利用Transcriptome分析技術了解點帶石斑以及龍膽石斑魚苗感染溶藻弧菌其先天免疫的調控機制

Abstract

點帶石斑 (Epinephelus coioides) 在台灣是一種極富經濟潛力的養殖魚種。然而在幼苗時期的石斑魚對細菌病原體如溶藻弧菌很容易受到感染。為了更加了解溶藻弧菌感染點帶石斑幼苗的免疫機制，我們使用了高通量次世代定序技術來研究感染時的基因表現，總讀值為114,851,002，共有9,687,355,560核苷酸，總計209,082個重疊群，平均長度為372bp。Gene ontology (GO) 分析在 transcriptome 中顯示出12組細胞組成 (cellular component)群集，16組分子功能 (molecular function)的群集，以及42組生物過程 (biological process)的群集 (P value < 0.05)。共有32664 點帶石斑的基因在京都基因與基因组百科全書 (KEGG)被比對出來。1504不同表現的基因 (DEGs)在12個群集中被辨識出來 (P value < 0.05)。弧菌感染造成的基因表現包含補體系統、凝血因子、金黃色葡萄球菌感染、吞噬體活性、抗原呈獻系統以及抗原表現途徑。我們總結出在先天性免疫反應中的補體路徑以及抗菌胜肽 hepicidin可能在點帶石斑對抗溶藻弧菌感染之時扮演了重要的角色，其免疫反應的啟動時間約在感染後四個小時左右，這些結果顯示石斑魚在早期發育階段，溶藻弧菌感染時可能誘發補體反應，增進了我們對弧菌感染點帶石斑其免疫機制的瞭解。 為了更進一步了解其他種類的石斑魚幼苗感染溶藻弧菌的情況，我們使用了具有高經濟價值的龍膽石斑 (Epinephelus lanceolatus) 幼苗進行高通量次世代定序技術，結果顯示，檢測出的總讀值為28,705,411，共有2,152,905,850 個base，組出的unigene數目共有100,848個，經過篩選後剩餘5,913 unigene (篩選方式為 FPKM>0.3, 2FC, p<0.05)。GO分析中，細胞組成 (cellular component)共有30個GO 數量，生物過程 (biological process)以及分子功能 (molecular function)分別各有58個GO數量。從中挑選出與免疫相關的群組，生物過程中參與免疫反應(immune response)群組的 unigene 總共有27個，免疫系統過程 (immune system process)有31個， 發炎反應 (inflammatory response)有9個，壓力反應 (response to stress)有43個 unigene。細胞組成中參與膜攻擊複合物 (membrane attack complex; MAC)有8個unigene。分子功能中參與趨化因子活性 (chemokine activity)以及趨化因子受體結合(chemokine receptor binding)分別各有9個，細胞因子 (cytokine activity)有13個，細胞因子受體結合 (cytokine receptor binding) 有12個unigene。而在KEGG的分析上，共有47個pathway。由於從 KEGG的分析資料中所解出的pathway 所參與的基因數量偏低，只有1到4個unigene參與其中，無法從KEGG pathway中挑選出可能與免疫相關的途徑。因此最終以GO 所分析出的免疫基因利用GeneSpring連結出可能的pathway。從Real-time PCR的比對中得知TLR5、IL-1β、IL-8、SAA以及hepcidin 都具有大量表現，時間平均分布在4到16小時之間，整理後比對參考文獻以及GeneSpring 規畫出可能的路徑圖，其結果顯示感染途徑可能由溶藻弧菌的鞭毛刺激TLR5的活化，由MyD88 經一連串的訊息傳遞，最後刺激NFκB 產生如IL-1β、IL-8 誘導前發炎反應 (proinflammatory)或是趨化性 (chemotactic)。另一方面也可能經由serum amyloid A 刺激嗜中性球 (neutrophils) 到感染部位分泌MMP9 對IL-8進行剪切與活化，活化後的IL-8則會增強嗜中性球的化學毒性。而C3、C6、C7、C8以及C9的基因表現也顯示出補體系統同時也會受到刺激，組成MAC分解細菌細胞膜，抗菌胜肽 hepcidin的大量表現也可能殺死溶藻弧菌。結果表明其免疫調控路徑可能是經由TLR5調控下游cytokine的表現。總結以上言論，點帶石斑幼苗感染溶藻弧菌會啟動補體系統以及抗菌胜肽hepcidin的表現，而龍膽石斑幼苗感染相同細菌除上述兩種途徑外，更發現溶藻弧菌可能其鞭毛會誘導TLR5的強烈反應，調控後續細胞因子以及發炎反應的進行。

Orange-spotted grouper (Epinephelus coioides) with protogynous hermaphroditic features are one of the most economically important aquaculture species in Taiwan. However, larvae stage grouper are susceptible to infection by the bacterial pathogen Vibrio alginolyticus. To better understand the molecular mechanisms of the immune response to V. alginolyticus in Epinephelus coioides larvae, we used high-throughput deep sequencing technology to study the effect of infection on gene expression. A total of 114,851,002 reads were assembled, consisting of 9,687,355,560 nucleotides; these were further assembled into 209,082 contigs with a mean length of 372 bp. Gene ontology (GO) analysis of the transcriptome revealed 12 cellular component subcategories, 16 molecular function subcategories, and 42 biological process subcategories (P value < 0.05). A total of 32,664 Epinephelus coioides genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG); 1,504 differentially expressed genes (DEGs) were subsequently identified, in 12 categories (P value < 0.05). Vibrio infection affected the expression of genes involved in complementation, coagulation cascades, pathogen (Staphylococcus aureus) infection, phagosome activity, antigen processing, and the antigen presentation pathway. We conclude that the complement pathway of innate immunity and the hepicidin antimicrobial peptide may play important roles in the defense of Epinephelus coioides larvae against V. alginolyticus, and the immune response may activate at 4h after bacterial infection. These results implicate the complement pathway signal pathway in immunity during V. alginolyticus infection at early developmental stages, enhancing our understanding of the mechanisms underlying the immune response to Vibrio infection in Epinephelus coioides. To realize other kind of grouper, we choose Epinephelus lanceolatus which were higher economic value then Epinephelus coioides, we have established and conducted first-round annotation of transcriptome profiles in V. alginolyticus -infected and non-infected Epinephelus lanceolatus larva. The result shown that total read value became to 28,705,411, 2,152,905,850 total base, 100,848 unigenes number, 5,913 unigenes number filter for FPKM>0.3, 2FC, p<0.05. On the GO analysis, total 30 GO numbers in cellular component, both biological process and molecular function are 58 GO numbers. We choose the cluster of immune pathway, the cluster of biological process contains 27 unigenes in immune response, 31 unigenes in immune system process, 9 unigenes in inflammatory response, and 43 unigenes in response to stress. Cellular component contain 8 unigenes in membrane attack complex. Both chemokine activity and chemokine receptors binding contain 9 unigenes in molecular function, cytokine activity obtained 13 and cytokine receptor binding contain 12 unigenes. On the KEGG analysis, contain 47 total pathways. Because of KEGG pathway involved lower then GO analysis, only include 1-4 genes. Finally we choose GO analysis became a main sign decided the pathway by GeneSpring software. The result of Real-time qPCR shown that TLR5, IL-1β, IL-8, SAA and hepcidin consisted strong expression between 4 to 16h, and indicate that V. alginolyticus probably stimulate TLR5 activity by bacterial flagellum, through by MyD88 dependent pathway, finally produce JunB, IL-1βand IL-8 from NFκB pathway induce pro-inflammatory effects or chemotactic effects. Otherwise possibility via by serum amyloid A stimulate neutrophils to infect tissue secret MMP9 then cleave IL-8 and to activate. IL-8 would enhance neutrophil chemotaxis. The express gene from C3, C6, C7, C8 and C9 indicate they would induction complement system and become a membrane attack complex to lysis bacteria membrane. Antimicrobial peptide contain highly expression possibility destroy the V. alginolyticus. The result indicate that immune pathway of V. alginolyticus infected Epinephelus lanceolatus probably via TLR5 regulate down- stream cytokine gene expression. Summarize two species of grouper, Epinephelus coioides infected by V. alginolyticus stimulated complement system and hepcidin expression, in Epinephelus lanceolatus both above-mentioned immune pathway also activation. Moreover, V. alginolyticus probably stimulated TLR5 by flagellum to mediate cytokine and inflammatory response.