Teleost-specific TLR23 in Takifugu rubripes recruits MyD88 to trigger ERK pathway and promotes antibacterial defense.

Takifugu rubripes is a highly valued cultured fish in Asia, while pathogen infections can result in severe diseases and lead to substantial economic losses. Toll-like receptors (TLRs), as pattern recognition receptors, play a crucial role on recognition pathogens and initiation innate immune response. However, the immunological properties of teleost-specific TLR23 remain largely unknown. In this study, we investigated the biological functions of TLR23 (TrTLR23) from T. rubripes, found that TrTLR23 existed in various organs. Following bacterial pathogen challenge, the expression levels of TrTLR23 were significantly increased in immune related organs. TrTLR23 located on the cellular membrane and specifically recognized pathogenic microorganism. Co-immunoprecipitation and antibody blocking analysis revealed that TrTLR23 recruited myeloid differentiation primary response protein (MyD88), thereby mediating the activation of the ERK signaling pathway. Furthermore, in vivo showed that, when TrTLR23 is overexpressed in T. rubripes, bacterial replication in fish tissues is significantly inhibited. Consistently, when TrTLR23 expression in T. rubripes is knocked down, bacterial replication is significantly enhanced. In conclusion, these findings suggested that TrTLR23 played a critical role on mediation TLR23-MyD88-ERK axis against bacterial infection. This study revealed that TLR23 involved in the innate immune mechanism, and provided the foundation for development disease control strategies in teleost.

The TAK1/JNK axis participates in adaptive immunity by promoting lymphocyte activation in Nile tilapia.

The transforming growth factor beta-activated kinase 1 (TAK1)/c-Jun N-terminal kinase (JNK) axis is an essential MAPK upstream mediator and regulates immune signaling pathways. However, whether the TAK1/JNK axis harnesses the strength in regulation of signal transduction in early vertebrate adaptive immunity is unclear. In this study, by modeling on Nile tilapia (Oreochromis niloticus), we investigated the potential regulatory function of TAK1/JNK axis on lymphocyte-mediated adaptive immune response. Both OnTAK1 and OnJNK exhibited highly conserved sequences and structures relative to their counterparts in other vertebrates. Their mRNA was widely expressed in the immune-associated tissues, while phosphorylation levels in splenic lymphocytes were significantly enhanced on the 4th day post-infection by Edwardsiella piscicida. In addition, OnTAK1 and OnJNK were significantly up-regulated in transcriptional level after activation of lymphocytes in vitro by phorbol 12-myristate 13-acetate plus ionomycin (P + I) or PHA, accompanied by a predominant increase in phosphorylation level. More importantly, inhibition of OnTAK1 activity by specific inhibitor NG25 led to a significant decrease in the phosphorylation level of OnJNK. Furthermore, blocking the activity of OnJNK with specific inhibitor SP600125 resulted in a marked reduction in the expression of T-cell activation markers including IFN-γ, CD122, IL-2, and CD44 during PHA-induced T-cell activation. In summary, these findings indicated that the conserved TAK1/JNK axis in Nile tilapia was involved in adaptive immune responses by regulating the activation of lymphocytes. This study enriched the current knowledge of adaptive immunity in teleost and provided a new perspective for understanding the regulatory mechanism of fish immunity.

Granzyme B secreted by T cells is involved in anti-bacterial immune response of tilapia.

Secreted by natural killer cells and cytotoxic T lymphocytes, Granzyme B is involved in regulating the adaptive immune response in vertebrates and plays a pivotal role in resisting virus invasion and removing pathogens. Although it had been extensively studied in mammals, the involvement of Granzyme B in adaptive immune response of early vertebrates remained elusive. In this study, we investigated the Granzyme B in Oreochromis niloticus (OnGrB), found that its function domain was conserved. Additionally, OnGrB was widely expressed in various tissues and could respond to T-cell activation in vitro at the transcriptional level. Furthermore, we prepared the recombinant OnGrB (rOnGrB) as an immunogen to develop a mouse anti-OnGrB monoclonal antibody (mAb). Using this anti-OnGrB mAb as a tool, we explored the expression of OnGrB in the adaptive immune response of tilapia. Our findings revealed that T cell was a significant source of OnGrB production, the expression of OnGrB at the protein level and the proportion of OnGrB + T cells increased after both T cell activation in vitro and infection with Edwardsiella piscicida in vivo. More importantly, our findings also preliminarily illuminated that p65 could regulate the transcriptional activity of OnGrB. These results indicated that OnGrB was involved in the adaptive immunity of tilapia and played a critical role in T cell function in teleost. Our study provided theoretical support and new perspectives for understanding adaptive immunity in teleost.

The Edwardsiella piscicida thioredoxin-like protein inhibits ASK1-MAPKs signaling cascades to promote pathogenesis during infection.

It is important that bacterium can coordinately deliver several effectors into host cells to disturb the cellular progress during infection, however, the precise role of effectors in host cell cytosol remains to be resolved. In this study, we identified a new bacterial virulence effector from pathogenic Edwardsiella piscicida, which presents conserved crystal structure to thioredoxin family members and is defined as a thioredoxin-like protein (Trxlp). Unlike the classical bacterial thioredoxins, Trxlp can be translocated into host cells, mimicking endogenous thioredoxin to abrogate ASK1 homophilic interaction and phosphorylation, then suppressing the phosphorylation of downstream Erk1/2- and p38-MAPK signaling cascades. Moreover, Trxlp-mediated inhibition of ASK1-Erk/p38-MAPK axis promotes the pathogenesis of E. piscicida in zebrafish larvae infection model. Taken together, these data provide insights into the mechanism underlying the bacterial thioredoxin as a virulence effector in downmodulating the innate immune responses during E. piscicida infection.

Identification and application of T3SS translocation signal in Edwardsiella piscicida attenuated carrier as a bivalent vaccine.

Type III secretion system (T3SS)-dependent translocation has been used to deliver heterologous antigens by vaccine carriers into host cells. In this research, we identified the translocation signal of Edwardsiella piscicida T3SS effector EseG and constructed an antibiotic resistance-free balanced-lethal system as attenuated vaccine carrier to present antigens by T3SS. Edwardsiella piscicida LSE40 asd gene deletion mutant was constructed and complemented with pYA3342 harbouring the asd (aspartate ß-semialdehyde dehydrogenase) gene from Salmonella. Fusion proteins composed of EseG N-terminal 1-108 amino acids and the TEM1-ß-lactamase reporter were inserted in plasmid pYA3342. The fusion protein could secrete into the cell culture, translocate into HeLa cells, and localize in the membrane fraction. Then, the double gene deletion mutant LSE40ΔasdΔpurA was constructed as an attenuated vaccine carrier, and Aeromonas hydrophila GapA (glyceraldehyde-3-phosphate dehydrogenase) was fused with the translocation signal, instead of the TEM1-ß-lactamase reporter. The bivalent vaccine could protect blue gourami (Trichogaster trichopterus) against E. piscicida and A. hydrophila, with the relative per cent survival of 80.77% and 63.83%, respectively. These results indicated that EseG N-terminal 1-108 amino acid peptide was the translocation signal of E. piscicida T3SS, which could be used to construct bivalent vaccines based on an attenuated E. piscicida carrier.

An Edwardsiella piscicida esaS mutant reveals contribution to virulence and vaccine potential.

Edwardsiella piscicida is a Gram-negative pathogen that causes disease in diverse aquatic organisms. The disease leads to extensive losses in commercial aquaculture species, including farmed U.S. catfish. The type III secretion system (T3SS) often contributes to virulence of Gram-negative bacteria. The E. piscicida esaS gene encodes a predicted T3SS export apparatus protein. In the current study, an E. piscicida esaS mutant was constructed and characterized to increase our understanding of the role of T3SS in E. piscicida virulence. Deletion of esaS did not significantly affect biofilm formation and hemolytic activity of E. piscicida, but it had significant effects on expression of hemolysis and T3SS effector genes during biofilm growth. EpΔesaS showed significantly (P < 0.05) reduced virulence in catfish compared to the parent strain. No mortalities occurred in fish infected with EpΔesaS at 6.3 × 105 and 1.26 × 106 CFU/fish compared to 26% mortality in fish infected with wild-type E. piscicida at 7.5 × 105 CFU/fish. Bioluminescence imaging indicated that EpΔesaS invades catfish and colonizes for a short period in the organs. Furthermore, catfish immunized with EpΔesaS at 6.3 × 105 and 1.26 × 106 CFU provided 47% and 87% relative percent survival, respectively. These findings demonstrated that esaS plays a role in E. piscicida virulence, and the deletion mutant has vaccine potential for protection against wild-type E. piscicida infection.

Pathogenicity and immunogenicity of Edwardsiella piscicida ferric uptake regulator (fur) mutations in zebrafish.

Edwardsiella piscicida is the etiological agent of edwardsiellosis in fish and causes severe economic losses in global aquaculture. Vaccination would be the most effective method to prevent infectious diseases and their associated economic losses. The ferric uptake regulator (Fur) is an important transcriptional global regulator of Gram-negative bacteria. In this study, we examined the regulatory function of Fur in E. piscicida. We designed a strain that displays features of the wild-type virulent strain of E. piscicida at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. Regulated delayed attenuation in vivo is based on the substitution of a tightly regulated araC ParaBAD cassette for the promoter of the fur gene such that expression of this gene is dependent on arabinose provided during growth. Thus, following E. piscicida mutant colonization of lymphoid tissues, the Fur protein ceases to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. We deleted the promoter, including all sequences that interact with activator or repressor proteins, for the fur gene, and substituted the improved araC ParaBAD cassette to yield an E. piscicida strain with the ΔPfur170:TT araC ParaBADfur deletion-insertion mutation (χ16012). Compared to the wild-type strain J118, χ16012 exhibited retarded growth and enhanced siderophore production in the absence of arabinose. mRNA levels of Fur-regulated genes were analyzed in iron deplete or replete condition in wild-type and fur mutant strains. We observed zebrafish immunized with χ16012 showed better colonization and protection compared to the Δfur (χ16001). Studies showed that E. piscicida strain χ16012 is attenuated and induces systemic and mucosal IgM titer in zebrafish. In addition, we found an increase in transcript levels of tnf-α, il-1ß, il-8 and ifn-γ in different tissues of zebrafish immunized with χ16012 compared to the unimmunized group. We conclude that, E. piscicida with regulated delayed attenuation could be an effective immersion vaccine for the aquaculture industry.

Characterization of a novel live attenuated Edwardsiella piscicida vaccine based on the overexpressed type III secretion system and systematic deletion of the associated effectors.

Edwardsiella piscicida causes edwardsiellosis in a variety of fish species and leads to tremendous economic losses in the global aquaculture industries. Thus, effective and safe prevention and control of this bacterium are urgently needed to combat the related infections. Live attenuated vaccines (LAVs) effectively prevent infectious diseases. However, most of the existing E. piscicida LAVs are based on the deletion of genes encoding the translocon components of the type III secretion system (T3SS), the core virulence system, which is the most prominent protective bacterial antigen with the strongest immunogenicity. In this study, we systematically deleted all of the 9 established T3SS effectors in E. piscicida (aka 9Δ) and the rpoS gene encoding the alternative sigma factor, the esrB repressor (10Δ), then we overexpressed esrB and T3SS in E. piscicida to obtain the recombinant strain 10Δ/esrBOE. The modified strains 10Δ and 10Δ/esrBOE exhibited severe attenuation and in vivo colonization defects. Additionally, vaccination by intraperitoneal injection with 10Δ and 10Δ/esrBOE could significantly upregulate the expression of the antigen recognition related gene (TLR5) and the adaptive immune response-related gene (MHC II) in the spleen/kidney of turbot fish, and it also enhanced the hosts' serum bactericidal capacity. Finally, vaccination with 10Δ/esrBOE led to increased immune protection against the challenge of wild type E. piscicida EIB202 in turbot fish. Collectively, these findings demonstrated that 10Δ/esrBOE was a novel LAV strain and therefore a potential novel strategy for the construction of LAVs against bacterial pathogens.

Characterization of the inflammasome component SmASC in turbot (Scophthalmus maximus).

Apoptosis-associated speck-like protein containing a C-terminal caspase recruit domain (ASC) is an important adapter protein in the inflammasome complex that mediates inflammatory caspase activation and host innate immunity in mammals. However, the function of inflammasome components in lower vertebrate remains poorly understood. In this study, full length of SmASC was cloned from turbot (Scophthalmus maximus). Through bioinformatic analysis, we found that SmASC shares relatively high identity with ASC in bony fish. Furthermore, we found that the intact SmASC can form an oligomeric speck-like structure, while the PYD segment of SmASC can form the filamentous structure. Moreover, expression of SmASC was induced after intraperitoneal injection of Edwardsiella piscicida (E. piscicida) in vivo. To further explore the role of SmASC during infection, we constructed SmASC knockdown and overexpression models by administration of siRNA and overexpression plasmids in vivo, respectively. Expression of SmASC decreased the propagation of E. piscicida in different immune organs. In summary, our results characterize the function of SmASC in S. maximus, suggesting that the SmASC plays a critical role in turbot immune responses.

Immunization of a novel outer membrane protein from Aeromonas hydrophila simultaneously resisting A. hydrophila and Edwardsiella anguillarum infection in European eels (Angullia angullia).

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, an expressed recombinant Outer membrane proteinⅡ (rOmpⅡ) from A. hydrophila was intraperitoneally injected into European eels (Angullia angullia). All examined eels were equally divided into three groups. One group was injected with PBS only (PBS group), one group was injected with 1:1 mixture of PBS and Freund's incomplete adjuvant (PBS + F, adjuvant group), and the third group was injected with 1:1 mixture of 1 mg mL-1 rOmpⅡ and Freund's incomplete adjuvant (rOmpⅡ+F, OmpⅡ group). The immunogenicity of OmpⅡ was studied by detecting the expression of 4 immune-related genes, stimulation index (SI) of the whole blood cell, serum antibody titer, lysozyme and Superoxide Dismutase (SOD) activity, and relative percent of survival (RPS) rate. The results showed that gene expression of MHC-Ⅱ, LysC, SOD and IgM in the OmpⅡ group significantly increased in liver, spleen, kidney and intestine. At 28 days post the immunization (dpi), the SI of whole blood cells in the OmpⅡ group increased significantly; at 14, 21, 28 and 42 dpi, the serum antibody titers against A. hydrophila and E. anguillarum in the OmpⅡ group were significantly higher than that of the PBS and the adjuvant group; the SOD in the OmpⅡ group was found increased significantly in liver, kidney, mucus and serum. On the 28 dpi, eels were challenged by A. hydrophila, E. anguillarum and V. vulnificus for cross protection study. The results showed that the RPS of the OmpⅡ group were 83.33%, 55.56% and 33.33% respectively. These results showed that the expressed OmpⅡ from A. hydrophila significantly improve the immune function of Europena eels and their resistance to the infection of A. hydrophila and E. anguillarum simultaneously.

Pattern analysis of conditional essentiality (PACE)-based heuristic identification of an in vivo colonization determinant as a novel target for the construction of a live attenuated vaccine against Edwardsiella piscicida.

Edwardsiella piscicida is the aetiological agent of fish edwardsiellosis, causing huge economic losses in aquaculture industries. The use of a live attenuated vaccine (LAV) will be an effective strategy to control the disease in farmed fish. Thus, methods facilitating exploration of targets used for construction of an LAV will be of great significance. Previously, we devised an algorithm termed pattern analysis of conditional essentiality (PACE) to perform genome-wide analysis of the temporal dynamic behaviour of E. piscicida mutants colonizing turbot. Here, we correlated the conditional essentiality patterns of the PACE-derived colonization determinants with that of the aroC gene encoding chorismate synthase, the established target for LAV construction in E. piscicida, and identified ETAE_0023 as a novel valuable LAV target. ETAE_0023 encodes an uncharacterized DcrB family protein. Deletion of ETAE_0023 dramatically impaired E. piscicida invasion capability in ZF4 cells as well as colonization in fish and resulted in in vivo clearance at ∼30 days post-infection. ΔETAE_0023 showed an ∼2500-fold higher 50% lethal dose (LD50) than that of the wild type strain. Vaccination with ΔETAE_0023 by intraperitoneal (i.p.) injection upregulated expression of immune factors, i.e., IL-1ß, IgM, MHC-I and MHC-II, and produced significantly high levels of E. piscicida-specific IgM as well as serum bactericidal capacities in turbot. Moreover, a single i.p. inoculation with ΔETAE_0023 generated significant protection comparable to the established WED LAV strain in turbot against challenge with the wild type strain after 5 weeks of vaccination. Taken together, we demonstrated a PACE-based method for heuristic identification of targets for LAV construction and presented ΔETAE_0023 as a new LAV candidate against edwardsiellosis.

Universal stress proteins contribute Edwardsiella piscicida adversity resistance and pathogenicity and promote blocking host immune response.

Universal stress proteins (Usps) exist ubiquitously in bacteria and other organisms. Usps play an important role in adaptation of bacteria to a variety of environmental stresses. There is increasing evidence that Usps facilitate pathogens to adapt host environment and are involved in pathogenicity. Edwardsiella piscicida (formerly included in E. tarda) is a severe fish pathogen and infects various important economic fish including tilapia (Oreochromis niloticus). In E. piscicida, a number of systems and factors that are involved in stress resistance and pathogenesis were identified. However, the function of Usps in E. piscicida is totally unknown. In this study, we examined the expressions of 13 usp genes in E. piscicida and found that most of these usp genes were up-regulated expression under high temperature, oxidative stress, acid stress, and host serum stress. Particularly, among these usp genes, usp13, exhibited dramatically high expression level upon several stress conditions. To investigate the biological role of usp13, a markerless usp13 in-frame mutant strain, TX01Δusp13, was constructed. Compared to the wild type TX01, TX01Δusp13 exhibited markedly compromised tolerance to high temperature, hydrogen peroxide, and low pH. Deletion of usp13 significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis showed that the inactivation of usp13 significantly impaired the ability of E. piscicida to invade into host cell and infect host tissue. Introduction of a trans-expressed usp13 gene restored the lost virulence of TX01Δusp13. In support of these results, host immune response induced by TX01 and TX01Δusp13 was examined, and the results showed reactive oxygen species (ROS) levels in TX01Δusp13-infected macrophages were significantly higher than those in TX01-infected cells. The expression level of several cytokines (IL-6, IL-8, IL-10, TNF-α, and CC2) in TX01Δusp13-infected fish was significantly higher than that in TX01-infected fish. These results suggested that the deletion of usp13 attenuated the ability of bacteria to overcome the host immune response to pathogen infection. Taken together, our study indicated Usp13 of E. piscicida was not only important participant in adversity resistance, but also was essential for E. piscicida pathogenicity and contributed to block host immune response to pathogen infection.

Synergistic effect of a combined live Vibrio anguillarum and Edwardsiella piscicida vaccine in turbot.

In aquaculture, more than one pathogen usually be isolated from the sick fish, creating an urgent need for developing combined vaccines to control fish disease caused by multiple pathogens simultaneously. In our previous work, two live attenuated vaccines against Vibrio anguillarum and Edwardsiella piscicida were vaccinated in turbot, exhibiting an efficient protection. However, some immunological processes such as antigenic competition, antigenic cross-reaction and antigen induced suppression during combined vaccination are unknown. In this study, we evaluated the effectiveness of the combined live vaccines and explored the immunological processes after vaccination. We found that the combined two live attenuated vaccines for V. anguillarum and E. piscicida induced a stronger immune response without existing antigen competition. Instead, a synergistic effect was observed not only for triggering innate immune response but for stimulation of adaptive immunity. Our study suggested that the two combined live vaccines against V. anguillarum and E. piscicida could be used simultaneously in the future.

Edwardsiella piscicida virulence effector trxlp promotes the NLRC4 inflammasome activation during infection.

Edwardsiella piscicida is an important pathogenic bacterium that causes hemorrhagic septicemia in fish. This bacterium could activate NLRC4 and NLRP3 inflammasomes via type III secretion system (T3SS), and inhibit NLRP3 inflammasome via type VI secretion system (T6SS) effector during infection in macrophages. However, the roles of other virulence factors in regulating inflammasome activation during E. piscicida infection remain poorly understood. In this study, we focused on clarification the role of ETAE_RS10155, a thioredoxin-like protein (Trxlp), during bacterial infection in macrophages. We found that mutation of this gene barely influences the bacteria growth and infection capability. Interestingly, the inflammasome activation was reduced in Δtrxlp-infected macrophages, compared with wild-type E. piscicida did. Moreover, Trxlp mainly promotes the NLRC4, but not NLRP3 inflammasome activation during E. piscicida infection. Finally, Trxlp-mediated NLRC4 inflammasome activation is crucial for host surveillance in vivo. Taken together, our results clarify the complex and contextual role of bacterial virulence effector in modulating inflammasome activation, and offer new insights into the warfare between the fish bacterial weapons and host innate immunological surveillance.

Construction and evaluation of type III secretion system mutants of the catfish pathogen Edwardsiella piscicida.

Catfish is the largest aquaculture industry in the United States. Edwardsiellosis is considered one of the most significant problems affecting this industry. Edwardsiella piscicida is a newly described species within the genus Edwardsiella, and it was previously classified as Edwardsiella tarda. It causes gastrointestinal septicaemia, primarily in summer months, in farmed channel catfish in the south-eastern United States. In the current study, we adapted gene deletion methods used for Edwardsiella to E. piscicida strain C07-087, which was isolated from a disease outbreak in a catfish production pond. Four genes encoding structural proteins in the type III secretion system (T3SS) apparatus of E. piscicida were deleted by homologous recombination and allelic exchange to produce in-frame deletion mutants (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT). The mutants were phenotypically characterized, and virulence and vaccine efficacy were evaluated. Three of the mutants, EpΔssaV, EpΔyscR and EpΔesaM, were significantly attenuated compared to the parent strain (p < .05), but EpΔescT strain was not. Vaccination of catfish with the four mutant strains (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT) provided significant protection when subsequently challenged with wild-type strain. In conclusion, we report methods for gene deletion in E. piscicida and development of vaccine candidates derived from a virulent catfish isolate.

Edwardsiella piscicida lacking the cyclic AMP receptor protein (Crp) is avirulent and immunogenic in fish.

Edwardsiella piscicida is a Gram-negative pathogen that generally causes lethal septicemia in marine and freshwater fish. We generated a E. piscicida CK216 Δcrp mutant to investigate various biological roles related to this organism, including pathogenesis. Lack of Crp in CK216 was demonstrated by immunoblotting using a Crp-specific antibody. Compared to the parental strain, the mutant exhibited changes in three biochemical phenotypes, including ornithine decarboxylation, citrate utilization, and H2S production. Complementation of crp deletion in trans rescued the phenotype of the parental strain. This study proved that hemolytic activity in E. piscicida is controlled by Crp. In addition, significantly reduced motility of E. piscicida CK216 was observed, which resulted from a lack of flagella synthesis. To examine the virulence in fish, E. piscicida cells were injected into the goldfish (Carassius auratus) via intraperitoneal route. The LD50 of CK216 was 9.25 × 108 CFU, while that of the CK108 parental strain was 9.24 × 105 CFU, attenuated 1000 fold in goldfish. Fish immunized with CK216 elicited IgM responses. Moreover, 80% of goldfish immunized with 1 × 106 CFU survived after administration of a lethal dose (1 × 107 CFU) of virulent E. piscicida CK41, suggesting the potential for E. piscicida CK216 to serve as a live attenuated vaccine in aquaculture.

Comprehensive gene expression profiling in Japanese flounder kidney after injection with two different formalin-killed pathogenic bacteria.

Pathogenic bacteria possess some components, that are recognized by the host immune receptors. Different components are recognized by distinct receptors, and thus it is speculated that these components may regulate different gene sets. To investigate the gene expression profiles regulated by different bacterial components in Japanese flounder Paralichthys olivaceus, Japanese flounder were intraperitoneally injected with formalin-killed bacterial cells (FKC) of Edwardsiella tarda and Streptococcus iniae. The numbers of differentially regulated genes were much larger in the fish injected with E. tarda than those with S. iniae. Comprehensive gene expression profiling showed that almost all of the genes differentially regulated by injections of E. tarda FKC were also differentially regulated by injections of S. iniae FKC. mRNA levels of inflammatory cytokines, including interleukin (IL)-1ß, IL-8, interferon γ and tumor necrosis factor were upregulated in both of the injected groups. Each of these mRNAs except for IL-8 mRNA were also much higher in the E. tarda FKC injected group than in the S. iniae FKC injected group. Furthermore, mRNA levels of IL-6 were strongly up-regulated in the E. tarda FKC injected group. The E. tarda FKC might induce higher inflammatory responses than S. iniae FKC.

ScRNA-seq reveals trained immunity-engaged Th17 cell activation against Edwardsiella piscicida-induced intestinal inflammation in teleost.

Mucosal immunity typically involves innate and adaptive immune cells, while the cellular mechanism of teleost's intestinal immune cells that engages gut homeostasis against bacterial infection remains largely unknown. Taking advantage of the enteric fish pathogen (Edwardsiella piscicida) infection-induced intestinal inflammation in turbot (Scophthalmus maximus), we find that ß-glucan training could mitigate the bacterial infection-induced intestinal inflammation. Through single-cell transcriptome profiling and cellular function analysis, we identify that E. piscicida infection could tune down the activation of intestinal Th17 cells, while ß-glucan-training could preserve the potential to amplify and restore the function of intestinal Th17 cells. Moreover, through pharmacological inhibitor treatment, we identify that Th17 cells are essential for ameliorating bacterial infection-induced intestinal inflammation in teleost. Taken together, these results suggest a new concept of trained immunity activation to regulate the intestinal Th17 cells' function, which might contribute to better developing strategies for maintaining gut homeostasis against bacterial infection.

Ly-6D of Japanese flounder (Paralichthys olivaceus) functions as a complement regulator and promotes host clearance of pathogen.

The Lymphocyte antigen-6 (Ly-6) superfamily has been considered to play an important role in the innate immunity of mammals. The functions of Ly-6 proteins are diverse since their low sequence homology. Currently, the function of Ly-6D, a member of Ly-6 family proteins, is completely unknown in teleost. In the present study, we identified and characterized a Ly-6D homologue (named PoLy-6D) from the teleost fish Paralichthys olivaceus and examined its immune function. PoLy-6D possesses a hydrophobic signal peptide, a LU domain including a conserved "LXCXXC" motif in N-terminus and a "CCXXXXCN" motif in C-terminus. Under normal physiological condition, PoLy-6D expression distributes in all the examined tissues, the highest three tissues are successively spleen, head kidney, and blood. When infected by extracellular and intracellular bacterial pathogens and viral pathogen, PoLy-6D expression was induced and the patterns vary with different types of microbial pathogens infection and different immune tissues. In vitro experiment showed recombinant PoLy-6D (rPoLy-6D) inhibited the lysis of rabbit red blood cells by serum and selectively improved bacterial survival in serum. After serum were treated by antibody of rPoLy-6D, bacteriostatic effect of serum was obviously enhanced. These results indicate the importance of PoLy-6D as a complement regulator. rPoLy-6D possessed the binding activity to multiple bacteria but did not exhibit antimicrobial activities. The interaction between rPoLy-6D and bacteria suggests that PoLy-6D is involved in host clearance of pathogens probably by serving as a receptor for pathogens. Overexpression of PoLy-6D in vivo promoted the host defense against invading E. piscicida. These findings add new insights into the regulation mechanism of the complement system in teleost and emphasize the importance of Ly-6D products for the control of pathogen infection.

Characterization and expression analysis of tandemly-replicated asc genes in the Japanese medaka, Oryzias latipes.

ASC is a component of the inflammasome playing crucial roles in the inflammatory response. In mammals, ASC induces pyroptosis and inflammatory cytokine production. In this study, three asc genes (asc1, asc2, and asc3) from the Japanese medaka (Oryzias latipes) were identified and characterized. These asc genes were tandem replicates on chromosome 16, and their exon-intron structures differed between them. All three ASCs conserved the pyrin and caspase-recruitment domains, which are important for inflammasome formation. In phylogenetic analysis, all ASCs clustered with those of other teleosts. The asc1 expression levels were significantly higher in several organs than those of asc2 and asc3, suggesting that asc1 may act as a dominant asc in the Japanese medaka. Expression of the three asc genes showed different patterns during Aeromonas hydrophila and Edwardsiella piscicida infections. Furthermore, their expression was adequately down-regulated in the medaka fin-derived cells stimulated with ATP for 12 h, while asc2 expression was statistically up-regulated after nigericin stimulation for 24 h. Moreover, the expression of asc2 and asc3 was significantly higher in the skin of ASC-1-knockout medaka than in that of the wild type medaka during A. hydrophila infection.