Transcriptome analysis of the effect of Vibrio alginolyticus infection on the innate immunity-related TLR5-mediated induction of cytokines in Epinephelus lanceolatus

Abstract

Epinephelus lanceolatus, considered to be an aquaculture fish species of high economic value in East Asia, is one of the largest groupers in the Epinephelus genus. Vibrio alginolyticus is a bacterial species that causes high morbidity in marine fish; infection can cause exophthalmia, ulcers, septicemia, and corneal opaqueness in fish. Epinephelus lanceolatus larvae infected with Vibrio alginolyticus were subjected to transcriptome analysis to study the immune regulation pathway. Grouper larvae were injected with 2.6 × 10(4) CFU/fish in 20 μl of V. alginolyticus and control larvae were injected with TSB; RNA samples were then collected at 4, 6, 8, 10, 12, 16, 24, and 48 h after infection. Extracted RNA was subjected to reverse transcription, and used to examine the immune gene response of E. lanceolatus by Real-time PCR. Samples taken at 6 h were subjected to next-generation sequencing, resulting in a total read value of 28,705,411 and total base number of 2,152,905,850. The unigene number was 100,848, and 5913 unigenes were filtered using FPKM>0.3, 2FC, p < 0.05. Gene Ontology (GO) analysis of the filtered genes revealed a total of 30 GO numbers in the cellular component, and 58 GO numbers for both biological processes and molecular functions. Of the GO group related to immune pathways, 27 unigenes related to biological processes involving the immune response, 31 related to the immune system, 9 related to the inflammatory response, and 43 related to the response to stress were identified. KEGG pathway analysis only detected 1 to 4 genes, and as such, we selected the GO analysis results for further analysis using GeneSpring. This demonstrated that V. alginolyticus probably stimulates TLR5 activity via the bacterial flagellum, through an MyD88-dependent pathway; the resulting production of IL-1β and IL-8 through the NFκB pathway induces pro-inflammatory and/or chemotactic effects. Alternatively, serum amyloid A may stimulate neutrophils that induce the secretion of MMP9 from infected tissues, resulting in the cleavage and activation of IL-8. IL-8, in turn, would enhance neutrophil chemotaxis. Infection also induced expression of genes encoding C3, C6, C7, C8, and C9, which induce the complement system and form the membrane attack complex to lyse the bacteria membrane. The qPCR results indicated that TLR5 is significantly increased between 10 and 16 h, IL-1β between 8 and 16 h, IL-8 between 8 and 12 h, and C6 between 4 and 16 h, as compared to levels in the control. One antimicrobial peptide, hepcidin, was also strongly expressed between 4 and 10 h in infected fish. The results indicate that V. alginolyticus infection probably induces an immune response via TLR5-mediated regulation of down-stream cytokine gene expression. A second possibility is that the complement system and hepcidin may be involved in the immune response. These results may be applied by examining the immune effects of feeding E. lanceolatus larvae on a recombinant protein mixture based on the up-regulated genes.

Keywords: Cytokine; Epinephelus lanceolatus; Infection; Transcriptome; Vibrio alginolyticus.