Pol-miR-363-3p plays a significant role in the immune defense of Japanese flounder Paralichthys olivaceus against bacterial and viral infection.

In this study, we examined the function of a Japanese flounder (Paralichthys olivaceus) microRNA (miRNA), pol-miR-363-3p. We found that pol-miR-363-3p targets an ubiquitin-specific protease (USP), USP32. USP is a family of deubiquitinating enzymes essential to the functioning of the ubiquitin proteasome system. In mammals, USP32 is known to be associated with cancer and immunity. In fish, the function of USP32 is unknown. We found that flounder USP32 (PoUSP32) expression was detected in the major tissues of flounder, particularly intestine. In vitro and in vivo studies showed that pol-miR-363-3p directly regulated PoUSP32 in a negative manner by interaction with the 3'UTR of PoUSP32. Overexpression of pol-miR-363-3p or interference with PoUSP32 expression in flounder cells significantly blocked Streptococcus iniae infection. Consistently, in vivo knockdown of pol-miR-363-3p or overexpression of PoUSP32 enhanced dissemination of S. iniae in flounder tissues, whereas in vivo knockdown of PoUSP32 inhibited S. iniae dissemination. In addition, pol-miR-363-3p knockdown also significantly promoted the tissue dissemination of the viral pathogen megalocytivirus, which, as well as S. iniae, regulated pol-miR-363-3p expression. Together these results revealed an important role of pol-miR-363-3p in flounder immune defense against bacterial and viral infection.

Characterization of Streptococcus iniae-induced microRNA profiles in Paralichthys olivaceus and identification of pol-3p-10740_175 as a regulator of antibacterial immune response.

MicroRNAs (miRNAs) are involved in many biological activities including immune defense against pathogens. In this study, we applied high-throughput sequencing technology to examine miRNAs in Japanese flounder (Paralichthys olivaceus) infected with Streptococcus iniae at different times. A total of 1038 miRNAs were identified, of which, 249 were novel miRNAs, and 81 showed differential expression (named DEmiRNAs) after S. iniae infection. Of the 81 DEmiRNAs identified, 34 and 58 occurred at 6 h and 24 h post-infection, respectively; most DEmiRNAs were strongly time-specific, and only 13.6% of the DEmiRNAs were shared between the two time points. A total of 9582 target genes were predicted for the 81 DEmiRNAs. The putative target genes were enriched in various GO and KEGG pathways of biological processes and molecular/cellular functions, in particular endocytosis, regulation of transcription, lysososme, and the signaling pathways of MAPK, ErbB, and AMPK. One of the DEmiRNAs, pol-3p-10740_175, was found to target dual specificity phosphatase 6 (Dusp6) and repress the expression of the latter. Transfection of flounder FG cells with pol-3p-10740_175 caused a significant inhibition on S. iniae invasion. The results of this study provided the first S. iniae-induced miRNA profile in Japanese flounder and indicated that flounder miRNAs play an important role in antibacterial immunity.

pol-miR-194a of Japanese flounder (Paralichthys olivaceus) suppresses type I interferon response and facilitates Edwardsiella tarda infection.

MicroRNAs (miRNAs) are a type of small non-coding RNAs that participate in diverse cellular processes including microbial invasion and immune defense. In a previous study, we identified a large amount of Japanese flounder (Paralichthys olivaceus) miRNAs responsive to megalocytivirus infection. In the present study, we examined the function of one of these miRNAs, pol-miR-194a, in association with the infectivity of Edwardsiella tarda, an intracellular bacterial pathogen to many fish species including flounder. We found that pol-miR-194a was induced in expression to a significant extent in the spleen, liver, and gill of Japanese flounder infected by E. tarda. Transfection of flounder cells with pol-miR-194a mimic significantly enhanced the intracellular replication of E. tarda. pol-miR-194a was able to interact specifically with the 3'UTR of IRF7 in a negative manner, resulting in inhibition of IRF7 expression. Consistently, pol-miR-194a significantly blocked the promoter activity of type Ⅰ interferon. Taken together, these results indicate that pol-miR-194a plays an important role in the regulation of flounder immune response as well as microbial infection, and that pol-miR-194a probably serves as a target for E. tarda to manipulate and escape host immune defense.

Integrin αvß1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection.

Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or ß1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and ß1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvß1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV.

A virulent Bacillus cereus strain from deep-sea cold seep induces pyroptosis in a manner that involves NLRP3 inflammasome, JNK pathway, and lysosomal rupture.

Recent studies indicate that the Bacillus species is distributed in deep-sea environments. However, no specific studies on deep-sea Bacillus cereus have been documented. In the present work, we isolated a B. cereus strain, H2, from the deep-sea cold seep in South China Sea. We characterized the pathogenic potential of H2 and investigated H2-induced death of different types of cells. We found that H2 was capable of tissue dissemination and causing acute mortality in mice and fish following intraperitoneal/intramuscular injection. In vitro studies revealed that H2 infection of macrophages induced pyroptosis and activation of the NLRP3 inflammasome pathway that contributed partly to cell death. H2 infection activated p38, JNK, and ERK, but only JNK proved to participate in H2-triggered cell death. Reactive oxygen species (ROS) and intracellular Ca2+ were essential to H2-induced activation of JNK and NLRP3 inflammasome. In contrast, lysosomal rupture and cathepsins were required for H2-induced NLRP3 inflammasome activation but not for JNK activation. This study revealed for the first time the virulence characteristics of deep-sea B. cereus and provided new insights into the mechanism of B. cereus infection.

Japanese flounder pol-miR-3p-2 suppresses Edwardsiella tarda infection by regulation of autophagy via p53.

MicroRNAs (miRNAs) are post-transcriptional regulators that play vital roles in diverse physiological processes including immunity. In this study, we investigated the regulatory mechanism and function of a novel Japanese flounder (Paralichthys olivaceus) miRNA, pol-miR-3p-2. pol-miR-3p-2 was responsive in expression to the infection of the bacterial pathogen Edwardsiella tarda. pol-miR-3p-2 negatively regulated the expression of p53 through interaction with the 3'UTR of p53. Overexpression of pol-miR-3p-2 promoted autophagy, resulting in augmented production of LC3-II, while knockdown of p53 increased the level of beclin, a key factor of autophagy. In vivo and in vitro studies showed that E. tarda infection induced autophagy in flounder, and pol-miR-3p-2 inhibited the infectivity of E. tarda. Together these results indicate that pol-miR-3p-2 regulates autophagy through the target gene p53, thus revealing a regulatory link between p53 and autophagy in teleost, and that pol-miR-3p-2 plays an important role in the immune defense against E. tarda.

The novel fish miRNA pol-miR-novel_171 and its target gene FAM49B play a critical role in apoptosis and bacterial infection.

MicroRNAs (miRNAs) are a type of small, non-coding RNAs that participate in many cellular and biological processes by regulating mRNA stability. In a previous study, we identified 96 Japanese flounder (Paralichthys olivaceus) miRNAs responsive to the infection of Edwardsiella tarda, a bacterial pathogen to fish as well as humans. In the current study, we examined the regulation and function of one novel miRNA, i.e., pol-miR-novel_171, from the above 96 miRNA pool. We found that pol-miR-novel_171 expression was regulated by E. tarda and megalocytivirus in a pathogen-specific manner, and that pol-miR-novel_171 targeted the gene of FAM49B (family with sequence similarity 49 member B) of flounder (named PoFAM49B) by negative interaction with the 3'-UTR of PoFAM49B. To date, the function fish FAM49B is unknown. We found that PoFAM49B expressed in multiple tissues of flounder, and recombinant PoFAM49B interacted with and inhibited the growth of Gram-negative bacterial pathogens. Interference with PoFAM49B expression in flounder cells promoted E. tarda infection. Similar effects on E. tarda infection were observed with pol-miR-novel_171 overexpression. Consistently, in vivo knockdown of PoFAM49B in flounder enhanced E. tarda dissemination in fish tissues. Furthermore, interference with PoFAM49B expression, or overexpression of pol-miR-novel_171, promoted apoptosis of flounder cells, while in vitro and in vivo knockdown of PoFAM49B augmented the expressions of key apoptosis-associated genes. These results revealed for the first time the immune function of fish FAM49B and the regulatory mechanism of a novel fish miRNA by demonstrating that pol-miR-novel_171, via PoFAM49B, played a critical role in apoptosis and anti-bacterial immunity.