriptomic analysis and immune-related gene expression of golden pompano (Trachinotus ovatus) after Amyloodinium ocellatum infection.

Amyloodiniosis is a severe disease of marine and brackish water fish caused by Amyloodinium ocellatum. Golden pompano (Trachinotus ovatus) is often repeatedly infected by A. ocellatum, leading to extensive mortality. However, little is known about the immune response mechanisms of the T. ovatus following reinfection with A. ocellatum. In this study, an extensive analysis at the transcriptome level of T. ovatus skin was carried out at 24 h post-infection by A. ocellatum. During the transcriptomic analysis, 1367 differentially expressed genes (DEGs) in the skin of T. ovatus under A. ocellatum infection and control conditions were obtained. In Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotated analyses, the DEGs were significantly enriched in the immune-related pathways. To better understand the immune-related gene expression dynamics, a quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to assess the primary and secondary infection groups of T. ovatus at different stages (3 h, 12 h, 24 h, 48 h and, 72 h post-infection) of infection with A.ocellatum. The results showed that innate immunity-related genes [interleukin (IL-8), chemokine ligand 3 (CCL3), toll-like receptor 7 (TLR7), and G-type lysosome (LZM g)] and adaptive immunity-related gene [major histocompatibility complex (MHC) alpha antigen I and MHC alpha antigen II] expression levels in the primary and secondary infection groups were significantly increased compared to the control group. The expression of MHC I and MHC II was more rapidly upregulated in the secondary infection group compared with the primary infection group after A.ocellatum infection. However, no significant differences of A.ocellatum load were observed in primary and secondary infection groups. In addition, the serum of the primary infection group had significantly higher concentrations of triglyceride (TG), higher alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) activities than the control group. This study contributes to understanding the defense mechanisms in fish skin against ectoparasite infection.

Effects of dietary black soldier fly (Hermetia illucens Linnaeus) on the disease resistance of juvenile grouper (Epinephelus coioides).

An experiment was performed to study the effects of dietary levels of black soldier fly larva meal (BSFLM) on the growth performance, immunity and disease resistance of juvenile grouper (Epinephelus coioides). Four isoproteic and isoenergetic diets were formulated with dietary BSFLM levels of 0 g/kg (T0), 25 g/kg (T2.5), 50 g/kg (T5) and 100 g/kg (T10). Each diet was randomly fed to triplicate groups, each containing 40 fish. The results of the 30-day study indicated that fish growth performance was not affected in the T2.5 and T5 groups compared with the T0 group. In the group with a dietary BSFLM level of 100 g/kg, the feed coefficient was significantly higher than that in the other three groups. The superoxide dismutase, catalase, glutathione peroxidase, lysozyme activity, and malondialdehyde content in the liver, and the interleukin-1 beta (IL-1ß), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α) and heat shock protein 70 (HSP70) expression in the gills, head kidney, liver and spleen remained consistent in all groups. In addition, no significant differences in the cumulative mortality or parasite abundance in groupers after Vibrio harveyi and Cryptocaryon irritans infection were observed. These results suggested that BSFLM supplemented diets did not inhibit disease resistance in groupers.

Production of monoclonal antibody against grouper (Epinephelus coioides) CD4-1 and the distribution of CD4-1+ cells.

CD4-a transmembrane glycoprotein molecule expressed on the surface of helper T (Th) cells-plays a central role in adaptive immune protection. In the current study, we developed a monoclonal antibody (mAb) against the grouper CD4-1. Western blotting and immunohistochemistry results revealed that the CD4-1 mAb could recognize the recombinant and natural protein of grouper CD4-1 as well as the CD4-1+ cells in the various tissues from grouper. Tissue distribution analyses revealed that the grouper CD4-1+ cells were expressed in all tissues tested in the healthy grouper, with greater localization in the thymus, head kidney, and spleen tissues. In addition, we tested the changes in the proportion of CD4-1+ cells in the thymus, head kidney, and the gills of grouper post the infection by C. irritans. Our data suggest that the CD4-1 mAb produced against grouper in the current study can be used as a tool to characterize CD4-1+ cells and to investigate the functions of the grouper CD4-1+ cells in the host response against pathogens infection.

Quantitative detection of parasitic ciliate Cryptocaryon irritans in seawater with an optimized sample processing method.

The protozoan Cryptocaryon irritans is one of the most important ectoparasites of marine fish, causing 'white spot disease' and mass mortality in aquaculture. To accurately predict disease outbreaks and develop prevention strategies, improved detection methods are required that are sensitive, convenient and rapid. In this study, a pair of specific primers based on the C. irritans 18S rRNA gene was developed and used in a real-time PCR (qPCR) assay. This assay was able to detect five theronts in 1 L of natural seawater. Furthermore, a linear model was established to analyse the log of Ct value and parasite abundance in seawater (y = -2.9623x + 24.2930), and the coefficient of determination (R2 ) value was 0.979. A lysis buffer was optimized for theront DNA extraction and used for storage sample. This method was superior to the commercial water DNA kit, and there was no significant degradation of DNA at room temperature for 24-96 hr. A dilution method was developed to manage qPCR inhibitors and used to investigate natural seawater samples in a net cage farm with diseased fish, and the findings were consistent with the actual situation. This study provides a valuable tool for assisting in the early monitoring and control of cryptocaryoniasis in aquaculture.

Study on the characterization of grouper (Epinephelus coioides) immunoglobulin T and its positive cells.

Immunoglobulins (Igs) play a vital role in the adaptive immunity of gnathostomes. IgT, a particular Ig class in teleost fishes, receives much attention concerning the mucosal immunity. While, the characteristic and function of Epinephelus coioides IgT is still unknown. In our study, a polyclonal antibody was first prepared with grouper IgT heavy chain recombinant protein. IgT was revealed to be polymeric in serum and mucus. In normal groupers, IgT had high expression level in head kidney and spleen, while little amount in gills, thymus, gut and liver. The number of IgT-positive cells in different tissues was in line with their IgT expression. Furthermore, IgT could coat fractional bacteria in the mucus. In conclusion, this research revealed the protein characteristic, basal expression and bacterial coverage of grouper IgT. This is the first study to identify the characteristic of grouper IgT and demonstrate the capacity of coating microbes.

Grouper (Epinephelus coioides) IRAK-4 regulates activation of NF-κB and expression of inflammatory cytokines in grouper spleen cells.

IRAK-4 is a serine/threonine kinase that can bind to interleukin-1 receptor induced by interleukin-1. It plays a key role in the Toll-like receptor signaling pathway and is involved in innate and adaptive immune responses. In this study, piscine IRAK-4 significantly activated nuclear factor (NF)-κB signaling in grouper spleen cells. Grouper (Epinephelus coioides) IRAK-4 (EcIRAK-4) co-localized with EcMyD88 and did not impair EcMyD88-dependent NF-κB activation. Different doses of EcIRAK-4 caused different degrees of nuclear translocation of the transcription factor NF-κB p65 subunit, and it induced transcription of multiple pro-inflammatory cytokines. Using expression vectors of deletion domains or mutations at important sites of EcIRAK-4, we found that the EcIRAK-4 kinase domain is necessary for its signal transduction function. The conserved amino acid sites performed functions similar to those in mammals, and grouper-specific amino acids such as E339 also played important roles. These findings provide information about the functional characteristics of IRAK-4 in lower vertebrates.

IκB kinase α-1 and -2 regulate cytokine expression in the orange-spotted grouper (Epinephelus coioides).

IκB kinase (IKK) is the core regulator of the nuclear factor-κB (NF-κB) pathway, which is involved in cellular development and proliferation, as well as the inflammatory response. IKKα is an important subunit of the IKK complex. In this study, two IKKαs (EcIKKα-1 and -2) were characterized in E. coioides. Similar to IKKα of other species, EcIKKα-1 and -2 contained a kinase domain, a leucine zipper, a helix-loop-helix domain and a beta NF-κB essential modulator-binding domain. Sequence alignment indicated that EcIKKα-1 and -2 shared high degrees of sequence identity with IKKs from other species (about 63%-96%). EcIKKα-1 and -2 are widely expressed in all tissues, but have different expression profiles in normal groupers. Additionally, EcIKKα-1 and -2 responded rapidly to Cryptocaryon irritans infection at the local infection site (i.e., gill tissue), but there was no significant change in EcIKKα-2 expression. In GS cells, EcIKKα-1 was uniformly distributed in the cytoplasm, while EcIKKα-2 was observed uniformly both in the cytoplasm and nucleus. Both EcIKKα-1 and -2 were found to activate NF-κB, but the luciferase activity of EcIKKα-2 was twice that of EcIKKα-1. In addition, EcIKKα-1 and -2 can regulate the expression of immune-related cytokines (IL-1ß, IL-6, IL-8, IL-12 [p35 subunit], and TNF-α). These findings should prove helpful to further elucidate the innate immunity function of IKKα in fish.

Distribution of Mpeg1+ cells in healthy grouper (Epinephelus coioides) and after Cryptocaryon irritans infection.

Cryptocaryon irritans is an extremely harmful ciliated obligate parasite that is responsible for large economic losses in aquaculture. C. irritans infection can cause an insect-resistant immune response in fish, and many immune cells can be observed in the local infection site. However, it is unclear whether macrophages are involved in the host defense against C. irritans infection. The Mpeg1 protein can form pores and destroy the cell membrane of invading pathogens, and is also used as a macrophage-specific marker in mammals. Therefore, a polyclonal antibody against grouper recombinant Mpeg1a was produced to mark macrophages in this study, which could recognize both isoforms of Mpeg1 (Mpeg1a/b). Immunofluorescence revealed that EcMpeg1 positive cells were mostly distributed in the head kidney and spleen in healthy grouper. Immunofluorescence and immunohistochemistry showed that the number of EcMpeg1 positive cells increased in the gills after infection with C. irritans, implying that EcMpeg1 positive cells may be involved in the process of grouper resistance against C. irritans infection.

Orange-spotted grouper (Epinephelus coioides) NADPH oxidase: Cloning and expression analysis after Cryptocaryon irritans infection.

Phagocytic cells are activated to produce a large amount of reactive oxygen species (ROS) that kill pathogens quickly and efficiently through oxidation. NADPH oxidase is the main source of intracellular ROS. In the present study, five subunits of the phagocytic NADPH oxidase complex were identified in orange-spotted grouper (Epinephelus coioides). The open reading frame of grouper gp91phox, p22phox, p67phox, p47phox, and p40phox were 1,698 bp, 564 bp, 1,497 bp, 1,290 bp, and 1,050 bp, respectively, and encoded 565, 187, 498, 429, and 349 amino acids. Evolutionary analysis indicated that these proteins are evolutionarily homologous to the corresponding proteins of other fish and mammals, and contain conserved functional domains and sites that are important in mammals. In addition, real-time polymerase chain reaction analysis showed that the expression of these five genes was higher in immune-related tissues in normal grouper, and that these genes were up-regulated in gill and spleen after C. irritans infection, which suggests that these genes may be involved in the defense against C. irritans infection.

Effects of short-term fasting on the resistance of Nile tilapia (Oreochromis niloticus) to Streptococcus agalactiae infection.

Short-term feed deprivation or fasting is commonly experienced by aquaculture fish species and may be caused by seasonal variations, production strategies, or diseases. To assess the effects of fasting on the resistance of Nile tilapia to Streptococcus agalactiae infection, vaccinated and unvaccinated fish were fasted for zero, one, three, and seven days prior to infection. The cortisol levels of both vaccinated and unvaccinated fish first decreased and then increased significantly as fasting time increased. Liver glycogen, triglycerides, and total cholesterol decreased significantly after seven days of fasting, but glucose content did not vary significantly between fish fasted for three and seven days. Hexokinase (HK) and pyruvate kinase (PK) activity levels were lowest after seven days of fasting, while phosphoenolpyruvate carboxykinase (PEPCK) activity levels varied in opposition to those of HK and PK. Serum superoxide dismutase (SOD) and catalase (CAT) activity levels first increased and then decreased as fasting time increased; SOD activity was highest after three days of fasting. Interleukin-1beta (IL-1ß) and IL-6 mRNA expression levels first increased and then decreased significantly, peaking after three days of fasting. However, suppressor of cytokine signaling-1 (SOCS-1) mRNA expression levels were in opposition to those of IL-1ß and IL-6. Specific antibody levels did not vary significantly among unvaccinated fish fasted for different periods. Although specific antibody level first increased and then decreased in the vaccinated fish as fasting duration increased, there were no significant differences in the survival rates of fasted vaccinated fish after challenge with S. agalactiae. The final survival rates of vaccinated fish fasted for zero, one, three, and seven days were 86.67 ±â€¯5.44%, 80.00 ±â€¯3.14%, 88.89 ±â€¯6.28%, and 84.44 ±â€¯8.32%, respectively. Among the unvaccinated fish, the survival rate was highest (35.56 ±â€¯3.14%) in the fish fasted for three days and lowest (6.67 ±â€¯3.14%) in the fish fasted for seven days. Therefore, our results indicated that short-term fasting (three days) prior to an infection might increase the resistance of unvaccinated Nile tilapia to S. agalactiae.

Transcriptome analysis provides insights into molecular immune mechanisms of rabbitfish, Siganus oramin against Cryptocaryon irritans infection.

The rabbitfish Siganus oramin is resistant to the ciliate parasite Cryptocaryon irritans. L-amino acid oxidase (LAAO) protein from rabbitfish can kill C. irritans in vitro, however, other immune defence mechanisms against C. irritans remains unknown. Here, we generated transcriptomes of rabbitfish skin at 12 h post infection (PI) by C. irritans. The transcriptomes contained 238, 504, 124 clean reads were obtained and then assembled into 258,869 unigenes with an average length of 621 bp and an N50 of 833 bp. Among them, we obtained 418 differentially expressed genes (DEGs) in the skin of rabbitfish under C. irritans infection and control conditions, including 336 significantly up-regulated genes and 82 significantly down-regulated genes. Seven immune-related categories with 32 differentially expressed immune genes were obtained using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. DEGs included innate immune molecules, such as LAAO, antimicrobial peptide, lysozyme g, as well as complement components, chemokines and chemokine receptors, NOD-like receptor/Toll-like receptor signaling pathway molecules, antigen processing and T/B cell activation and proliferation molecules. We further validated the expression results of nine immune-related DEGs using quantitative real-time PCR. This study provides new insights into the early immune response of a host that is resistant to C. irritans.

Molecular characteristics and functional study of tumor necrosis factor receptor-associated factor 2 from the orange-spotted grouper (Epinephelus coioides).

In mammals, tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial intracellular adaptor protein, which performs a vital role in numerous signaling pathways that activate NF-κB, MAPKs, and IRFs. In the present study, three TRAF2 sequences were identified from the orange-spotted grouper (Epinephelus coioides), and named EcTRAF2-1, EcTRAF2-2, and EcTRAF2-3. These sequences contained conserved structure features that were similar to those of mammals. EcTRAF2-1 shared relatively low sequence identity with the other two EcTRAF2s. In healthy E. coioides, EcTRAF2s were widely expressed in all tissues tested, but with distinct expression profiles. After infection with Cryptocaryon irritans, EcTRAF2s was markedly upregulated in the gill and head kidney at most time points, implying that EcTRAF2s may be involved in host defense against C. irritans infection. In HEK293T cells, EcTRAF2s were scattered in the cytoplasm. EcTRAF2-1 and EcTRAF2-2 increased the activity of NF-κB, while EcTRAF2-3 reduced NF-κB activation mediated by EcTRAF2-1 implying that EcTRAF2-3 might be a negative regulator of EcTRAF2-1.

GroEL, a novel vaccine candidate of piscine Streptococcus agalactiae identified by immunoproteome.

Streptococcus agalactiae is the major etiological agent of streptococcosis, which is responsible for huge economic losses in fishery, particularly in tilapia (Oreochromis niloticus) aquaculture. A research priority to control streptococcosis is to develop vaccines, so we sought to figure out the immunogenic proteins of S. agalactiae and screen the vaccine candidates for streptococcosis in the present study. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting and mass spectrometry (MS), was employed to investigate the immunogenic proteins of S. agalactiae THN0901. Whole-cell soluble proteins were separated using 2-DE, and the immunogenic proteins were detected by western blotting using rabbit anti-S. agalactiae sera. A total of 17 immunoreactive spots on the soluble protein profile, corresponding to 15 different proteins, were identified by MALDI-TOF/TOF MS. Among the immunogenic proteins, GroEL attracted our attention as it was demonstrated to be immunogenic and protective against other streptococci. Nevertheless, to date, there have been no published reports on the immunogenicity and protective efficacy of GroEL against piscine S. agalactiae. Therefore, recombinant GroEL (rGroEL) was expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. Immunization of tilapia with rGroEL resulted in an increase in antibody titers and conferred protection against S. agalactiae, with the relative percentage survival of 68.61 ±â€¯7.39%. The immunoproteome in the present study narrows the scope of vaccine candidates, and the evaluation of GroEL immunogenicity and protective efficacy shows that GroEL forms an ideal candidate molecule in subunit vaccine against S. agalactiae.

Grouper (Epinephelus coioides) Mpeg1s: Molecular identification, expression analysis, and antimicrobial activity.

Macrophage expressed gene 1 (Mpeg1) is a molecule that can form pores and destroy the cell membrane of invading pathogens. In this study, we identified two Mpeg1 isoforms from the orange-spotted grouper (Epinephelus coioides) and named them EcMpeg1a and EcMpeg1b. Predicted proteins of the two EcMpeg1s contained a signal peptide, a conserved membrane attack complex/perforin (MACPF) domain, a transmembrane segment, and an intracellular region. Sequence alignment demonstrated that two EcMpeg1 proteins share a high sequence identity with that of other teleosts. Tissue distribution analysis showed that EcMpeg1s were expressed in all tissues tested in healthy grouper, with the highest expression in the head kidney and spleen. After infection with the ciliate parasite Cryptocaryon irritans, expression of the two EcMpeg1s was significantly upregulated in the spleen and gills. Furthermore, the recombinant EcMpeg1a showed antiparasitic and antibacterial activity against Gram-negative and -positive bacteria, whereas EcMpeg1b had an inhibitory effect only against Gram-positive bacteria. These results indicated that EcMpeg1s play an important role in the host response against invading pathogens.

Grouper (Epinephelus coioides) MyD88 adaptor-like (Mal): Molecular cloning, expression, and functionality.

Initiation of the innate immune response requires recognition of pathogen-associated molecular patterns by pathogen recognition receptors such as Toll-like receptors (TLRs). MyD88 adaptor-like (Mal) is an adaptor that responds to TLR activation and acts as a bridging adaptor for MyD88. In the present study, the open reading frame of Mal was identified in orange-spotted grouper (Epinephelus coioides), and named EcMal. It contained 831 bp encoding 276 aa, and was encoded by a 1299 bp DNA sequence with three exons and two introns. EcMal and the Mal sequence of other species shared different degrees of sequence identity, and clustered into the same group. EcMal was distributed in all tissues tested in healthy grouper, with the highest expression level in the head kidney. After infection with Cryptocaryon irritans, the expression level of EcMal was up-regulated in the gill and spleen. In addition, EcMal exhibited global cytosolic and nucleus localization, and could significantly activate NF-κB activity in grouper spleen cells.

Molecular characteristics and function study of TNF receptor-associated factor 5 from grouper (Epinephelus coioides).

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is a key adapter molecule that participates in numerous signaling pathways. The function of TRAF5 in fish is largely unknown. In the present study, a TRAF5 cDNA sequence (EcTRAF5) was identified in grouper (Epinephelus coioides). Similar to its mammalian counterpart, EcTRAF5 contained an N-terminal RING finger domain, a zinc finger domain, a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. The EcTRAF5 protein shared relatively low sequence identity with that of other species, but clustered with TRAF5 sequences from other fish. Real-time PCR analysis revealed that EcTRAF5 mRNA was broadly expressed in numerous tissues, with relatively high expression in skin, hindgut, and head kidney. Additionally, the expression of EcTRAF5 was up-regulated in gills and head kidney after infection with Cryptocaryon irritans. Intracellular localization analysis demonstrated that the full-length EcTRAF5 protein was uniformly distributed in the cytoplasm; while a deletion mutant of the coiled-coil domain of EcTRAF5 was observed uniformly distributed in the cytoplasm and the nucleus. After exogenous expression in HEK293T cells, TRAF5 significantly activated NF-κB. The deletion of the EcTRAF5 RING domain or of the zinc finger domain dramatically impaired its ability to activate NF-κB, implying that the RING domain and the zinc finger domain are required for EcTRAF5 signaling.

Virulence regulation of cel-EIIB protein mediated PTS system in Streptococcus agalactiae in Nile tilapia.

Streptococcus agalactiae is a major pathogen of tilapia causing significant economic losses for the global aquatic industry yearly. To elucidate the role of cel-EIIB protein-mediated phosphotransferase systems (PTS) in the virulence regulation of S. agalactiae, cel-EIIB gene deletion in a virulent strain THN0901 was achieved by homologous recombination. The cellobiose utilization of △cel-EIIB strain was significantly decreased relative to S.a.THN0901 strain incubating in LB with 10 mg/ml cellobiose (p < 0.05). The biofilm formation ability of △cel-EIIB strain was also significantly decreased when cultured in BHI medium (p < 0.05). Under a lower infection dose, the accumulative mortality of tilapia caused by △cel-EIIB strain was dramatically decreased (20%), of which S.a.THN0901 strain and △cel-EIIB::i strain were 53.33% and 50%, respectively. The competition experience using tilapia model indicated the invasion and colonization ability of △cel-EIIB strain was significantly weaker than that of S.a.THN0901 strain (p < 0.05). Compared to △cel-EIIB::i strain, the mRNA expression of csrS, csrR, rgfA, rgfC, bgrR and bgrS was significantly downregulated in △cel-EIIB strain (p < 0.05). In conclusion, cel-EIIB protein-mediated cel-PTS not only contributes to biofilm formation and virulence regulation, but also plays an important role in the invasion and colonization of S. agalactiae.