摘要:白色念珠菌具有正常的菌落,但它會轉變形成伺機性病原體,較易引起死亡,而易導致較高的發病率。C 型凝集素受體(CLR)為一種可辨識碳氫化合物的蛋白質,因這些蛋白質可與病原體細胞壁中不同醣蛋白或醣脂質相互親合鍵結,而可對病原體具有辨識、清除之功能外,尚對免疫細胞間之交互作用具有調節角色。白色念珠菌的細胞壁主要成份為甘露聚醣,β-聚醣與幾丁質三種glycans。目前在一些已被發現CLR 成員如CLEC1、2、2B、4A、4B、4C、4D、4K、4L、4M、4N、5A、7A、9A、10A、12A、及13A 中,只有dectin-1 (CLEC7A)與 DC-SIGN (CLEC4L)較被廣泛研究。先前研究發現dectin-1 可辨識β-1,3-聚醣,藉由Syk 與CARD9 路徑去誘導NF- κB 路徑之活化。甘露聚醣會被DC-SIGN 辨識,可藉由觸使RAF1 活化,而誘導NF- κB 的次單位(subunit) p65 磷酸化。最近由研究發現,白色念珠菌細胞壁中第三種多醣-幾丁質,會被甘露醣受器 (mannose receptor)與 Dectin-1 所辨識,但在辨識後所引發相關訊號傳遞仍不清楚。因此有關CLRs 如何調控先天免疫系統仍舊是一個未知但確十分重要的研究課題。在抵抗不同病原體所誘發的發炎疾病中, IL-1β 是一個主要的媒介物。IL-1β產生過程通常需要兩個訊號來調控,第一個訊號是與NF-κB 訊息傳遞有關,可以促進pro-IL-1β合成,第二個訊號是要引起包含pro-caspase-1 在內的inflammasome 複合體的活化,促使pro-IL-1β 經由蛋白水解,產生具有活性的IL-1β分泌到細胞外。目前,對於CLR 被活化後,如何誘導及調控pro-IL-1β合成與inflammasome 活化等方面沒有相關深入的報告。因為有些疾病會caspase-1 活性失常與IL-1β分泌失控等有關,因此了解inflammasome 路徑對疾病的發病原因可望找到新的治療切入點,對因IL-1β過量產生所引起發炎疾病的療法上能有新的突破。這個計劃主要是探討不同CLR 在白色念珠菌所誘導發炎的IL-1β產生上所扮演角色,包括在人及小鼠單核細胞,巨噬細胞及人體表皮角質細胞。CLR 與其相對配體ligand)專一性親合力與對inflammasomes 活化所需相關訊號傳遞之分子特性將被探討。此研究主要目標為(1)確定CLR 成員對甘露聚醣、β-聚醣及幾丁質之專一性親合能力。關於此部分實驗,我們利用從白色念珠菌細胞壁粗萃取出來的聚醣,與以異源表現系統所純化出具Fc 標記重組CLR融合蛋白等為材料,藉由已建立的enzyme-linked assay (EIA)方法,來做親合力方面的實驗。目前,我們利用17 種CLR 融合蛋白來做EIA,由先前研究結果發現,有些受體會分別對甘露聚醣、β-聚醣及幾丁質具有親合力。(2)確定CLR 在人及小鼠單核細胞,巨噬細胞誘導IL-1β基因轉錄的作用機制。(3)確定CLR 在人及小鼠單核細胞,巨噬細胞誘導inflammasome 活化的訊號傳遞路徑及分子機制。(4)探討白色念珠菌誘導inflammasome 活化參與角質細胞產生生理及病理調解的角色及作用機制。此研究成果相信能給CLR 一個新的認知,了解其對IL-1β基因轉錄及inflammasome 活化的調控作用機制。此外亦可明瞭inflammasome 活化在白色念珠菌感染的病理過程的所扮演的角色。我們所建立的CLR protein array 對於尋找病源物質的作用分子也提供簡便快速有價值的資訊,這對於學術研究及研發新的治療感染症藥物有極大的好處。
Abstract: Candida albicans is a normal flora, but can become an opportunistic pathogen and cause the high incidence of mortality. C-type lectin receptors (CLRs) are carbohydrate recognizing proteins that mediate pathogen recognition/clearance, and immune cell interaction. These proteins are responsible for binding to a variety of glycoproteins or glycolipids present in the cell wall of pathogens. Three main glycan components in the cell wall of Candida albicans are mannan, β-glucan, and chitin. Currently among CLR members which have been identified, such as CLEC1, 2,2B, 4A, 4B, 4C, 4D, 4K, 4L, 4M, 4N, 5A, 7A, 9A, 10A, 12A, and 13A, only Dectin-1 (CLEC7A) and DC-SIGN (CLEC4L) have been more extensively investigated thus far. Previous studies showed that Dectin-1 can recognize β-1,3-glucan and through Syk and CARD9-dependent pathways activate NF-κB pathway. Mannan is recognized by DC-SIGN, which can trigger the activation of RAF1 andinduce the phosphorylation of NF- κB subunit p65. Recently, it has been known that chitin, the third kind of polysaccharide of the fungal cell wall, is recognized by mannose receptor and Dectin-1. But the possible signaling cascade after reorganization is still unclear. Therefore, it is still a blank but crucial subject to clarify the whole picture of innate immunity mediated by CLRs. IL-1β is a central orchestrator of immunity against various classes of pathogens, and a key trigger of inflammatorydiseases. Two signals are required for IL-1β production: an NF-κB-dependent signal that induces the synthesis of pro-IL-1β, and a second signal that triggers proteolytic pro-IL-1β processing and IL-1β maturation via pro-caspase-1-containing multiprotein complexes called inflammasomes. Currently how CLRs activation leading to inflammasome-dependent pro-IL-1β processing has not been reported. Since several diseases are associated with dysregulated activation of caspase-1 and secretion of IL-1β, understanding inflammasome pathways may provide insight into disease pathogenesis that might identify potential targets for therapeutic intervention. The main theme of this project is to explore the function of CLRs in Candida-induced inflammatory IL-1β production. The ligand-CLR binding specificity and molecular nature of signal pathways for inflammasome activation will be characterized in macrophages, monocytes and keratinocytes. The specific aims of this project are (1) Using EIA protein array to determine the binding specificity of mannan, β-glucan and chitin to CLR members. To this end, we have established the enzyme-linked assay (EIA) by isolation and purification of glycans from C. albicans cell wall as well as construction, expression and purification of Fc-tagged recombinant CLR fusion proteins. Currently we have generated seventeen CLR fusion proteins for EIA, and our preliminary data has observed some novel binding receptors for mannan, β-glucan and chitin separately. (2)Understand mechanisms that relay CLRs-induced signaling to pro-IL-1β gene expression in monoctyes and macrophages. (3) Understand signal transduction pathways of CLRs leading toinflammasome activation in monocytes and macrophages. (4) Explore the molecular mechanisms and pathophysiological roles of Candida-activated inflammasome in keratinocytes. The information of this study is believed to provide mechanistic details for CLRs on the transcriptional regulation of IL-1β, and also reveal novel functions of CLRs in inflammasome activation. It is obvious that this study will greatly advance the current knowledge on CLRs and inflammasome in candidasis. Moreover, the binding data obtained by this array screening is helpfulto understand the action targets of pathogens and thus favor future studies in pathogenesis-related signaling as well as in developing therapeutic agents.