Orientia tsutsugamushi是引起恙蟲病的病原體，是一種絕對細胞內寄生菌，先前的研究已將完整的Orientia tsutsugamushi 基因序列確定，然而，了解Orientia
tsutsugamushi 如何侵入哺乳類細胞的早期訊息機轉卻還是個挑戰，在本研究中，我們證明並比較Orientia tsutsugamushi 三個主要膜外表面蛋白TSA56, TSA47 和
TSA22貼附細胞的能力。透過表達和純化O. tsutsugamushi的特異性抗原56-kDa (包括特異性抗原56-kDa 的抗原片段ADI 和抗原片段ADIII),特異性抗原47-kDa 和特異性抗原22-kDa三個特異性表面抗原蛋白，以及藉由二十二位臨床上確定感染恙蟲病之病人血清進行免疫墨點法實驗和利用大腸桿菌系統大量表達表面蛋白後進
行細胞貼附試驗，進而評估病人血清中的抗體反應和反應強度，以及這些表面蛋白影響細菌貼附細胞的能力。實驗結果顯示，病人血清對TSA56 表面蛋白的抗體反應及反應強度，皆明顯高於TSA47 和TSA22，在細胞貼附試驗中，表達TSA56表面蛋白的大腸桿菌對於細胞的貼附能力，亦是顯著大於表達TSA47 和TSA22 表面蛋白的大腸桿菌，進一步，透過免疫點墨法，我們更發現TSA56 的抗原片段ADI在恙蟲病人血清中有比TSA56 的抗原片段ADIII 更顯著的抗體反應，而透過細胞貼附試驗,也發現表達TSA56 抗原片段ADI 的大腸桿菌有著比表達TSA56 抗原片段ADIII的大腸桿菌更高的細胞貼附能力，而之前研究指出TSA56抗原片段ADIII會透過與fibronectin 之間的反應促進O. tsutsugamushi 侵入宿主細胞，綜合以上
實驗結果，可以得知O. tsutsugamushi 能夠利用主要表面蛋白TSA56 來增加對宿主細胞的貼附能力，並引起較強的免疫反應，而TSA56 抗原片段ADI 可能是除了
TSA56抗原片段ADIII外另一個促進O. tsutsugamushi貼附宿主細胞進而侵入的位置。

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. Recent studies show that the complete genome sequence of
Orientia tsutsugamushi have been determined. However, the early signaling events involved in the entry of O.tsutsugamushi into mammalian cells remains a challenge. In this study, we demonstrate that adherence ability and comparison of three major outer membrane protein TSA56, TSA47 and TSA22 of O.tsutsugamushi. Through expression and purification of three type-specific antigen 56-kDa (include TSA56-antigen domain I, TSA56-antigen domain III), 47-kDa and 22-kDa of O. tsutsugamushi , antiserum immunoblots from 22 clinical O. tsutsugamushi-infected patients and in vitro adhesion
assay of E.coli overexpression outer membrane protein of O. tsutsugamushi , the antiserum titer and adherence ability of bacterial outer membrane proteins are determined. The data show that antiserum titer against three major outer membrane
proteins of O. tsutsugamushi was markedly higher in TSA56 compared to TSA47 and TSA22. In adhesion assay, adhesion of host cells by TSA56 was readily than TSA47 and TSA22. Furthermore, adhesion experiment and antiserum titer against antigen-domain I (ADI) region (19-114 aa) in the extracellular domain of TSA56 was also significantly higher than previously reported antigen-domain III(ADIII) region (237-366 aa) which facilitates the invasion of O. tsutsugamushi through interaction with fibronectin .Taken together, these results clearly indicate that O. tsutsugamushi exploits TSA56-mediated bacterial adhesion, abundant antiserum titer and ADI region of TSA56 may draw another adhesion site (except for previously reported ADIII) to invade eukaryotic host cells.

Abstract in Chinese…………………………….. i
Abstract in English…………………………....... iii
Introduction……………………………………....1
Specific Aims…………………………………….7
Materials and Methods………………………… 8
Results……………………………………………13
Conclusion and Discussion……………………19
Future works……………………………………... 23
References……………………………………......24
Figures……………………………………………..33
Appendix…………………………………………...38

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