Intestinal epithelial cells of Japanese flounder (Paralichthys olivaceus) as an in vitro model for studying intestine immune function based on transcriptome analysis.

Japanese flounder (Paralichthys olivaceus) is an economically crucial marine species, but diseases like hemorrhagic septicemia caused by Edwardsiella tarda have resulted in significant economic losses. E. tarda infects various hosts, and its pathogenicity in fish is not fully understood. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and are representative of typical PAMP molecules that cause activation of the immune system. The PoIEC cell line is a newly established intestinal epithelial cell line from P. olivaceus. In order to investigate whether it can be used as an in vitro model for studying the pathogenesis of E. tarda and LPS stimulation, we conducted RNA-seq experiments for the PoIECs model of E. tarda infection and LPS stimulation. In this study, transcriptome sequencing was carried out in the PoIEC cell line after treatment with LPS and E. tarda. A total of 62.52G of high-quality data from transcriptome sequencing results were obtained in nine libraries, of which an average of 87.96% data could be aligned to the P. olivaceus genome. Data analysis showed that 283 and 414 differentially expressed genes (DEGs) in the LPS versus Control (LPS-vs-Con) and E. tarda versus Control groups (Et-vs-Con), respectively, of which 60 DEGs were shared in two comparation groups. The GO terms were predominantly enriched in the extracellular space, inflammatory response, and cytokine activity in the LPS-vs-Con group, whereas GO terms were predominantly enriched in nucleus and positive regulation of transcription by RNA polymerase II in the Et-vs-Con group. KEGG analysis revealed that three immune-related pathways were co-enriched in both comparison groups, including the Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and Cytokine-cytokine receptor interaction. Five genes were randomly screened to confirm the validity and accuracy of the transcriptome data. These results suggest that PoIEC cell line can be an ideal in vitro model for studies of marine fish gut immunity and pathogenesis of Edwardsiellosis.

Role of TRAF6 from obscure puffer (Takifugu obscurus) in immune response against Edwardsiella tarda infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a vital molecule of inflammatory signaling pathways in innate immune response against pathogens. To elucidate its role in defense against Edwardsiella tarda infection in teleost fish, TRAF6 homologue was identified from obscure puffer (Takifugu obscurus) and functionally analyzed in this study. The obscure puffer TRAF6 (ToTRAF6) is a protein of 565 amino acids containing conserved RING domain, zinc finger-TRAF and MATH_TRAF6 domain. ToTRAF6 mRNA distributed in various healthy tissues of obscure puffer and was upregulated in the immune related tissues after E. tarda infection. ToTRAF6 protein was localized in the cytoplasm and aggregate as dots around the nuclei in FHM cells. The overexpression of ToTRAF6 in FHM cells decreased the quantity of E. tarda and induced the significant upregulation of downstream MAPK signaling pathway genes. These data suggest that ToTRAF6 is a key molecule of MAPK signaling pathway in defense against E. tarda infection.

Two novel teleost calreticulins PoCrt-1/2, with bacterial binding and agglutination activity, are involved in antibacterial immunity.

Calreticulin (Crt), a conserved lectin-like pleiotropic protein, plays crucial roles in mammalian immune response. In fish, the immunological function of Crt is limited investigated. Herein, we studied the antibacterial immunity of two type of Crt homologues (i.e. PoCrt-1 and PoCrt-2) in Japanese flounder (Paralichthys olivaceus). PoCrt-1 and PoCrt-2 are composed of 419 and 427 amino acid residues respectively, with 69.09% overall sequence identities with each other. Both PoCrt-1 and PoCrt-2 contain a signal peptide and three functional domains i.e. N-, P- and C-domains. Both PoCrt-1 and PoCrt-2 were constitutively expressed at various tissues with highest expression level in liver, and obviously regulated by Edwardsiella tarda and Vibrio harveyi. Furthermore, recombinant PoCrt-1 and PoCrt-2 (rPoCrt-1 and rPoCrt-2) could bind to different Gram-negative bacteria with highest binding index with E. tarda. At same time, in vitro rPoCrt-1 and rPoCrt-2 could agglutinate E. tarda, V. harveyi, and Vibrio anguillarum, and inhibit the bacterial growth. Similarly, in vivo rPoCrt-1 and rPoCrt-2 could significantly suppress the dissemination of E. tarda. Overall, these observations add new insights into the antibacterial immunity of Crt in P. olivaceus.

Transcriptome analysis of turbot (Scophthalmus maximus) kidney responses to inactivated bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda.

Turbot (Scophthalmus maximus) is a commercially important marine flatfish for global aquaculture. With intensive farming, turbot production is limited by several diseases, in which Aeromonas salmonicida and Edwardsiella tarda are two main causative agents. Vaccination is an effective and safe alternative to disease prevention compared to antibiotic treatment. In the previous study, we developed an inactivated bivalent vaccine against A. salmonicida and E. tarda with relative percent survival (RPS) of 77.1 %. To understand the protection mechanism in molecular basis of the inactivated bivalent vaccine against A. salmonicida and E. tarda, we use RNA-seq to analyze the transcriptomic profile of the kidney tissue after immunization. A total of 391,721,176 clean reads were generated in nine libraries by RNA-seq, and 96.35 % of the clean reads were mapped to the reference genome of S. maximus. 1458 (866 upregulated and 592 downregulated) and 2220 (1131 upregulated and 1089 downregulated) differentially expressed genes (DEGs) were obtained at 2 and 4 weeks post-vaccination, respectively. The DEGs were enriched in several important immune-related GO terms, including cytokine activity, immune response, and defense response. In addition, the analysis of several immune-related genes showed upregulation and downregulation, including pattern recognition receptors, complement system, cytokines, chemokines and immune cell surface markers. Eight DEGs (ccr10, calr, casr, mybpha, cd28, thr18, cd20a.3 and c5) were randomly selected for qRT-PCR analysis, which confirmed the validity of the RNA-seq. Our results provide valuable insight into the immune mechanism of inactivated bivalent vaccine against A. salmonicida and E. tarda in Scophthalmus maximus.

Alterations in certain immunological parameters in the skin mucus of the carp, Cirrhinus mrigala, infected with the bacteria, Edwardsiella tarda.

The bacterial fish pathogen Edwardsiella tarda causes heavy stock mortality, severely hampering fish production, resulting in great economic loss to the farming industry. The first biological barriers that confer immune protection against pathogen entry are the fish mucosal surfaces. The present study was undertaken to investigate the influence of E. tarda on certain enzymatic and non-enzymatic parameters in the skin mucous secretions of the fish Cirrhinus mrigala using spectrophotometry and zymography. Fish were randomly divided into three groups: control, vehicle control, and infected. A sublethal dose of E. tarda (2.2 × 106 CFU/fish) suspended in 50 µL of PBS was injected intra-peritoneally at 0 day (d). Subsequently, mucus samples were collected at 2 d, 4 d, 6 d and 8 d post-infection. The activities of lysozyme (LYZ), protease (PROT), alkaline phosphatase (ALP), acid phosphatase (ACP), catalase (CAT), peroxidase (PER), superoxide dismutase (SOD), and glutathione S-transferase (GST) decreased significantly in the skin mucus of the challenged fish, indicating the suppressed immune system and decreased antioxidant capacity of C. mrigala to E. tarda infection. Lipid peroxidation (LPO) and total nitrate-nitrite were significantly higher at several time points post-infection, suggesting that physiological functions have been impaired following pathogen challenge. The present findings could be relevant for fish aquaculture and underline the importance of skin mucus not only for assessing fish immune status but also for identifying early warning signals of disease caused by pathogens.

The regulatory function of GATA3 on immune response in Japanese flounder (Paralichthys olivaceus).

GATA3 belongs to the GATA family, and it could interact with the target gene promoter. It has been reported to play a central role in regulating lymphocyte differentiation. In this study, the GATA3 cDNA sequence was identified by a homologous clone and the RACE technology from Japanese flounder (Paralichthys olivaceus). The full-length of the GATA3 cDNA sequence was 2904 bp, including 1332 bp open reading frame (ORF), 265 bp 5 '-untranslated region (5' UTR), and 1308 bp 3 '-UTR, encoding 443 amino acids. GATA3 protein sequence was conserved in vertebrates and invertebrates, including two zinc finger domains. qRT-PCR showed that the expression of GATA3 was high in the gill, kidney, and spleen. Expression of GATA3 slowly increased at the earlier stages and culminated at the late gastrula and somatic stages. Immunohistochemistry (IHC) results showed that the GATA3 protein was expressed in lymphocyte cells, undifferentiated basal and pillar cells of the gills, as well as lymphocyte cells and melanin macrophages of the kidney. The expression of GATA3 was significantly regulated in tissues and different types of lymphocytes after stimulation with Edwardsiella tarda. Dual-luciferase reporter assay indicated that the GATA3 protein could directly interact with promoters of target genes involved in the immune response. These findings suggested that GATA3 plays a major role in regulating the immune response. This study provided a theoretical basis for the immune response mechanism of teleost and a useful reference for later research on fish immunology.

Genome-wide identification of TNF receptor-associated factors in Japanese flounder (Paralichthys olivaceus) and functional analysis of resistance to temperature and Edwardsiella tarda stress.

Tumor necrosis factor receptor-associated factors (TRAFs), as the signaling mediators of the tumor necrosis factor (TNFR) superfamily, toll-like receptors (TLR) and interleukin-1 receptor (IL-1R) superfamily, can activate downstream signal transduction pathways and play an important role in the body's immune process. In this study, six TRAF genes, namely PoTRAF2a, PoTRAF2b, PoTRAF3, PoTRAF4, PoTRAF6 and PoTRAF7, were identified and annotated in Japanese flounder by using bioinformatics methods. Phylogenetic analysis confirmed that TRAF genes can be divided into seven groups. Analysis of motif composition and gene structure demonstrated that all PoTRAF members were evolutionarily conserved. The expression patterns of PoTRAF genes were then further investigated in six different developmental stages and eleven tissues of healthy fish, and it was found that there were spatial and tissue specificities among the members. To investigate the immune response of Japanese flounder to abiotic and biotic stresses, we further analyzed the expression profile of PoTRAFs after temperature stress and pathogen challenge. The result showed that PoTRAF3 and PoTRAF4 were observably differentially expressed under temperature stress, indicating that they were involved in the immune response after temperature stress. The expression of PoTRAF2a, PoTRAF2b and PoTRAF4 was significantly different after E. tarda infection, suggesting that they might have antibacterial effects. These results would help to clarify the molecular roles of PoTRAF genes in the regulation of immune and inflammatory responses in Japanese flounder.

Identification of the matrix metalloproteinase (MMP) gene family in Japanese flounder (Paralichthys olivaceus): Involved in immune response regulation to temperature stress and Edwardsiella tarda infection.

The Matrix metalloproteinase (MMP) gene family is responsible for regulating the degradation of Extra Cellular Matrix (ECM) proteins, which are important for physiological processes such as wound healing, tissue remodeling, and stress response. Although MMPs have been studied in many species, their role in immune response in Japanese flounder (Paralichthys olivaceus) is still not fully understood. This study conducted a comprehensive analysis of MMPs in flounder, including gene structures, evolutionary relationships, conserved domains, molecular evolution, and expression patterns. Analysis revealed that MMP genes could be grouped into 17 subfamilies and were evolutionarily conserved and functionally-constrained. Meanwhile, MMP genes were found to express in different embryonic and larval stages and might play the role of sentinel in healthy tissues. Furthermore, expression profiling showed that MMPs had diverse functions in environmental stress, with 60% (9/15) and 73% (11/15) of MMPs showing differential expression patterns under temperature stress and Edwardsiella tarda (E. tarda) infection, respectively. These findings provide a useful resource for understanding the immune functions of MMP genes in Japanese flounder.

A novel teleost microRNA regulates autophagy and NF-κB activation during bacterial infection.

MicroRNAs (miRNAs) are a class of non-coding RNAs with regulatory functions in many cellular processes, including immune defense. In this study, we identified novel-m0089-3p, a novel miRNA with unknown function, in the teleost fish Japanese flounder (Paralichthys olivaceus) and investigated its immune function. Novel-m0089-3p was found to target the autophagy-associated gene ATG7 and negatively regulate ATG7 expression via interaction with the 3' UTR of ATG7. During the infection of the bacterial pathogen Edwardsiella tarda, novel-m0089-3p expression was induced in flounder, which in turn repressed ATG7 expression. Overexpression of novel-m0089-3p or blocking ATG7 expression inhibited autophagy and promoted the intracellular replication of E. tarda. Novel-m0089-3p overexpression, as well as E. tarda infection, activated NF-κB and stimulated the expression of inflammatory cytokines. Together these results revealed an important role of novel-m0089-3p in response to bacterial infection.

Characterization of Japanese flounder (Paralichthys olivaceus) STAT members: An immune-related gene family involved in Edwardsiella tarda and temperature stress.

The signal transducer and activator of transcription (STAT) family members are not only the transcriptional activators, but also play important roles in regulating inflammatory response. Some members have been reported to be involved in innate bacterial and antiviral immunity in aquatic organisms. However, no systematic research on STATs has been found in teleost. In this present study, we characterized six STAT genes in Japanese flounder based on bioinformatics methods, namely PoSTAT1, PoSTAT2, PoSTAT3, PoSTAT4, PoSTAT5 and PoSTAT6. The phylogenetic analysis of STATs in fish indicated that STATs were highly conserved and revealed an absence of STAT5 in a few species. Further analysis of gene structures and motifs showed STAT proteins shared a similar structure and probably had similar functionality in Japanese flounder. The expression profiles of different development stages and tissues demonstrated that PoSTATs exhibited specificity in temporality and spatiality as well as PoSTAT4 was highly expressed in gill. The transcriptome data analysis of E. tarda and temperature stress showed that PoSTAT1 and PoSTAT2 were more respective to these two kinds of stress. In addition, the results also demonstrated that these PoSTATs might regulate immune response in different ways, manifested by up-regulation in E. tarda infection and down-regulation in temperature stress. In a word, this systematic analysis of PoSTATs would provide valuable information about the phylogenetic relationship of STATs in fish species and help understand the role of STAT genes in the immune response of Japanese flounder.

Identification of olive flounder (Paralichthys olivaceus) toll-like receptor genes: Involvement in immune response to temperature stress and Edwardsiella tarda infection.

Toll-like receptor (TLR) genes are best known for their roles in the innate immune defense. However, studies focusing on the reaction mechanisms of TLR genes in olive flounder (Paralichthys olivaceus) immune responses are still limited. In this study, 11 TLR family members (PoTLRs) were identified and classified from P. olivaceus genome. Phylogenetic analysis showed that PoTLRs were highly conserved in olive flounder. The analysis of motif prediction and gene structure indicated that TLRs had high sequence similarity. The expression patterns in developmental stages and different tissues showed that TLR members were spatially and temporally specific. RNA-Seq analysis of temperature stress and Edwardsiella tarda infection suggested that TLR members were involved in inflammatory responses, PoTLR5b and PoTLR22 showed significant differences in response to both temperature stress and E. tarda stress, indicating their potential immune functions. The results of this study suggested that TLR genes played important roles in the innate immune response of olive flounder, and would provide a solid basis for further study of their functions.

Cathepsin H and cathepsin B of Cynoglossus semilaevis are involved in anti-bacterial immunity against Edwardsiella tarda.

Cathepsin H and Cathepsin B are two lysosomal cysteine proteases participating in various physiological processes including immune responses. In fish, the functional roles of Cathepsin H and Cathepsin B during bacterial infection are less understood. In a previous work, we characterized a Cathepsin B homologue (CsCatB) of half-smooth tongue sole (Cynoglossus semilaevis), an economically valuable fish species in China. In this report, we identified a Cathepsin H homologue (CsCatH) from C. semilaevis. In healthy tongue sole, the transcriptional expression of CsCatH was detected in nine different tissues. Laser scanning confocal microscopic analysis showed that ectopically expressed CsCatH and CsCatB were co-localized with the lysosome. Upon infection by Edwardsiella tarda, a significant fish pathogen which caused a severe fish disease termed edwardsiellosis, the expressions of CsCatH and CsCatB were remarkedly upregulated. The knockdown of CsCatH and CsCatB significantly increased the replication of E. tarda and mitigated E. tarda-induced apoptosis in tongue sole tissues. These findings revealed the importance of CsCatH and CsCatB in anti-bacterial immunity of tongue sole.

Genome-wide identification of olive flounder (Paralichthys olivaceus) SOCS genes: Involvement in immune response regulation to temperature stress and Edwardsiella tarda infection.

The suppressors of cytokine signaling (SOCS) gene family participates in development and immunity through negative regulation of cytokine signaling pathways. Although the immune response of SOCS gene family members has been extensively characterized in teleost, no similar study has been reported in olive flounder yet. In our present study, a total of 13 SOCSs in olive flounder were identified and characterized systematically. By querying the SOCS sequences of ten teleost fish species, we found there were exactly more members of SOCSs in fish than mammals, which indicated that there were more duplication events occurred in fish than in higher vertebrates. Phylogenetic analysis clearly illuminated that SOCS genes were highly conserved. The analysis of gene structure and motif showed SOCS proteins of olive flounder shared a high level of sequence similarity strikingly. The expression profiles of tissues and developmental stages indicated that SOCS members had a kind of specificity in temporality and spatiality. RNA-Seq analysis of temperature stress and E. Tarda infection demonstrated SOCS members were involved in inflammatory response. In a word, our results would provide a further reference for understanding the mechanism of SOCS genes in olive flounder.

Matrix metalloproteinase 9 modulates immune response along with the formation of extracellular traps in flounder (Paralichthys olivaceus).

MMP-9 belongs to the Matrix Metalloprotease family, which is mainly involved in the protein hydrolysis process of extracellular matrix and plays important roles in many biological processes, such as embryogenesis, tissue remodeling, angiogenesis, inflammatory processes and wound healing. In this study, we described the sequence characteristics of the MMP-9 gene in flounder (PoMMP-9). PoMMP-9 was highly homologous to MMP-9 from turbot, medaka, and Fugu rubripes. The mRNA of PoMMP-9 was constitutively expressed in all tested tissues of healthy flounder with the highest expression levels in the head kidney and spleen. A time-dependent expression pattern of PoMMP-9 in the head kidney and spleen was found after the bacterial and virus challenge. This indicates that PoMMP-9 is inducible and involved in immune responses. Indirect immunofluorescence assay showed that the PoMMP-9 was co-localization in the extracellular traps (ETs) released by the leukocytes. After overexpression, PoMMP-9 can recruit more inflammatory cells and play a broad immune process from pathogen elimination to wound healing at the inflammatory site through ETs. In summary, this study provided new insights into the biological function of MMP-9 in teleost.

A novel C1qDC (PoC1qDC) with a collagen domain in Paralichthys olivaceus mediates complement activation and against bacterial infection.

Complement C1q domain containing protein (C1qDC) is a vital recognition molecule and has an important effect on immunity. The C1qDCs exhibit opsonic activity in fish, while the mechanisms of C1qDCs in activation complement still remain unclear. This study explored immunological characteristics of a C1qDC from Japanese flounder (Paralichthys olivaceus) (PoC1qDC). PoC1qDC consists of 296 amino acid residues, possessing a collagen domain and a C1q domain. According to our results, PoC1qDC was expressed in 9 diverse tissue samples and showed up-regulation after bacterial challenge. Recombinant PoC1qDC (rPoC1qDC) activated normal serum bactericidal and hemolytic activities by interaction with Japanese flounder IgM, but not enhanced the complement activity of C3-depeleted serum. rPoC1qDC was significantly bound to various bacterial species and agglutination activity against Edwardsiella piscicida and Streptococcus iniae. Furthermore, rPoC1qDC showed direct interaction with peripheral blood leucocytes while enhancing phagocytic and chemotactic activity. When PoC1qDC was overexpressed in Japanese flounder before E. piscicida infection, bacterial replication was significantly inhibited in fish tissues. Consistently, when PoC1qDC expression in Japanese flounder was knocked down, bacterial replication was significantly enhanced. The above findings first suggested the role of PoC1qDC in teleost in mediating complement activation by interaction with IgM, which can positively influence bacterial infection.

Identification and functional analysis of dual-specificity phosphatases (DUSP) genes in Japanese flounder (Paralichthys olivaceus) against temperature and Edwardsiella tarda stress.

Dual-specificity Phosphatases (DUSPs) are not only the key regulators of dephosphorylating and inactivating mitogen-activated protein kinases (MAPKs), but play a crucial role in the immune response. However, the role of DUSP genes in Japanese flounder (PoDUSPs) is still unclear. In this study, 28 DUSP genes in Japanese flounder were identified and classified based on the whole genome database. Phylogenetic analysis and protein structure analysis revealed that DUSPs had highly conserved domains in teleosts. Molecular evolution analysis indicated that the PoDUSP genes were conservative during evolution and were functional-constrained. Meanwhile, PoDUSP genes were found to express in different embryonic and larval stages which might play the role of sentinel in healthy organisms. Furthermore, PoDUSP genes' expression profiles after temperature stress and Edwardsiella tarda (E. tarda) infection were determined in Japanese flounder without precedent, and the results demonstrated that Podusp1, Podusp2 and Podusp16 were more respective to temperature variation whereas Podusp1 and Podusp6 were more respective to E. tarda infection. In summary, our results provide useful resources for understanding the immune responsibilities of DUSP genes in flatfish.

Taurine promotes Oreochromis niloticus survival against Edwardsiella tarda infection.

Edwardsiella tarda represents one of the most important pathogens that infects a variety of hosts including aquatic animals and humans. The outbreak of E. tarda infection is frequently reported in aquaculture that causes huge economic loss. Due to the widespread of antibiotic resistance, available antibiotics to treat bacterial infection are limited. Therefore, enhancing aquatic animals to survive upon E. tarda infection become an urgent issue. In this study, we profiled the metabolomic change of tilapia in-between the dying and survival fish upon E. tarda infection. The dying and survival fish mounts differential metabolic response, from which we identify a key metabolite, taurine, whose abundance is increased in both the survival group and the dying group but is more significant in the survival group. Exogenous taurine increases tilapia survival rate by 37.5% upon E. tarda infection. Further quantitative PCR analysis demonstrate taurine increases the expression of immune genes in liver, spleen and head kidney. Therefore, our study shows a new strategy to enhance fish immune response against bacterial infection.

Transcriptome analysis and protein-protein interaction in resistant and susceptible families of Japanese flounder (Paralichthys olivaceus) to understand the mechanism against Edwardsiella tarda.

Edwardsiella tarda is one of the most harmful bacterial pathogens for aquaculture flatfish. After artificial infection of 47 Japanese flounder (Paralichthys olivaceus) families, resistant and susceptible families were identified in this study. High-throughput sequencing was performed on the liver transcriptome of uninfected groups (PoRU and PoSU) and infected groups (PoRC and PoSC). Through assembly and annotation, a total of 3012 and 1386 differentially expressed genes (DEGs) were identified in PoRU vs. PoSU and PoRC vs. PoSC. The significant enrichment pathways between PoRU and PoSU were mainly in metabolic and biosynthesis pathways. A total of thirty dominant enrichment pathways between PoRC and PoSC mainly focused on some immune-related pathways, including the hematopoietic cell lineage, cytokine-cytokine receptor interaction, complement and coagulation cascades, antigen processing and presentation, the intestinal immune network for immunoglobulin A (IgA) production and T/B cell receptor signaling pathway. Under the protein-protein interaction (PPI) analysis, hub genes, including CD molecules, complement component factors and chemokines, were identified in the network, and 16 core genes were differentially expressed in resistant and sustainable families in quantitative polymerase chain reaction (qPCR) validation. This study represents the first transcriptome analysis based on resistant and susceptible families and provides resistant genes to understand the potential molecular mechanisms of antibacterial function in marine fish. The results obtained in this study provide crucial information on gene markers for resistant breeding of Japanese flounder.

Acid resistance system CadBA is implicated in acid tolerance and biofilm formation and is identified as a new virulence factor of Edwardsiella tarda.

Edwardsiella tarda is a facultative intracellular pathogen in humans and animals. The Gram-negative bacterium is widely considered a potentially important bacterial pathogen. Adaptation to acid stress is important for the transmission of intestinal microbes, so the acid-resistance (AR) system is essential. However, the AR systems of E. tarda are totally unknown. In this study, a lysine-dependent acid resistance (LDAR) system in E. tarda, CadBA, was characterized and identified. CadB is a membrane protein and shares high homology with the lysine/cadaverine antiporter. CadA contains a PLP-binding core domain and a pyridoxal phosphate-binding motif. It shares high homology with lysine decarboxylase. cadB and cadA are co-transcribed under one operon. To study the function of the cadBA operon, isogenic cadA, cadB and cadBA deletion mutant strains TX01ΔcadA, TX01ΔcadB and TX01ΔcadBA were constructed. When cultured under normal conditions, the wild type strain and three mutants exhibited the same growth performance. However, when cultured under acid conditions, the growth of three mutants, especially TX01ΔcadA, were obviously retarded, compared to the wild strain TX01, which indicates the important involvement of the cadBA operon in acid resistance. The deletion of cadB or cadA, especially cadBA, significantly attenuated bacterial activity of lysine decarboxylase, suggesting the vital participation of cadBA operon in lysine metabolism, which is closely related to acid resistance. The mutations of cadBA operon enhanced bacterial biofilm formation, especially under acid conditions. The deletions of the cadBA operon reduced bacterial adhesion and invasion to Hela cells. Consistently, the deficiency of cadBA operon abated bacterial survival and replication in macrophages, and decreased bacterial dissemination in fish tissues. Our results also show that the expression of cadBA operon and regulator cadC were up-regulated upon acid stress, and CadC rigorously regulated the expression of cadBA operon, especially under acid conditions. These findings demonstrate that the AR CadBA system was a requisite for the resistance of E. tarda against acid stress, and played a critical role in bacterial infection of host cells and in host tissues. This is the first study about the acid resistance system of E. tarda and provides new insights into the acid-resistance mechanism and pathogenesis of E. tarda.

A Novel microRNA of Japanese Flounder Regulates Antimicrobial Immunity Involving a Bacteria-Binding CSF3.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate diverse biological processes including immunity. In a previous high-throughput RNA sequencing study, a novel miRNA, pol-miR-novel_642, was identified from Japanese flounder (Paralichthys olivaceus), a farmed fish species with important economic value. In this study, we investigated the regulatory mechanism and the function of pol-miR-novel_642 and its target gene. We found that pol-miR-novel_642 targeted, in a sequence-specific manner, a flounder gene encoding an uncharacterized protein that is a structural homologue of murine granulocyte colony stimulating factor 3 (CSF3). The expression of pol-miR-novel_642 and its target gene (named PoCSF3-1) was regulated, in different manners, by the bacterial pathogen Edwardsiella tarda and the viral pathogen megalocytivirus. Overexpression of pol-miR-novel_642 or interference with PoCSF3-1 expression in flounder cells strongly potentiated E. tarda infection. Consistently, in vivo knockdown of PoCSF3-1 enhanced bacterial dissemination in flounder tissues but blocked viral replication, whereas in vivo overexpression of PoCSF3-1 inhibited bacterial dissemination and facilitated viral infection. Overexpression/knockdown of PoCSF3-1 and pol-miR-novel_642 also affected the activation of autophagy. Recombinant PoCSF3-1 (rPoCSF3-1) interacted with and inhibited the growth of Gram-negative bacteria in a manner relying on a PoCSF3-1-characteristic structural motif that is absent in mouse CSF3. rPoCSF3-1 also regulated the proliferation, inflammatory response, and immune defense of flounder head kidney leukocytes in a structure-dependent fashion. Together, these results reveal the function of a novel miRNA-CSF3 regulatory system of flounder, and add new insights into the role and mechanism of fish miRNA and CSF3 in antimicrobial immunity.