埃及斑蚊Spätzle-6 在免疫反應和發育之雙重角色

Dual roles of Spätzle-6 in the immune responses and development in the mosquito, Aedes aegypti

黃俊豪(Chun-Hao Huang)

指導教授 ： 蕭信宏

國立臺灣大學/醫學院/微生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00444

摘要

蚊子是許多傳染性疾病的重要媒介，例如瘧疾、登革熱、絲蟲病以及日本腦炎…等疾病。針對這些病媒蚊傳播的疾病，各國的研究團隊除了致力於發展新藥及開發疫苗之外，以遺傳工程技術控制病媒傳病能力的新策略也漸漸受到重視。因此，研究蚊子體內對抗外來病原的相關基因及調控機制能提供此一策略新的目標。本實驗室之前的研究發現埃及斑蚊Inhibitor of Apoptosis-2(IAP2)參與病媒蚊抗菌蛋白合成之調控，而利用岡比亞瘧蚊(Anopheles gambiae)的cDNA microarray分析結果發現，當岡比亞瘧蚊被混和的細菌(包含Staphylococcus aureus 和Escherichia coli)或Plasmodium berghei 誘發免疫反應時，有多個免疫反應基因會隨著IAP2 表現量增加而大量表現，其中我們發現Spätzle-6(Spz6)的功能有待深入探討。在果蠅(Drosophila melanogaster)的研究顯示，果蠅的Toll pathway 可以藉由Spz 活化Toll receptor，進而促進Toll pathway 的訊號傳遞，引發抗菌蛋白的產生。同時，果蠅的Spz6 也被證實參與早期胚胎發育。因此，我們想更進一步研究Spz6 在埃及斑蚊的免疫反應及發育所扮演的角色。首我們藉由RNAinterference(RNAi)的方式來抑制埃及斑蚊Spz6 的表現，再分別去感染Beauveriabassiana, Staphylococcus aureus 和Escherichia coli，最後計算埃及斑蚊之存活率。實驗結果顯示埃及斑蚊的Spz6 被抑制之後，感染E. coli 後的存活率有明顯的上升，因此我們推測Spz6 參與埃及斑蚊體內的IMD pathway 的調控且可能是扮演負調控子的角色。此外，我們也分析埃及斑蚊Spz6 被抑制的時候，分別感染S.aureus 和E. coli 後，再去偵測IMD pathway 下游基因Cecropin A 和Defencin A的表現，實驗結果顯示，在Spz6 被抑制時，再感染E. coli 的情況下，所偵測到的Cecropin A 和Defencin A 表現量和控制組比較起來都是過量表現。接著我們再進一步去分析Spz6 在埃及斑蚊體內不同組織表現的情形，在埃及斑蚊分別感染S. aureus 和E. coli 後，以Real-Time PCR 去偵測Spz6 在不同組織的表現量，實驗結果顯示當細菌感染蚊子之後，Spz6 在蚊子中腸的表現量有顯著的升高。此外，我們也利用RT-PCR測詴Spz6 在發育時期的表現，發現Spz6 會在black pupa時期有大量表現，於是我們也利用RNAi 的方式，在white pupae 時期去抑制Spz6的表現，結果我們發現蚊子的羽化率和羽化時間分別有明顯的降低及延遲，因此我們推測Spz6 在蚊子的發育可能也扮演重要的角色。根據以上的實驗結果，我們發現Spz6 在埃及斑蚊體內扮演者兩樣功能的角色，一個是免疫調控，另一個則是調控其發育。未來，我們希望釐清Spz6 在IMD signal pathway 與其他因子的相對關係以及Spz6 在轉譯表現的模式，並去瞭解Spz6 在變態(Metamorphosis)方面所扮演的角色。

關鍵字

埃及斑蚊 ； Sp&auml ； tzle-6 ； 卵黃生成 ； 變態

並列摘要

Mosquitoes are major vectors of several devastating human diseases, such as malaria, Dengue fever, West Nile encephalitis, Japanese encephalitis and filariasis. In addition to developing new drugs and vaccines and controlling these diseases researcher have also been devoted to combat the vector-borne diseases by developing new genetic engineering approaches. Therefore, studies of the regulation machinery of mosquito innate immunity against pathogens and development provide new insights on vector-born disease control. In this study, we use the major vector of Dengue fever, the mosquito Aedes aegypti, as our experimental model. Microarray analysis in Anopheles gambiae showed that several immune responsive genes, including Spätzle-6(Spz6), were up-regulated upon immune stimulation including Inhibitor of Apoptosis-2(IAP2). IAP2 was shown to be an important component participated in the regulation of antimicrobial peptides in the mosquito. In addition, studies in Drosophila melanogaster showed that Spz gene family plays important roles in egulating Toll signaling pathway and the production of antimicrobial peptides. These studies trigger our interest to study the role of Spz6 in the mosquito Aedes aegypti. To understand the role of Aedes aegypti Spz6 in response to invading pathogens, we used RNA interference(RNAi) approach to inhibit the expression of Aedes aegypti Spz6. Survival assay was performed following the challenge of Beauveria bassiana, Staphylococcus aureus or Escherichia coli. The results showed that Aedes aegypti were significantly resistant to E. coli challenge in the absence of Spz6, suggesting that Spz6 acted as an inhibitory regulator in the IMD pathway. Next, we are intrigued to know the effects of Spz6 to the downstream targets of IMD pathway. Therefore, we examined the transcriptional expression profiles of Cecropin A and Defencin A in the absence of Spz6. Interestingly, both Cecropin A and Defencin A showed relatively higher expressional pattern upon S. aureus and E. coli challenge in the absence of Spz6. We further analyzed the Spz6 expressional pattern in different tissues. To our surprise, Spz6 was highly expressed in the mosquito midgut, whereas other Spz members were expressed in the mosquito ovary. On the other hand, we found that Spz6 is highly expressed in the black pupae stage. Therefore, we performed RNAi approach to inhibit the expression of the Spz6 in white pupae stage. We observed the significant reduction of the emerging rate of the adult mosquito in the absence of Spz6. Interestingly, we found that Spz6 process alternative splicing during metamorphosis. The oviposition rate was also reduced in the absence of Spz6. Taken together, our results showed that Spz6 is a negative regulator in the IMD pathway and participate in the vitellogenesis and development. Detail analysis of Spz6 will benefit the development of new strategies for the control of the vector-borne disease.

並列關鍵字

Aedes aegypti ； Sp&auml ； tzle-6 ； Vitellogenesis ； Metamorphosis

參考文獻

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