Antibacterial Activity and Mechanisms of TroHepc2-22, a Derived Peptide of Hepcidin2 from Golden Pompano (Trachinotus ovatus).

Hepcidin, a cysteine-rich antimicrobial peptide, has a highly conserved gene structure in teleosts, and it plays an essential role in host immune response against various pathogenic bacteria. Nonetheless, few studies on the antibacterial mechanism of hepcidin in golden pompano (Trachinotus ovatus) have been reported. In this study, we synthesized a derived peptide, TroHepc2-22, from the mature peptide of T. ovatus hepcidin2. Our results showed that TroHepc2-22 has superior antibacterial abilities against both Gram-negative (Vibrio harveyi and Edwardsiella piscicida) and Gram-positive (Staphylococcus aureus and Streptococcus agalactiae) bacteria. Based on the results of a bacterial membrane depolarization assay and propidium iodide (PI) staining assay in vitro, TroHepc2-22 displayed antimicrobial activity by inducing the bacterial membrane depolarization and changing the bacterial membrane permeability. Scanning electron microscopy (SEM) visualization illustrated that TroHepc2-22 brought about membrane rupturing and the leakage of the cytoplasm for the bacteria. In addition, TroHepc2-22 was verified to have hydrolytic activity on bacterial genomic DNA in view of the results of the gel retardation assay. In terms of the in vivo assay, the bacterial loads of V. harveyi in the tested immune tissues (liver, spleen, and head kidney) were significantly reduced in T. ovatus, revealing that TroHepc2-22 significantly enhanced the resistance against V. harveyi infection. Furthermore, the expressions of immune-related genes, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1-ß (IL-1ß), IL-6, Toll-like receptor 1 (TLR1), and myeloid differentiation factor 88 (MyD88) were significantly increased, indicating that TroHepc2-22 might regulate inflammatory cytokines and activate immune-related signaling pathways. To summarize, TroHepc2-22 possesses appreciable antimicrobial activity and plays a vital role in resisting bacterial infection. The observation of our present study unveils the excellent application prospect of hepcidin as a substitute for antibiotics to resist pathogenic microorganisms in teleosts.

Macrophage migration inhibitory factor (MIF) of golden pompano (Trachinotus ovatus) is involved in the antibacterial immune response.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with a unique structure involved in immune regulation and inflammation. In the present study, we identified a MIF from Trachinotus ovatus (golden pompano) and analyzed its function. TroMIF shares high homology (58.26%-94.78%) with the other known MIF sequences of vertebrates. TroMIF is most closely related to large yellow croaker (Larimichthys crocea). The expression of TroMIF was most abundant in the liver and head kidney, and was significantly up-regulated after Edwardsiella tarda infection. The subcellular localization of TroMIF was mostly distributed in the cytoplasm. In vitro results revealed that the recombinant protein rTroMIF could inhibit the migration of head kidney lymphocytes (HKLs) and macrophages (HKMs) and enhance the phagocytic activity of HKMs. As a pro-inflammatory cytokine, rTroMIF could increase the expression levels of some pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1ß), IL-6, IL-8, and interferon-gamma (IFN-γ) and decrease the expression of IL-10. The rTroMIF was proved to have enzymatic redox activity in vitro. Furthermore, overexpression of TroMIF in the head kidney cell line of golden pompano could significantly enhance its ability to resist E. tarda infection from 1 h to 4 h. The knockdown of TroMIF expression induced a significant increase in the number of bacteria after E. tarda infection at 1, 2, and 4 hpi. Our results suggest that TroMIF is an essential effector of the innate immune system and plays a pivotal role in antibacterial immunity.

R848 Is Involved in the Antibacterial Immune Response of Golden Pompano (Trachinotus ovatus) Through TLR7/8-MyD88-NF-κB-Signaling Pathway.

R848 is an imidazoquinoline compound that is a specific activator of toll-like receptor (TLR) 7/8 and is often used in immunological research in mammals and teleosts. However, the immune responses initiated by R848 through the TLR7/8 pathway in response to bacterial infection remain largely unexplored in teleosts. In the current study, we investigated the antibacterial response and the participating signaling pathway initiated by R848 in golden pompano (Trachinotus ovatus). We found that R848 could stimulate the proliferation of head kidney lymphocytes (HKLs) in a dose-dependent manner, enhance the survival rate of HKLs, and inhibit the replication of bacteria in vivo. However, these effects induced by R848 were significantly reduced when chloroquine (CQ) was used to blocked endosomal acidification. Additionally, an in vivo study showed that R848 strengthened the antibacterial immunity of fish through a TLR7/8 and Myd88-dependent signaling pathway. A cellular experiment showed that Pepinh-MYD (a Myd88 inhibitor) significantly reduced the R848-mediated proliferation and survival of HKLs. Luciferase activity analysis showed that R848 enhanced the nuclear factor kappa B (NF-κB) activity, whereas this activity was reduced when CQ and Pepinh-MYD were present. Additionally, when an NF-κB inhibitor was present, the R848-mediated pro-proliferative and pro-survival effects on HKLs were significantly diminished. An in vivo study showed that knockdown of TLR7, TLR8, and Myd88 expression in golden pompano via siRNA following injection of R848 resulted in increased bacterial dissemination and colonization in fish tissues compared to that of fish injection of R848 alone, suggesting that R848-induced antibacterial immunity was significantly reduced. In conclusion, these results indicate that R848 plays an essential role in the antibacterial immunity of golden pompano via the TLR7/8-Myd88-NF-κB- signaling pathway.

Construction and characterization of a DNA vaccine encoding the SagH against Streptococcus iniae.

Streptococcus iniae is an important aquaculture pathogen that is associated with disease outbreaks in wild and cultured fish species. Streptolysin S has been identified as an important virulence factor of S. iniae. With an aim to develop effective vaccines against S. iniae for Japanese flounder (Paralichthys olivaceus), in this study, we constructed a DNA vaccine based on the sagH gene, which belongs to the streptolysin S-associated gene cluster. In fish vaccinated with pSagH, the transcription of sagH was detected in tissues and SagH protein was also detected in the muscles of pSagH-vaccinated fish by immunohistochemistry. The immunoprotective effect of SagH showed that fish vaccinated with pSagH at one and two months exhibited a high relative percent survival (RPS) of 92.62% and 90.58% against S. iniae serotype I, respectively. In addition, SagH conferred strong cross protection against S. iniae serotype II and resulted in an RPS of 83.01% and 80.65% at one and two months, respectively. Compared to the control group, fish vaccinated with pSagH were able to induce much stronger respiratory burst activity, and higher titer of specific antibodies. The results of quantitative real-time PCR demonstrated that pSagH upregulated the expression of several immune genes that are possibly involved in both innate and adaptive immune responses. These results indicate that pSagH is a candidate DNA vaccine candidate against S. iniae serotype I and II infection in Japanese flounder in aquaculture.

Latent infection of low-virulence anaerobic bacteria in degenerated lumbar intervertebral discs.

BACKGROUND: The existence of latent low-virulence anaerobic bacteria in degenerated intervertebral discs (IVDs) remains controversial. In this study, the prevalence of low-virulence anaerobic bacteria in degenerated IVDs was examined, and the correlation between bacterial infection and clinical symptoms was analysed. METHODS: Eighty patients with disc herniation who underwent discectomy were included in this study. Under a stringent protocol to ensure sterile conditions, 80 disc samples were intraoperatively retrieved and subjected to microbiological culture. Meanwhile, tissue samples from the surrounding muscle and ligaments were harvested and cultured as contamination markers. The severity of IVD degeneration and the prevalence of Modic changes (MCs) were assessed according to preoperative MRI analysis. RESULTS: Of the 80 cultured discs, 54 were sterile, and 26 showed the presence of bacteria: Propionibacterium acnes (21 cases) and coagulase-negative staphylococci (5 cases). MRI revealed that the presence of bacteria was significantly associated with MCs (P<0.001). However, there was no significant association between bacterial infection and the severity of IVD degeneration (P = 0.162). CONCLUSIONS: Our findings further validated the presence of low-virulence anaerobic bacteria in degenerated IVDs, and P. acnes was the most frequent bacterium. In addition, the latent infection of bacteria in IVDs was associated with Modic changes. Therefore, low-virulence anaerobic bacteria may play a crucial role in the pathophysiology of MCs and lumbar disc herniation.

Characterization of Spleen Transcriptome of Schizothorax prenanti during Aeromonas hydrophila Infection.

Schizothorax prenanti (S. prenanti) is an indigenous fish species and is popularly cultured in southwestern China. In recent years, intensive farming of S. prenanti and water quality deterioration has increased the susceptibility of this fish to various pathogens, including Aeromonas hydrophila (A. hydrophila), which has caused severe damage to S. prenanti production. However, the understanding of molecular immune response of S. prenanti to A. hydrophila infection is still lacking. In order to better comprehend the S. prenanti time series immune response process against A. hydrophila, we conducted the first transcriptomic comparison in S. prenanti spleen at 4, 24, and 48 h after the infection challenge of A. hydrophila against their control counterparts. In total, 628 million clean reads were obtained from 18 libraries and assembled into 262,745 transcripts. After eliminating sequence redundancy, 69,373 unigenes with an average length of 1476 bp were obtained. Comparative analysis revealed 1890 unigenes with significantly differential expression, including 172, 455, 589 upregulated and 27, 676, 551 unigenes downregulated genes for 4, 24, and 48 h post-infection, respectively. Differentially expressed genes (DEGs) were validated using qPCR for 15 randomly selected genes. Enrichment and pathway analysis of DEGs was carried out to understand the functions of the immune-related genes. Our results revealed that many important functional genes relating to complement and coagulation cascades, chemokine signaling pathway, toll-like receptor signaling pathway, NOD-like receptor signaling pathway and leukocyte transendothelial migration were regulated during the infection of A. hydrophila, and the expression of those genes reflected the transcriptome profiles during the challenging stages.

De novo assembly of Schizothorax waltoni transcriptome to identify immune-related genes and microsatellite markers.

Schizothorax waltoni (S. waltoni) is one kind of the subfamily Schizothoracinae and an indigenous economic tetraploid fish to Tibet in China. It is rated as a vulnerable species in the Red List of China's Vertebrates, owing to overexploitation and biological invasion. S. waltoni plays an important role in ecology and local fishery economy, but little information is known about genetic diversity, local adaptation, immune system and so on. Functional gene identification and molecular marker development are the first and essential step for the following biological function and genetics studies. For this purpose, the transcriptome from pooled tissues of three adult S. waltoni was sequenced and analyzed. Using paired-end reads from the Illumina Hiseq4000 platform, 83 103 transcripts with an N50 length of 2337 bp were assembled, which could be further clustered into 66 975 unigenes with an N50 length of 2087 bp. The majority of the unigenes (58 934, 87.99%) were successfully annotated by 7 public databases, and 15 KEGG pathways of immune-related genes were identified for the following functional research. Furthermore, 19 497 putative simple sequence repeats (SSRs) of 1-6 bp unit length were detected from 14 690 unigenes (21.93%) with an average distribution density of 1 : 3.28 kb. We identified 3590 unigenes (5.36%) containing more than one SSR, providing abundant potential polymorphic markers in functional genes. This is the first reported high-throughput transcriptome analysis of S. waltoni, and it would provide valuable genetic resources for the functional genes involved in multiple biological processes, including the immune system, genetic conservation, and molecular marker-assisted breeding of S. waltoni.

Identification of Immune-Related Genes and Development of SSR/SNP Markers from the Spleen Transcriptome of Schizothorax prenanti.

Schizothorax prenanti (S. prenanti) is mainly distributed in the upstream regions of the Yangtze River and its tributaries in China. This species is indigenous and commercially important. However, in recent years, wild populations and aquacultures have faced the serious challenges of germplasm variation loss and an increased susceptibility to a range of pathogens. Currently, the genetics and immune mechanisms of S. prenanti are unknown, partly due to a lack of genome and transcriptome information. Here, we sought to identify genes related to immune functions and to identify molecular markers to study the function of these genes and for trait mapping. To this end, the transcriptome from spleen tissues of S. prenanti was analyzed and sequenced. Using paired-end reads from the Illumina Hiseq2500 platform, 48,517 transcripts were isolated from the spleen transcriptome. These transcripts could be clustered into 37,785 unigenes with an N50 length of 2,539 bp. The majority of the unigenes (35,653, 94.4%) were successfully annotated using non-redundant nucleotide sequence analysis (nt), and the non-redundant protein (nr), Swiss-Prot, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. KEGG pathway assignment identified more than 500 immune-related genes. Furthermore, 7,545 putative simple sequence repeats (SSRs), 857,535 single nucleotide polymorphisms (SNPs), and 53,481 insertion/deletion (InDels) were detected from the transcriptome. This is the first reported high-throughput transcriptome analysis of S. prenanti, and it provides valuable genetic resources for the investigation of immune mechanisms, conservation of germplasm, and molecular marker-assisted breeding of S. prenanti.

(-)-Epigallocatechin-3-gallate inhibits osteosarcoma cell invasiveness by inhibiting the MEK/ERK signaling pathway in human osteosarcoma cells.

The notorious lung metastatic capability of osteosarcoma aggravates patient mortality and remains the primary challenge to be overcome. We investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on the metastasis capability of osteosarcoma cells. We performed cytotoxicity assays (MTT) to determine the appropriate concentration of EGCG for experiments. Migration, invasion, wound-healing, and adhesion assays were performed to assess the effect of EGCG on the metastasis of osteosarcoma. Changes in the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway were investigated using Western blot analyses. In addition, a MEK inhibitor (U0126) was used in invasion assays to determine the effect of the MEK/ERK signaling pathway. We found that EGCG may markedly inhibit the migration and invasion capacity of osteosarcoma cells, which occurred concurrently with inhibition of the expression of phospho-MEK and phospho-ERK. Inhibitors of MEK inhibited the invasion of osteosarcoma cells, and this effect could be enhanced by EGCG. We also detected the expression of c-Jun N-terminal kinase, p38, and their respective phospho-proteins, but did not find any meaningful changes. Taken together, our results demonstrated that EGCG could inhibit the metastasis capability of osteosarcoma cells by inhibiting MEK/ERK signaling activity and may provide new therapeutic value for osteosarcoma.