Vibrio alginolyticus PEPCK Mediates Florfenicol Resistance through Lysine Succinylation Modification.

Protein succinylation modification is a common post-translational modification (PTM) that plays an important role in bacterial metabolic regulation. In this study, quantitative analysis was conducted on the succinylated proteome of wild-type and florfenicol-resistant Vibrio alginolyticus to investigate the mechanism of succinylation regulating antibiotic resistance. Bioinformatic analysis showed that the differentially succinylated proteins were mainly enriched in energy metabolism, and it was found that the succinylation level of phosphoenolpyruvate carboxyl kinase (PEPCK) was highly expressed in the florfenicol-resistant strain. Site-directed mutagenesis was used to mutate the lysine (K) at the succinylation site of PEPCK to glutamic acid (E) and arginine (R), respectively, to investigate the function of lysine succinylation of PEPCK in the florfenicol resistance of V. alginolyticus. The detection of site-directed mutagenesis strain viability under florfenicol revealed that the survival rate of the E mutant was significantly higher than that of the R mutant and wild type, indicating that succinylation modification of PEPCK protein may affect the resistance of V. alginolyticus to florfenicol. This study indicates the important role of PEPCK during V. alginolyticus antibiotic-resistance evolution and provides a theoretical basis for the prevention and control of vibriosis and the development of new antibiotics.

The immune function of heme oxygenase-1 (HO-1) from Nile tilapia (Oreochromis niloticus) in response to bacterial infection.

Heme oxygenase-1 (HO-1), an inducible rate-limiting metabolic enzyme, exerts critical immunomodulatory functions by potential anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Although accumulative studies have focused on the immune functions of HO-1 in mammals, the roles in fish are poorly understood, and the reports on involvement in the defensive and immune response are very limited. In this study, On-HO-1 gene from Oreochromis niloticus was successfully cloned and identified, which contained an open reading frame (ORF) of 816 bp and coded for a protein of 271 amino acids. The On-HO-1 protein phylogenetically shared a high homology with HO-1 in other teleost fish (76.10%-98.89 %) and a lowly homology with HO-1 in mammals (38.98%-41.55 %). The expression levels of On-HO-1 were highest in the liver of healthy tilapias and sharply induced by Streptococcus agalactiae or Aeromonas hydrophila. Besides, On-HO-1 overexpression significantly increased non-specific immunological parameters in serum during bacterial infection, including LZM, SOD, CAT, ACP, and AKP. It also exerted anti-inflammatory and anti-apoptotic effects in response to the immune response of the infection with S. agalactiae or A. hydrophila by upregulating anti-inflammatory factors (IL-10, TGF-ß), autophagy factors (ATG6, ATG8) and immune-related pathway factors (P65, P38), and down-regulating pro-inflammatory factors (IL-1ß, IL-6, TNF-α), apoptotic factors (Caspase3, Caspase9), pyroptosis factor (Caspase1), and inflammasome (NLRP3). These results suggested that On-HO-1 involved in immunomodulatory functions and host defense in Nile tilapia.

A CD6 homolog of Nile tilapia (Oreochromis niloticus) conserved binding bacteria involved in the regulation of Streptococcus agalactiae induced inflammation.

As a lymphocyte-specific surface receptor belonging to the cysteine-rich superfamily of scavenger receptors, CD6 acts as a pattern recognition receptor for microbial components and is involved in the regulation of inflammatory responses. However, the characteristics and functions of CD6 molecules in lower vertebrates represented by teleost fish are unknown. In this study, a CD6 homolog (designated OnCD6) was characterized from Nile tilapia (Oreochromis niloticus), and establishing its role as a PRRs that participates in immune recognition. OnCD6 contains an open reading frame of 1872 bp that encodes a peptide of 623 amino acids, and contains two conserved SR domain. Multiple sequence alignment revealed that OnCD6 shares a relatively high level of identity with those of other species. Transcriptional expression analysis revealed that OnCD6 was constitutively expressed in immunes tissues such as head kidney and thymus. The expression level of OnCD6 in mainly immune tissues were found significantly upregulated after the injection of Streptococcus agalactiae (S. agalactiae). Moreover, OnCD6 protein was located in the head kidney and brain, mainly over the plasma membrane of lymphocytes in these immune tissues. In vitro experiments showed that CD6 extracellular protein bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of bacterial surface conserved components LPS and LTA etc. In vivo experiments demonstrated that overexpression OnCD6 before S. agalactiae challenge significantly improved tilapia survival, and this was concomitant with reduced bacterial load and pro-inflammatory cytokines (IL-1ß and TNF-α). Taken together, our results illustrated the function of CD6 molecular pattern recognition receptors (PRRs) is conserved and plays an important role in antibacterial infection.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

The role of HMGB2 in the immune response of Nile tilapia (Oreochromis niloticus) to streptococcal infection.

High mobility group protein B2 (HMGB2) is an abundant chromatin-associated protein with pivotal roles in transcription, cell proliferation, differentiation, inflammation, and tumorigenesis. However, its immune function in Nile tilapia (Oreochromis niloticus) remains unclear. In this study, we identified a homologue of HMGB2 from Nile tilapia (On-HMGB2) and investigated its functions in the immune response against streptococcus infection. The open reading frame (ORF) of On-HMGB2 spans 642 bp, encoding 213 amino acids, and contains two conserved HMG domains. On-HMGB2 shares over 80 % homology with other fish species and 74%-76 % homology with mammals. On-HMGB2 was widely distributed in various tissues, with its highest transcript levels in the liver and the lowest in the intestine. Knockdown of On-HMGB2 promoted the inflammatory response in Nile tilapia, increased the bacterial load in the tissues, and led to elevated mortality in Nile tilapia following Streptococcus agalactiae infection. Taken together, On-HMGB2 significantly influences the immune system of Nile tilapia in response to streptococcus infection.

Molecular characteristics and functional analysis of non-specific cytotoxic cell receptor (NCCRP1) in golden pompano (Trachinotus ovatus).

Non-specific cytotoxic cells (NCCs) are cytotoxic cell population found in innate immune system of teleost, playing crucial role in immune defense. Non-specific cytotoxic cell receptor protein 1 (NCCRP1) is responsible for recognizing target cells and activating NCCs. That said, since the studies regarding NCCs' role in fish during pathogen infection are few, it is necessary to conduct more comprehensive studies. In this study, we identified NCCRP1 from Trachinotus ovatus (ToNCCRP1). The open reading frame of ToNCCRP1 was found to be 702 bp, encoding a protein of 233 amino acids. Additionally, ToNCCRP1 contained a conserved F-box-associated domain and exhibited more than 61 % similarity to NCCRP1 in other fish species. Quantitative real-time PCR analysis showed that ToNCCRP1 mRNA was generally expressed in all tissues, with the highest level expressed in the liver. Furthermore, the expression of ToNCCRP1 was significantly upregulated following infection with Streptococcus iniae. In vitro experiments demonstrated that recombinant ToNCCRP1 possessed bacterial agglutination and binding capabilities, suggesting its antibacterial function. Additionally, we investigated the immune response of head kidney leukocytes (HKLs) to ToNCCRP1. The challenge experiments revealed that ToNCCRP1 played a role in the immune response by influencing the inflammatory response, regulating signaling pathways and apoptosis in HKLs. These findings suggest that NCCRP1 is involved in the immune defense against pathogenic infections in golden pompano, providing insights into the immune mechanisms of teleost.

Functional properties of ATPIF1 in the orange-spotted grouper (Epinephelus coioides) in response to viral infection.

ATP synthase inhibitory factor 1 (ATPIF1) can activate mitochondrial autophagic pathway and mediates immune response by regulating ATP synthase activity. However, the role of fish ATPIF1 on viral infection is still unknown. In this study, we identified an ATPIF1 homolog (Ec-ATPIF1) from orange-spotted grouper (Epinephelus coioides). Ec-ATPIF1 is mainly expressed in the kidney and liver. The expression of Ec-ATPIF1 was significantly up-regulated after RGNNV stimulation in vitro. Further experiments showed that overexpression of Ec-ATPIF1 inhibited the expression of viral genes (CP and RdRp) and intracellular ATP synthesis. Ec-ATPIF1 overexpression also promoted the expression of mitophagy related genes (PINK1, Parkin, BNIP3, NIX, FUNDC1, LC3), inflammation-related factors (IL-1ß, IL-6, IL-8, IL-10, TNF-α, TLR2) and interferon pathway factors (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5, TRIF). While the knockdown of Ec-ATPIF1 exhibited the opposite effects on the expression of viral genes and immune-related factors above. These data suggest that Ec-ATPIF1 can impact viral infection by regulating mitophagy, ATP synthesis, the expression of inflammatory factors and interferon pathway factors. These findings will be beneficial to better explore the immune regulatory mechanisms of fish respond to viral infection.

Characterization and functional analysis of SOCS9 from orange-spotted grouper (Epinephelus coioides) during virus infection.

The suppressor of cytokine signaling (SOCS) proteins family have twelve members including eight known mammalian SOCS members (CISH, SOCS1-7) and four new discovery members (SOCS3b, SOCS5b, SOCS8 and SOCS9) that is regarded as a classic feedback inhibitor of cytokine signaling. Although the function of the mammalian SOCS proteins have been well studied, little is known about the roles of SOCS in fish during viral infection. In this study, the molecular characteristics of SOCS9 from orange-spotted grouper (Epinephelus coioides, EcSOCS9) is investigated. The EcSOCS9 protein encoded 543 amino acids with typical SH2 (389-475aa) and SOCS_box (491-527aa), sharing high identities with reported fish SOCS9. EcSOCS9 was expressed in all detected tissues and highly expressed in kidney. After red-spotted grouper nervous necrosis virus (RGNNV) infection, the expression of EcSOCS9 was significantly induced in vitro. Furthermore, EcSOCS9 overexpression enhanced RGNNV replication, promoted virus-induced mitophagy that evidenced by the increased level of LC3-Ⅱ, BCL2, PGAM5 and decreased level of BNIP3 and FUNDC1. Besides, EcSOCS9 overexpression suppressed the expression levels of ATP6, CYB, ND4, ATP level and induced ROS level. The expression levels of interferon (IFN) related factors (IRF1, IRF3, IRF7, P53), inflammatory factors (IL1-ß, IL8, TLR2, TNF-α) and IFN-3, ISRE, NF-κB, AP1 activities were also reduced by overexpressing EcSOCS9. These date suggests that EcSOCS9 impacts RGNNV infection through modulating mitophagy, regulating the expression levels of IFN- related and inflammatory factors, which will expand our understanding of fish immune responses during viral infection.

Identification and characterization of four Bacillus species from the intestine of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂), their antagonistic role on common pathogenic bacteria, and effects on intestinal health.

As an alternative to the criticized antibiotics, probiotics have been adopted for their eco-friendly nature and ability to enhance host growth and immunity. Nevertheless, reports suggest ineffectiveness in commercially available probiotics since most are from non-fish sources; thus, this study was envisaged to isolate and characterize new Bacillus spp. from the gut of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) which could serve as potential probiotics. The isolation and characterization were performed based on their morphological and biochemical properties, and 16S rRNA sequencing homology analysis. A subsequent 30-day in vivo biosafety feeding trial was conducted to ascertain isolates' non-pathogenicity, as well as their effects on fish growth, and intestinal mucosal microvilli via scanning electron microscopy (SEM) analysis. Four Bacillus spp. strains, namely, B. velezensis strain PGSAK01 (accession number OQ726606), B. stercoris strain PGSAK05 (accession number OQ726607), B. velezensis strain PGSAK17 (accession number OQ726601), and B. subtilis strain PGSAK19 (accession number OQ726605), were identified and characterized in the current study. The strains showed promising probiotic properties such higher adhesion capability, higher thermotolerance, displaying higher survivability to 0.5 % bile, lower pH tolerance, γ-haemolytic activity, and multispecies characteristics. Among the 24 antibiotics tested, while all isolates showed susceptibility to 21, the PGSAK01 strain showed resistance to furazolidone antibiotics. None of the isolates showed possession of i) virulence factor genes encoding enterotoxigenic (hblA, hblC, hblD, nheA, nheB, and entFM) and emetic (cereulide synthetase gene, ces) genes, and ii) streptomycin resistance gene (vat c), ampicillin-resistant genes (mecA and bla), and vancomycin-resistant gene (van B). Nevertheless, the PGSAK01 and PGSAK17 strains showed possession of tek K, cat, and ant(4')-Ia (adenylyltransferase) (except the PGSAK01) resistant genes. All isolates displayed better antimicrobial effects against pathogenic bacteria Streptococcus agalactiae, S. iniae, Vibrio harveyi, and V. alginolyticus. The in vivo biosafety trial involved hybrid grouper fish being grouped into five (average weight 32 ± 0.94 g), namely, the group fed the basal diet void of isolate's supplementation (control), and the remaining four groups fed the basal diet with 1 × 108 CFU/g diet of individual strain PGSAK01, PGSAK05, PGSAK17, and PGSAK19 supplementation. At the end of the study, a significantly higher WGR, K (except the PGSAK01 group), VSI; lysozyme (except PGSAK01 group), total antioxidant activity, alkaline phosphatase, superoxide dismutase enzyme activities; highly dense intestinal mucosal villi (based on the scanning electron microscopy analysis); and significantly lower malondialdehyde levels were witnessed in the isolated treated groups compared to the control, supporting the results obtained in the auto-aggregation and cell-surface hydrophobicity test. This work's results have provided thought-provoking targets; thus, studies involving extensive genome sequencing and functional annotation analysis will be explored to offer unfathomable insights into their mechanisms of action and potential health benefits, further establishing the four Bacillus strains' (PGSAK01, PGSAK05, PGSAK17, and PGSAK19) potential role in probiotic fields and functional foods.

A novel study of brain microvascular endothelial cells induced by astrocyte conditioned medium for constructing blood brain barrier model in vitro: A promising tool for meningitis of teleost.

The blood-brain barrier (BBB) is mainly composed of specialized endothelial cells, which can resist harmful substances, transport nutrients, and maintain the stability of the brain environment. In this study, an endothelial cell line from tilapia (Oreochromis niloticus) named TVEC-01 was successfully established. During the earlier establishment phase of the cell line, the TVEC-01 cells were persistently exposed to an astrocyte-conditioned medium (ACM). TVEC-01 cells were identified as an endothelial cell line. TVEC-01 cells retained the multiple functions of endothelial cells and were capable of performing various experiments in vitro. Furthermore, TVEC-01 cells efficiently expressed BBB-related tight junctions and key efflux transporters. From the results of the qRT-PCR, we found that the TVEC-01 cell line did not gradually lose BBB characteristics after persistent and repetitive passages, which was different from the vast majority of immortalized endothelial cells. The results showed that ACM induced up-regulation of the expression levels of multiple BBB-related genes in TVEC-01 cells. We confirmed that Streptococcus agalactiae was capable of invading the TVEC-01 cells and initiating a series of immune responses, which provided a theoretical basis for S. agalactiae to break through the BBB of teleost through the transcellular traversal pathway. In summary, we have successfully constructed an endothelial cell line of teleost, named TVEC-01, which can be used in many experiments in vitro and even for constructing BBB in vitro. Moreover, it was confirmed that S. agalactiae broke through the BBB of teleost through the transcellular traversal pathway and caused meningitis.

Immune response and protective efficacy of Streptococcus agalactiae vaccine coated with chitosan oligosaccharide for different immunization strategy in nile tilapia (Oreochromis niloticus).

In the past decade, the outbreak of Streptococcus agalactiae has caused significant economic losses in tilapia farming. Vaccine immunization methods and strategies have gradually evolved from single-mode to multi-mode overall prevention and control strategies. In this study, an inactivated vaccine of S. agalactiae with a chitosan oligosaccharide (COS) adjuvant was constructed using different administration methods: intraperitoneal injection (Ip), immersion combined with intraperitoneal injection (Im + Ip), immersion combined with oral administration (Im + Or), and oral administration (Or). Safety analysis revealed no adverse effects on tilapia, and the vaccine significantly promoted fish growth and development when administered through Im + Or or Or immunization. Following vaccination, innate immunity parameters including SOD, ACP and CAT activities were all significantly enhanced. Additionally, specific serum IgM antibodies reached their highest level at the 6th week post vaccination. Skin and intestinal mucus IgT antibodies reached peaked at the 6th and 7th week post vaccination, respectively. The relative peak expression values for IL-8, IL-12, MHC-I, MHC-II, IgM, IgT, CD4, CD8, TNFα, IFNγ from Im + Ip group were significantly higher than those in Ip group, Im + Or group and Or group in most cases (p < 0.05). Importantly, the relative protection survival of Im + Ip group was the highest (78.6%), followed by the Ip group (71.4%), the Or group (64.3%) and the Im + Or group (57.1%). In summary, this study encourages further research on multi-channel immunization strategies of other kinds of vaccines in other aquatic economic animals to improve their disease resistance.

Effects of Chinese herbal medicines on growth performance, intestinal flora, immunity and serum metabolites of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatu♂).

The immunomodulatory roles of Chinese herbal Medicine (CHM) in aquatic animals have been well-recorded. However, how CHM impacts the intestinal microbiota and serum metabolism is not fully understood. In this study, the effects of different additive levels of CHM on the growth performance, immunity, intestinal flora and serum metabolism of hybrid grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatu) were investigated. The addition of 0.5%, 1.0%, 1.5% and 2.0% Chinese herbal medicine compound to feed could significantly improve the weight gain rate (WGR), specific growth rate (SGR) and survival rate (SR) of grouper, reduced feed coefficient, while had no significant difference on morphometric parameter. The most significant improvement for the parameters above was observed in 1.5% group. Different addition levels of CHM could also significantly enhance the activities of ACP, AKP, SOD, CAT and LZM in serum. Accordingly, the supplementation of CHM significantly induced up-regulation of immune genes such as IL-8, IL-1ß, TNF-α, Nrf2, Lzm in the liver, spleen and head kidney of grouper, improved the resistance of grouper to V. harveyi as well. The intestinal flora analysis showed that at the phylum level, the main dominant species of intestinal microorganisms were Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota, Gemmatimonadota, Desulfobacterota, Fusobacteriota and Myxococcota. At the genus level, the high abundance was Lactobacillus, Streptococcus, Bacteroides, Escherichia, Romboutsia, Sphingomonas and Muribaculaceae. The abundance of probiotics (such as Lachnospiraceae, Lactobacillaceae, Streptococcaceae, etc) in CHM-supplement groups were higher (highest in 1.5% group) compared with control group. Moreover, a total of 11 common differential metabolic pathways were screened by LC-MS metabolism analysis of serum, they were Neuroactive ligand-receptor interaction, Purine metabolism, Linoleic acid, Glycerophospholipid metabolism, Taurine and hypotaurine metabolism, Arginine and proline metabolism, ABC transporters, Aminoacyl-tRNA biosynthesis, Arachidonic acid metabolism, Drug metabolism-cytochrome P450, alpha-Linolenic acid metabolism. Also, three common differential metabolites (PI(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/22:1(13Z)), PC(22:0/20:4(5Z,8Z,11Z,14Z)) associated with intestinal health, growth and disease resistance was found. These data will contributes to a comprehensive understand for the regulatory roles of CHM on fish, which is also beneficial for the disease control and sustainable development of aquaculture.

Molecular characterization and functional analysis of CD209E from Nile Tilapia (Oreochromis Niloticus) involved in immune response to bacterial infection.

CD209 plays significant roles in pathogen recognition, innate and adaptive immunity, and cell-cell interactions. In the present study, a CD209 antigen-like protein E from Nile tilapia (Oreochromis niloticus) (designated as OnCD209E) was identified and characterized. OnCD209E contains an open reading frame (ORF) of 771 bp encoding a 257 amino acid protein, as well as the carbohydrate recognition domain (CRD). Multiple sequence analysis exhibits that the amino acid sequence of OnCD209E was relatively high homologous to that of partial fish, especially the highly conserved CRD, in which four conserved disulfide-bonded cysteine residues, WIGL conserved motif and two Ca2+/carbohydrate-binding sites (EPD and WFD motifs) were founded. Quantitative real-time PCR and Western Blot revealed that OnCD209E mRNA/protein is generally expressed in all tissues examined, but with wealth in head kidney and spleen tissues. The mRNA expression of OnCD209E was significantly increased in brain, head kidney, intestine, liver, and spleen tissues in response to the stimulation with polyinosinic-polycytidylic acid, Streptococcus agalactiae and Aeromonas hydrophila in vitro. Recombinant OnCD209E protein exhibited detectable bacterial binding and agglutination activity against different bacteria as well as inhibited the proliferation of tested bacteria. Subcellular localization analysis revealed that OnCD209E was mostly localized in the cell membrane. Moreover, overexpression of OnCD209E could activate nuclear factor-kappa B reporter genes in HEK-293T cells. Collectively, these results demonstrated that CD209E may potentially involve in immune response of Nile tilapia against bacterial infection.

Apolipoprotein Eb (On-ApoEb) protects Oreochromis niloticus against Streptococcus agalactiae infection.

Apolipoprotein E (ApoE), a critical targeting protein, has been found to play an essential role in the protection against infection and inflammation. However, the immune functions of ApoE against bacterial infection in fish have not been investigated. In this study, a full-length cDNA for ApoE, named On-ApoEb was cloned from Oreochromis niloticus. The predicted cDNA sequence was 831bp in length and coded for a protein of 276 amino acid residues, which shared 63.87%-98.55% identity with ApoEb from other fishes, and about 22% identity with ApoEb from mammals. On-ApoEb from O. niloticus was highly expressed in the liver and could be activated in the tissues (liver, spleen, brain, and intestine) after infection with Streptococcus agalactiae. Moreover, the results revealed that On-ApoEb could decrease the expression levels of pro-inflammatory factors, immune-related pathways, and apoptosis, while increasing the expression levels of anti-inflammatory factors. Furthermore, On-ApoEb was noted to improve the survival rate and reduce the bacterial load in the liver and spleen. These results suggested that On-ApoEb was connected with immune response and had anti-inflammation and anti-apoptosis activities.

A novel perlucin-like protein (PLP) protects Litopenaeus vannamei against Vibrio harveyi infection.

C-type lectins (CTLs), as pattern recognition receptors (PRRs), play an important role in the innate immunity of Litopenaeus vannamei. In this study, a novel CTL, named perlucin-like protein (PLP), was identified from L. vannamei, which shared homology sequences of PLP from Penaeus monodon. PLP from L. vannamei was expressed in the hepatopancreas, eyestalk, muscle and brain and could be activated in the tissues (hepatopancreas, muscle, gill and intestine) after infection with the pathogen Vibrio harveyi. Bacteria (Vibrio alginolyticus, V. parahaemolyticus, V. harveyi, Streptococcus agalactiae and Bacillus subtilis) could be bound and agglutinated by the PLP recombinant protein in a Ca2+-dependent manner. Moreover, PLP could stabilise the expression of the immune-related genes (ALF, SOD, HSP70, Toll4 and IMD) and apoptosis gene (Caspase2). The RNAi of PLP could remarkably affect the expression of antioxidant gene, antimicrobial peptide genes, other CTLs, apoptosis genes, Toll signaling pathways, and IMD signaling pathways. Moreover, PLP reduced the bacterial load in the hepatopancreas. These results suggested that PLP was involved in the innate immune response against V. harveyi infection by recognising bacterial pathogens and activating the expression of immune-related and apoptosis genes.

Apolipoprotein A-I (ApoA-I) protects Nile tilapia (Oreochromis niloticus) against bacterial infection.

Apolipoprotein A-I (ApoA-I) is a lipoprotein involved in a variety of physiological and pathological processes. However, the immunomodulatory functions of ApoA-I in fish are not well understood. In this study, ApoA-I from Nile tilapia (Oreochromis niloticus) (On-ApoA-I) was identified, and its function in bacterial infection was investigated. The open reading frame of On-ApoA-I is 792 bp, which codes for a protein containing 263 amino acids. On-ApoA-I shared over 60% sequence similarity with other teleost fish and more than 20% with mammalian ApoA-I. On-ApoA-I was found to be highly expressed in the liver and significantly induced during Streptococcus agalactiae infection by qRT‒PCR analysis. Furthermore, invivo studies revealed that recombinant On-ApoA-I protein could suppress inflammation and apoptosis and improve the likelihood of surviving bacterial infection. Additionally, On-ApoA-I showed invitro antimicrobial properties against Gram-positive and Gram-negative bacteria. These findings offer a theoretical basis for further investigations into the role of ApoA-I in fish immunology.

Immune response and protective efficacy of mannosylated polyethylenimine (PEI) as an antigen delivery vector, administered with a Streptococcus agalactiae DNA vaccine in Nile tilapia (Oreochromis niloticus).

This study examined the effectiveness of a DNA vaccine for S. agalactiae that was delivered by mannose-based polyethyleneimine (Man-PEI). The results showed that Man-PEI/pcDNA-Sip stimulated a higher serum antibody titer compared to control or other vaccine groups (p < 0.05). Additionally, it induced higher expression of immune-related genes, and increased activities of superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Furthermore, the Man-PEI/pcDNA-Sip group showed an improved relative percent survival (RPS) of 85.71%. These results demonstrate the potential value of Man-PEI as a vaccine delivery vehicle, and suggest that it can be effective in boosting the immune protective rate induced by pcDNA-Sip vaccines.

A novel C-type lectin for Litopenaeus vannamei involved in the innate immune response against Vibrio infection.

C-type lectins (CTLs), as a member of pattern recognition receptors, play a vital role in the innate immune response of invertebrates to eliminate micro-invaders. In this study, a novel CTL of Litopenaeus vannamei, namely, LvCTL7, was successfully cloned, with an open reading frame of 501 bp and a capability to encode 166 amino acids. Blast analysis showed that the amino acid sequence similarity between LvCTL7 and MjCTL7 (Marsupenaeus japonicus) was 57.14%. LvCTL7 was mainly expressed in hepatopancreas, muscle, gill and eyestalk. Vibrio harveyi can significantly affect LvCTL7 expression level in hepatopancreases, gills, intestines and muscles (p < 0.05). LvCTL7 recombinant protein can bind to Gram-positive bacteria (Bacillus subtilis) and Gram-negative bacteria (Vibrio parahaemolyticus and V. harveyi). It can cause the agglutination of V. alginolyticus and V. harveyi, but it had no effect on Streptococcus agalactiae and B. subtilis. The expression levels of SOD, CAT, HSP 70, Toll 2, IMD and ALF genes in the challenge group added with LvCTL7 protein were more stable than those in the direct challenge group (p < 0.05). Moreover, knockdown of LvCTL7 by double-stranded RNA interference downregulated the expression levels of genes (ALF, IMD and LvCTL5) that protect against bacterial infection (p < 0.05). These results indicated that LvCTL7 had microbial agglutination and immunoregulatory activity, and it was involved in the innate immune response against Vibrio infection in L. vannamei.

Involvement of Nrf2 in the immune regulation of Litopenaeus vannamei against Vibrio harveyi infection.

NF-E2-related factor-like-2 (Nrf2) is a transcription factor that belongs to the Cap'n'Collar transcription factor family and plays a role in regulating inflammation, autophagy, metabolism, proteostasis, and cancer prevention. However, its influence on Vibrio spp infection in L. vannamei remains uncertain. In this study, the effects of Nrf2 on the immune response in Vibrio spp infection was determined by RT-PCR and histopathological analysis. The results showed that RNAi of Nrf2 significantly decreased the expression of antioxidant-related genes (CAT, SOD and GST; p < 0.05), and significantly up-regulated inflammation-related genes (IMD, pro-PO, P38, Toll, Hsp70, NFκB and RAB6A; p < 0.05) and the apoptosis gene (caspase3). Under the infection of V. harveyi, histopathological analysis showed that after RNAi of Nrf2, the hepatopancreas of shrimp has an abnormal arrangement of hepatic tubules and vacuolization of hepatocyte; The basement membrane is peeled off and the epithelial cells are massively necrotic. Compared with the RNAi of Nrf2 group, the tissue damage in the SFN group was much lessened, and there were fewer apoptosis signals in the TUNEL assay. In conclusion, this experiment indicated that Nrf2 is involved in the regulation of inflammatory response, oxidative stress,and apoptosis induced by V. harveyi in L. vannamei.

Characterization of CD166 from Nile tilapia (Oreochromis niloticus) displays a broad pathogen recognition spectrum and involved the immune response to microbial aggression.

CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules, and its mediated adhesion plays a crucial role in different physiological and pathological phenomena, especially related to leukocyte extravasation, immune synapse stability, T cell activation and proliferation. In this study, CD166 was identified from Nile tilapia (Oreochromis niloticus, OnCD166). OnCD166 contains an open reading frame of 1671 bp that encodes a peptide of 556 amino acids, and contains five consecutive extracellular immunoglobulin domains. It's tissue distribution and expression patterns after S. agalactiae challenge were also investigated. OnCD166 is widely distributed in various tissues of healthy tilapia. After Streptococcus agalactiae challenge, OnCD166 expressions were significantly up-regulated in all tested immune tissues. Meanwhile, the recombinant OnCD166 (rOnCD166E) protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Moreover, rOnCD166E could promote phagocytosis of macrophages. Taken together, our results illustrated that OnCD166 might as a receptor involved in the immune recognition and phagocytosis against invading pathogen, which play important roles in the immune responses of Nile tilapia against bacterial pathogens.