Aggregation-induced emission of TTCPy-3: A novel approach for eradicating Nocardia seriolae infections in aquatic fishes.

Aquatic fishes are threatened by the strong pathogenic bacterium Nocardia seriolae, which challenges the current prevention and treatment approaches. This study introduces luminogens with aggregation-induced emission (AIE) as an innovative and non-antibiotic therapy for N. seriolae. Specifically, the AIE photosensitizer, TTCPy-3 is employed against N. seriolae. We evaluated the antibacterial activity of TTCPy-3 and investigated the killing mechanism against N. seriolae, emphasizing its ability to aggregate within the bacterium and produce reactive oxygen species (ROS). TTCPy-3 could effectively aggregate in N. seriolae, generate ROS, and perform real-time imaging of the bacteria. A bactericidal efficiency of 100% was observed while concentrations exceeding 4 µM in the presence of white light irradiation for 10 min. In vivo, evaluation on zebrafish (Danio rerio) confirmed the superior therapeutic efficacy induced by TTCPy-3 to fight against N. seriolae infections. TTCPy-3 offers a promising strategy for treating nocardiosis of fish, paving the way for alternative treatments beyond traditional antibiotics and potentially addressing antibiotic resistance.

Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination of DNA.

Adenosine-to-inosine tRNA-editing enzyme has been identified for more than two decades, but the study on its DNA editing activity is rather scarce. We show that amphioxus (Branchiostoma japonicum) ADAT2 (BjADAT2) contains the active site 'HxE-PCxxC' and the key residues for target-base-binding, and amphioxus ADAT3 (BjADAT3) harbors both the N-terminal positively charged region and the C-terminal pseudo-catalytic domain important for recognition of substrates. The sequencing of BjADAT2-transformed Escherichia coli genome suggests that BjADAT2 has the potential to target E. coli DNA and can deaminate at TCG and GAA sites in the E. coli genome. Biochemical analyses further demonstrate that BjADAT2, in complex with BjADAT3, can perform A-to-I editing of tRNA and convert C-to-U and A-to-I deamination of DNA. We also show that BjADAT2 preferentially deaminates adenosines and cytidines in the loop of DNA hairpin structures of substrates, and BjADAT3 also affects the type of DNA substrate targeted by BjADAT2. Finally, we find that C89, N113, C148 and Y156 play critical roles in the DNA editing activity of BjADAT2. Collectively, our study indicates that BjADAT2/3 is the sole naturally occurring deaminase with both tRNA and DNA editing capacity identified so far in Metazoa.

Efficient nitrogen removal of a novel Pseudomonas chengduensis strain BF6 mainly through assimilation in the recirculating aquaculture systems.

Biological nitrogen removal has received increasing attention in wastewater treatment. A bacterium with excellent nitrogen removal performance was isolated from biofilters of recirculating aquaculture systems (RAS) and identified as Pseudomonas chengduensis BF6. It was indicated that inorganic nitrogen is transformed into gaseous and biological nitrogen by the metabolic pathways of denitrification, anammox, and assimilation, which is the main nitrogen removal pathway of strain BF6. The strain BF6 could effectively remove nitrogen within 24 h under the conditions of ammonia, nitrate, nitrite, and mixed nitrogen sources with maximum total nitrogen removal efficiencies reaching 97.00 %, 61.40 %, 79.10 %, and 84.98 %, respectively. The strain BF6 exhibited total nitrogen removal efficiency of 91.14 %, altered the microbial diversity and enhanced the relative abundance of Pseudomonas in the RAS biofilter. These findings demonstrate that Pseudomonas sp. BF6 is a highly efficient nitrogen-removing bacterium with great potential for application in aquaculture wastewater remediation.

"Two birds with one stone" strategy for the lung cancer therapy with bioinspired AIE aggregates.

Aggregation-induced emission luminogens (AIEgens) have emerged as novel phototherapeutic agents with high photostability and excellent performance to induce photodynamic and/or photothermal effects. In this study, a zwitterion-type NIR AIEgens C41H37N2O3S2 (named BITT) with biomimetic modification was utilized for lung cancer therapy. The tumor-associated macrophage (TAM)-specific peptide (CRV) was engineered into the lung cancer cell-derived exosomes. The CRV-engineered exosome membranes (CRV-EM) were obtained to camouflage the BITT nanoparticles (CEB), which targeted both lung cancer cells and TAMs through homotypic targeting and TAM-specific peptide, respectively. The camouflage with CRV-EM ameliorated the surface function of BITT nanoparticles, which facilitated the cellular uptake in both cell lines and induced significant cell death in the presence of laser irradiations in vitro and in vivo. CEB showed improved circulation lifetime and accumulations in the tumor tissues in vivo, which induced efficient photodynamic and photothermal therapy. In addition, CEB induced the tumor microenvironment remodeling as indicated by the increase of CD8 + and CD4 + T cells, as well as a decrease of M2 TAM and Myeloid-derived suppressor cells (MDSCs). Our work developed a novel style of bioinspired AIE aggregates, which could eliminate both lung cancer cells and TAMs, and remodel the tumor environments to achieve an efficient lung cancer therapy. To the best of our knowledge, we are the first to use this style of bioinspired AIE aggregates for photo-mediated immunotherapy in lung cancer therapy.

Characterization of the core gut microbiota of Nile tilapia (Oreochromis niloticus): indication of a putative novel Cetobacterium species and analysis of its potential function on nutrition.

The genus Cetobacterium has been considered a dominant group of gut bacteria in many freshwater fish, and members of this genus contribute to anaerobic metabolism. Because of its significant place in the gut of freshwater fish, many studies on Cetobacterium were performed. Those studies mostly focused on the temporal and spatial changes of its abundance in fish intestine, which were affected by food or other environmental conditions. However, only a few studies isolated strains from genus Cetobacterium and reported their characteristics. In the present study, we performed 16S rRNA sequencing of the intestinal mucosa of Nile tilapia (Oreochromis niloticus) and found that Cetobacterium sp. existed widely in the foregut, midgut and hindgut mucosa, and a strain of Cetobacterium was successfully isolated from the gut of tilapia. We sequenced its whole genome and predicted it to be a novel candidate species of Cetobacterium sp. and named it NK01. The size of its genome was 3,095,946 bp, with a guanine + cytosine content of 28.8%. Among the identified genes, 2855 were predicted to be coding DNA sequences, 84 were tRNA and 34 were rRNA. We found that NK01 produced amino acids, including leucine, isoleucine, valine, glycine, alanine, phenylalanine and proline. Strain NK01 could use starch, sucrose, maltose, glucose, and mannose and synthesize and utilize glycogen. INV, GPI, malQ, malZ, sacA, scrK, glgC, glgA and glk, which were related to carbohydrate metabolism, were detected. yiaY and adhE, which oxidize ethanol to acetaldehyde and participate in a variety of metabolic pathways, were also present in the genome. No coding genes directly involved in acetate or butyrate production were detected. NK01 could also catabolize a variety of vitamins, and all genes involved in folate synthesis were detected, including folP, folC, folA and eutT, which converted vitamin B12s into vitamin B12 coenzyme. Here, we investigated the draft genome and in vitro function of Cetobacterium isolated from the intestinal tract of Nile tilapia. The results provided a preliminary understanding of the core microbiota of fish gut.

Versatile Nano-PROTAC-Induced Epigenetic Reader Degradation for Efficient Lung Cancer Therapy.

Recent evidence has indicated that overexpression of the epigenetic reader bromodomain-containing protein 4 (BRD4) contributes to a poor prognosis of lung cancers, and the suppression of its expression promotes cell apoptosis and leads to tumor shrinkage. Proteolysis targeting chimera (PROTAC) has recently emerged as a promising therapeutic strategy with the capability to precisely degrade targeted proteins. Herein, a novel style of versatile nano-PROTAC (CREATE (CRV-LLC membrane/DS-PLGA/dBET6)) is developed, which is constructed by using a pH/GSH (glutathione)-responsive polymer (disulfide bond-linked poly(lactic-co-glycolic acid), DS-PLGA) to load BRD4-targeted PROTAC (dBET6), followed by the camouflage with engineered lung cancer cell membranes with dual targeting capability. Notably, CREATE remarkably confers simultaneous targeting ability to lung cancer cells and tumor-associated macrophages (TAMs). The pH/GSH-responsive design improves the release of dBET6 payload from nanoparticles to induce pronounced apoptosis of both cells, which synergistically inhibits tumor growth in both subcutaneous and orthotopic tumor-bearing mouse model. Furthermore, the efficient tumor inhibition is due to the direct elimination of lung cancer cells and TAMs, which remodels the tumor microenvironment. Taken together, the results elucidate the construction of a versatile nano-PROTAC enables to eliminate both lung cancer cells and TAMs, which opens a new avenue for efficient lung cancer therapy via PROTAC.

Nile tilapia TLR3 recruits MyD88 and TRIF as adaptors and is involved in the NF-κB pathway in the immune response.

TLR3 plays a crucial role in innate immunity. In the present study, OnTLR3 was identified in the Nile tilapia Oreochromis niloticus, with a conserved LRR domain and a C-terminal TIR domain. OnTLR3 was broadly expressed in all tissues tested, with the highest expression levels in the blood and the lowest in the kidney. TLR3 mRNA could be detected from pharyngula (2.5 dpf) to late larva (8.5 dpf) during embryonic and larval development. Moreover, the expression level of OnTLR3 was clearly altered in all five tissues after Streptococcus agalactiae infection in vivo and could be induced by LPS, poly(I:C), S. agalactiae WC1535 and △CPS in Nile tilapia macrophages. When OnTLR3 was overexpressed in 293 T cells, it was distributed in the cytoplasm and could significantly increase NF-κB activation. The pulldown assays showed that OnTLR3 interacted with both OnMyD88 and OnTRIF. The binding assays revealed the specificity of OnTLR3 for pathogen-associated molecular patterns (PAMPs) and bacteria that included S. agalactiae, Aeromonas hydrophila and poly(I:C), LPS and PGN. Taken together, these findings suggest that OnTLR3, as a pattern recognition receptor (PRR), might play an important role in the immune response to pathogen invasion.

Isolation, identification, and pathogenic characteristics of Nocardia seriolae in largemouth bass Micropterus salmoides.

The largemouth bass Micropterus salmoides is an important freshwater aquaculture fish in China. Recently, largemouth bass at a fish farm in Guangdong province experienced an outbreak of a serious ulcer disease. As part of the investigations conducted to identify the aetiology and identify potentially effective control measures, we isolated a pathogenic bacterium (NK-1 strain) from the diseased fish. It was identified as Nocardia seriolae through morphological observation, physiological and biochemical analysis, and molecular identification, and its pathogenicity was verified by experimental infection. Pathological changes in the diseased fish included granulomatous lesions in the liver and spleen, destruction of renal tubules, necrosis of intestinal epithelial cells, infiltration of inflammatory cells in the brain, vacuolation of cells, and swelling and cracking of the mitochondria and endoplasmic reticulum. Bacterial detection using qPCR showed that the spleen and intestine were the main organs targeted by N. seriolae. The mortality of largemouth bass experimentally infected with N. seriolae at 21°C was significantly lower than that in fish infected at higher temperatures between 24 and 33°C; there were no significant differences in the levels of mortality at these higher temperatures. The level of mortality of largemouth bass infected with N. seriolae was lowest at a neutral water pH of 7 but increased significantly at higher and lower pH. Of the tested Chinese herbal medicines, Chinese sumac Galla chinensis and Chinese skullcap Scutellaria baicalensis exhibited the best antibacterial effects. This study lays a foundation for the clinical diagnosis and scientific control of ulcer disease in largemouth bass.

Apigenin suppresses tumor angiogenesis and growth via inhibiting HIF-1α expression in non-small cell lung carcinoma.

Tumor angiogenesis inhibitors such as Bevacizumab, Ramucirumab and Endostar have been applied to the therapy of non-small cell lung carcinoma (NSCLC) patients, especially for lung adenocarcinoma (LUAD). However, several safe concerns such as neutropenia, febrile neutropenia and hypertension pulmonary hemorrhage limit their further development. And they often showed poor efficacy and serious side effect for lung squamous cell carcinoma (LUSC) patient. Thus, identification of effective and safe tumor angiogenesis inhibitor for NSCLC therapy is warranted. Apigenin is a bioflavonoid with potential anti-tumor effect and perfect safety, but its effect on tumor angiogenesis and underlying mechanism are still unclear. Herein, we found that apigenin not merely suppressed endothelial cells related motilities but also reduced pericyte coverage. Further research showed that apigenin had strong suppressive activity against HIF-1α expression and its downstream VEGF-A/VEGFR2 and PDGF-BB/PDGFßR signaling pathway. Apigenin also reduced microvessel density and pericyte coverage on the xengraft model of NCI-H1299 cells, leading to suppression of tumor growth. Moreover, apigenein showed perfect anti-angiogenic effect in xengraft model of LUSC cell NCI-H1703 cells, indicating it may be developed into a potential angiogenesis inhibitor for LUSC patient. Collectively, our study provides new insights into the anti-tumor mechanism of apigenin and suggests that apigenin is a safe and effective angiogenesis inhibitor for NSCLC therapy.

Nile tilapia TRIM39 recruits I3K413 and I3KL45 as adaptors and is involved in the NF-κB pathway.

Tripartite motif (TRIM) proteins play a regulatory function in cancer, cell apoptosis and innate immunity. To understand the role of TRIM39 in Nile tilapia (Oreochromis niloticus), TRIM39 cDNA was isolated. The total length of TRIM39 cDNA was 5025 bp. The deduced OnTRIM39 protein contains 549 amino acids and has conserved domains of the TRIM family, which are the RING, B-box, coiled-coil and PRY-SPRY domains. OnTRIM39 mRNA was widely expressed in various tissues. After challenge with Streptococcus agalactiae and stimulation with polyinosinic polycytidylic acid [poly (I:C)] and lipopolysaccharides (LPS), the amount of OnTRIM39 transcript was changed in various tested tissues. OnTRIM39 overexpression increased NF-κB activity. OnTRIM39 was present in the cytoplasm. Mass spectrometry of proteins pulled down with recombinant OnTRIM39 showed that 250 proteins potentially interact with OnTRIM39. The authors selected I3K4I3 from the 250 candidate proteins to verify its interaction with TRIM39. They also selected I3KL45, a member of the same 14-3-3 protein family, to verify its interaction with TRIM39. The results of pull-down assays showed that OnTRIM39 interacted with both I3K413 and I3KL45. These results contribute to further study of the innate immune mechanism of tilapia.

TLR5 recognizes Aeromonas hydrophila flagellin and interacts with MyD88 in Nile tilapia.

Toll-like receptor 5 (TLR5) is responsible for bacterial flagellin recognition in vertebrates. In the present study, TLR5M was identified in the Nile tilapia Oreochromis niloticus (OnTLR5), containing a conserved LRR domain, a transmembrane region and a C-terminal TIR domain, similar to that of other fishes and mammals. OnTLR5 was broadly expressed in all the tissues examined, presenting the highest expression levels in the blood and the lowest in the kidney. OnTLR5 was detected from 2 d postfertilization (dpf) to 8 dpf during embryonic development. Moreover, expression levels of OnTLR5 were clearly altered in all five tissues examined in response to Streptococcus agalactiae infection in vivo. Overexpression of OnTLR5 in HEK293T cells revealed that OnTLR5 was distributed in the cytoplasm and significantly increased NF-κB activation. In response to cotransfection with OnMyd88, OnTLR5 significantly upregulated OnMyd88-induced NF-κB activation. Pulldown assays showed that OnTLR5 interacts with OnMyd88 and revealed an interaction between TLR5 and Aeromonas hydrophila flagellin. Taken together, these findings suggest that OnTLR5 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Genome survey of Misgurnus anguillicaudatus to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome.

BACKGROUND: The dojo loach Misgurnus anguillicaudatus is an important economic species in Asia because of its nutritional value and broad environmental adaptability. Despite its economic importance, genomic data for M. anguillicaudatus is currently unavailable. METHODS AND RESULTS: In the present study, we conducted a genome survey of M. anguillicaudatus using next-generation sequencing technology. Its genome size was estimated to be 1105.97 Mb by using K-mer analysis, and its heterozygosity ratio, repeat sequence content, GC content were 1.45%, 58.98%, and 38.03%, respectively. A total of 376,357 microsatellite motifs were identified, and mononucleotides, with a frequency of 42.57%, were the most frequently repeated motifs, followed by 40.83% dinucleotide, 7.49% trinucleotide, 8.09% tetranucleotide, and 0.91% pentanucleotide motifs. The AC/GT, AAT/ATT, and ACAG/CTGT repeats were the most abundant motifs among dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. Besides, the complete mitochondrial genome was sequenced. Based on the Maximum Likelihood and Bayesian inference analyses, M. anguillicaudatus yingde in this study was the "introgressed" mitochondrial type. Seventy microsatellite loci were randomly selected from detected SSR loci to test polymorphic, of which, 20 microsatellite loci were assessed in 30 individuals from a wild population. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) per locus ranged from 7 to 19, 0.400 to 0.933, and 0.752 to 0.938, respectively. All 20 loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.05). CONCLUSIONS: This is the first report of genome survey sequencing in M. anguillicaudatus, genome information, mitochondrial genome, and microsatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

DDX43 recruits TRIF or IPS-1 as an adaptor and activates the IFN-ß pathway in Nile tilapia (Oreochromis niloticus).

DDX43 is one of the members of the DExD/H-box protein family, and emerging data suggest that it may play an important role in antiviral immunity across mammals. However, little is known about DDX43 in the fish immune response. In this study, we isolated the cDNA sequence of ddx43 in Nile tilapia (Oreochromis niloticus). The ddx43 gene was 2338 bp in length, contained an open reading frame (ORF) of 2064 bp and encoded a polypeptide of 687 amino acids. The predicted protein of OnDDX43 has three conserved domains, including the RNA binding domain KH, DEAD-like helicase superfamily DEXDc and C-terminal HELICc domain. In healthy Nile tilapia, the Onddx43 transcript was broadly expressed in all examined tissues, with the highest expression levels in the muscle and brain and the lowest in the liver. After challenge with Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C), the expression level of Onddx43 mRNA was upregulated or downregulated in all of the tissues tested. Overexpression of OnDDX43 in 293 T cells showed that it has a positive regulatory effect on IFN-ß. The subcellular localization showed that OnDDX43 was expressed in the cytoplasm. We performed further pull-down assays and found that OnDDX43 interacted with both interferon-ß promoter stimulator1 (IPS-1) and TIR domain-containing adaptor inducing interferon-ß (TRIF).

TLR1 in Nile tilapia: The conserved receptor cannot interact with MyD88 and TIRAP but can activate NF-κB in vitro.

Toll-like receptors (TLRs) play a critical role in the innate immune response of fish. In this study, we isolated the cDNA sequence of Nile tilapia TLR1 (OnTLR1). The deduced OnTLR1 protein contains a signal peptide, 7 leucine-rich repeats (LRRs), a C-terminal LRR (LRR-CT), a transmembrane region and a highly conserved TIR domain. In healthy Nile tilapia, the OnTLR1 transcript was broadly expressed in all examined tissues, with the highest expression levels in the spleen. After infection with Streptococcus agalactiae, the OnTLR1 transcripts were upregulated in the gill and kidney. After stimulation with polyinosinic-polycytidylic acid (poly(I:C)), the expression levels of OnTLR1 were significantly downregulated in the intestine, whereas OnTLR1 transcripts were significantly upregulated in the kidney. After challenge with lipopolysaccharide (LPS), the expression levels of OnTLR1 were significantly upregulated in the spleen and kidney. The subcellular localization showed that OnTLR1 was expressed in the cytoplasm. TLR1 significantly increased MyD88-dependent NF-κB activity. However, the results of a pull-down assay showed that OnTLR1 did not interact with MyD88 or TIRAP. Binding assays revealed the specificity of OnTLR1 for pathogen-associated molecular patterns (PAMPs) and bacteria that included S. agalactiae, Aeromonas hydrophila and poly(I:C) and LPS. Taken together, these findings suggest that OnTLR1, as a pattern recognition receptor (PRR), might play an important role in the immune response to pathogen invasion.

Efficient Inhibition of Streptococcus agalactiae by AIEgen-Based Fluorescent Nanomaterials.

Streptococcus agalactiae, referred to as group B streptococcus (GBS), is a prominent co-pathogenic bacterium causing the onset and death of human, animal, and aquatic products. Although antibiotics are efficient against GBS, antibiotic resistance through antibiotic overuse is an equally serious problem. Therefore, the treatment of GBS infection appears strongly dependent on nonantibiotic therapy, such as photodynamic therapy. Different from other photosensitizers (PSs), luminogens with aggregation-induced emission (AIEgen) can efficiently generate fluorescence and reactive oxygen species (ROS). Herein, TBP-1, an efficient AIE PSs, is chosen to resist GBS, and its antibacterial activity and the killing mechanism toward GBS are investigated. The ROS generation performance and the images of GBS treated with TBP-1 in the dark or under white light irradiation were investigated. TBP-1 with its high ROS generation ability can efficiently kill GBS and serve as a novel treatment strategy against GBS infection.

Distant hybridization and gynogenesis between Nile tilapia Oreochromis niloticus and Jaguar cichlid Parachromis managuensis.

To investigate the distant hybridization and gynogenesis between Nile tilapia Oreochromis niloticus and Jaguar cichlid Parachromis managuensis, reciprocal crossing was first performed between the two species. No offspring, however, were viable when there were these hybridizations. Gynogenesis was induced in O. niloticus and P. managuensis using ultraviolet (UV)-irradiated spermatozoa from P. managuensis and O. niloticus, respectively. The morphology during embryonic development indicated gynogenetic offspring of both O. niloticus and the P. managuensis were normal and deformed, and the results from flow cytometric analysis indicated normal fry were diploid and deformed fry were haploid. Gynogenetic O. niloticus and P. managuensis had the same DNA content and chromosome number as their species of origin, indicating that gynogenetic individuals were produced in both species. The presence of only females for both gynogenetic P. managuensis and O. niloticus was indicative of an XX genotype in the female P. managuensis and O. niloticus. Results from studies on genetic diversity indicated the average heterozygosity of the gynogenetic diploid population of O. niloticus were less than that of the cultured population, but the genetic homozygosity of the gynogenetic diploid population of O. niloticus was greater than that of the cultured population after one generation of gynogenesis, which achieved the goal of rapidly establishing genetic homozygosity.

Nile tilapia Toll-like receptor 7 subfamily: Intracellular TLRs that recruit MyD88 as an adaptor and activate the NF-κB pathway in the immune response.

Toll-like receptor 7 (TLR7) subfamily members are important pattern recognition receptors that participate in the recognition of pathogen-associated molecular patterns. In the present study, three TLR family members, OnTLR7, OnTLR8 and OnTLR9, were identified in the Nile tilapia Oreochromis niloticus. TLR7-, TLR8-and TLR9-deduced proteins have typical structural characteristics of TLRs, including Toll/interleukin-1 receptor (TIR), leucine-rich repeat (LRR) and transmembrane region (TM). OnTLR7, OnTLR8 and OnTLR9 were broadly expressed in all of the tissues tested, with the highest expression levels in the brain (TLR7) and spleen (TLR8 and TLR9). Moreover, the expression levels of OnTLR7, OnTLR8 and OnTLR9 were significantly increased in most tested tissues after Streptococcus agalactiae infection in vivo. After LPS stimulation, OnTLR7 and OnTLR9 mRNA expression levels were downregulated in the intestine and upregulated in the liver, spleen and kidney; however, OnTLR8 mRNA expression levels were upregulated in the kidney only after LPS stimulation for 5 d. After Poly I:C stimulation, OnTLR7 and OnTLR9 mRNA expression levels were upregulated in the intestine, liver, spleen and kidney, and the highest expression was found in the liver, while OnTLR8 mRNA expression levels were upregulated in the intestine, liver and kidney and downregulated in the spleen. Subcellular localization of OnTLR7, OnTLR8, and OnTLR9 in 293T cells showed that OnTLR9 was distributed in both the cytoplasm and nucleus while OnTLR8 and OnTLR7 were distributed mainly in the cytoplasm. Overexpression of OnTLR7, OnTLR8 and OnTLR9 in 293T cells had no significant effect on the activity of NF-κB, but they could significantly enhance MyD88-mediated NF-κB activity after cotransfection with MyD88. Pulldown assays showed that OnTLR7, OnTLR8, and OnTLR9 could interact with OnMyD88. Taken together, these results indicate that TLR7 subfamily genes play a role in the immune response to pathogen invasion of Nile tilapia.

Structurally diverse genes encode TLR13 in Nile tilapia: The two receptors can recognize Streptococcus 23S RNA and conduct signal transduction through MyD88.

Toll-like receptors (TLRs) play a crucial role in the innate immune system, which is the first line of defence against pathogens and pathogenic products in fish. In the present study, we cloned the full-length cDNA and genome sequences of two TLR13 s (OnTLR13a, OnTLR13b) from Nile tilapia (Oreochromis niloticus). TLR family motifs, i.e., the leucine-rich repeat (LRR) domains and Toll/interleukin (IL)-1 receptor (TIR) domains, were conserved in the putative proteins OnTLR13a and OnTLR13b, with fifteen LRR domains and one TIR domain. Four exons and three introns were identified in the OnTLR13a genome sequence, and three exons and two introns were identified in the OnTLR13b genome sequence. In healthy Nile tilapia tissues, OnTLR13a and OnTLR13b were ubiquitously expressed in all 11 tested tissues/organs. The highest expression levels were observed in the spleen (OnTLR13a) and blood (OnTLR13b), and the lowest expression levels were observed in the liver (OnTLR13a) and stomach (OnTLR13b). The expression level of OnTLR13b at 5.5 days postfertilization (dpf) was significantly higher than that at the other 8 time points (2.5, 3.5, 4.5, 5, 6, 6.5, 7.5 and 8.5 dpf). Upon stimulation with an intraperitoneal injection of 200 µL (107 CFU/mL) Streptococcus agalactiae, the expression levels of OnTLR13a and OnTLR13b were significantly upregulated in the intestine and gill. After cotransfection with MyD88, OnTLR13a significantly increased MyD88-dependent NF-κB activation in 293 T cells. However, OnTLR13b significantly impaired MyD88-dependent NF-κB activation. In addition, TLR13a slightly increased MyD88-dependent AP-1 activation, and TLR13b significantly increased MyD88-dependent AP-1 activation. TLR13a significantly increased MyD88-dependent interferon-ß (IFN-ß) activation, and TLR13b had no effect on MyD88-dependent IFN-ß activation. These findings suggest that although the deduced protein structure of OnTLR13 is evolutionarily conserved between OnTLR13 and other TLR members, its signal transduction function is markedly different. Co-immunoprecipitation (Co-IP) assays showed that both OnTLR13a and OnTLR13b could interact with OnMyD88. RNA pulldown assays showed that TLR13a and TLR13b could combine with the 23S rRNA of S. agalactiae. These results indicate that TLR13a and TLR13b play important roles in the innate immune response against bacterial infection in Nile tilapia.

Molecular characterization, expression and functional analysis of IRAK1 and IRAK4 in Nile tilapia (Oreochromis niloticus).

Interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 are critical signalling mediators and play pivotal roles in the innate immune and inflammatory responses mediated by TLR/IL-1R. In the present study, two IRAK family members, OnIRAK1 and OnIRAK4, were identified in the Nile tilapia Oreochromis niloticus with a conserved N-terminal death domain and a protein kinase domain, similar to those of other fishes and mammals. The gene structures of OnIRAK1 and OnIRAK4 are organized into fifteen exons split by fourteen introns and ten exons split by nine introns. OnIRAK1 and OnIRAK4 were broadly expressed in all of the tissues tested, with the highest expression levels being observed in the blood and the lowest expression levels being observed in the liver. Both genes could be detected from 2 d post-fertilization (dpf) to 8 dpf during embryonic development. Moreover, the expression levels of OnIRAK1 and OnIRAK4 were clearly altered in all five tissues after Streptococcus agalactiae infection in vivo and could be induced by LPS, Poly I: C, S. agalactiae WC1535 and △CPS in Nile tilapia macrophages. The overexpression of OnIRAK1 and OnIRAK4 in 293T cells showed that they were both distributed in the cytoplasm and could significantly increase NF-κB activation. Interestingly, after transfection, OnIRAK1 significantly upregulated OnMyd88-induced NF-κB activation, while OnIRAK4 had no effect on OnMyd88-induced NF-κB activation. Co-immunoprecipitation (Co-IP) assays showed that OnMyd88 did not interact with either OnIRAK1 or OnIRAK4 and that OnIRAK1 did not interact with OnIRAK4. Taken together, these findings suggest that OnIRAK1 and OnIRAK4 could play important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Bacillus velezensis LF01: in vitro antimicrobial activity against fish pathogens, growth performance enhancement, and disease resistance against streptococcosis in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is a major pathogen causing streptococcosis. To prevent and control this bacterial disease, antagonistic bacteria have become a new research hotspot. This study evaluated the probiotic potential of Bacillus velezensis LF01 strain, which is antagonistic to S. agalactiae. The active compounds produced by LF01 showed antimicrobial activity against a broad spectrum of fish pathogens, including S. agalactiae, Streptococcus iniae, Aeromonas hydrophila, Edwardsiella tarda, Edwardsiella ictaluri, Aeromonas schubertii, Aeromonas veronii, Aeromonas jandaei, and Vibrio harveyi. The antimicrobial compounds were heat stable, pH stable, UV stable, resistant to proteases, and could be stored for a long time. To evaluate the probiotic function of LF01 in Nile tilapia, juveniles were divided into three treatment groups: a control group, an interval feeding group, and a continuous feeding group. Tilapia fed with LF01-supplemented diets (1.0 × 109 CFU/g) showed significantly better growth performances than those of the control group (P < 0.05). Tilapia fed with LF01-supplemented diets significantly increased lysozyme (LZY) and superoxide dismutase (SOD) activities. The expression of three immune-related genes (C3, lyzc, and MHC-IIß) was higher in the intestine, head kidney, and gill of tilapia from the continuous feeding group than in those from the control group (P < 0.05). Tilapia fed with LF01-supplemented diets showed remarkably improved survival rates after S. agalactiae infection, and analysis of their intestinal tract pathogens revealed that the abundance of Edwardsiella and Plesiomonas had significantly decreased compared with the control group. Our findings demonstrate that LF01 is an effective antagonist against various fish pathogens and has potential for controlling infections by Streptococcus spp. and other pathogens in tilapia.