溫度逆境下表孔珊瑚內生桿菌中第三型分泌系統效應蛋白異檸檬酸裂解酶之表現

Abstract

Endozoicomonas montiporae CL-33T是一株從癭葉表孔珊瑚上所分離出的珊瑚共生菌。根據前人研究發現，E. montiporae的基因體當中編碼有格蘭氏陰性菌特有的第三型分泌系統以及第三型分泌系統效應蛋白。從基因體分析中發現E. montiporae當中一個第三型分泌系統效應蛋白屬於乙醛酸循環中重要酵素之一的異檸檬酸裂解酶。根據其他的文獻指出，當珊瑚處於高溫逆境之下，其體內乙醛酸循環相關的基因表現量也會增加，例如編碼異檸檬酸裂解酶之基因等，這樣的現象推測與調節珊瑚對環境的適應性有關。在本篇研究最初，我們利用專一性抗體透過西方墨點法證明E. montiporae確實具有產生異檸檬酸裂解酶的能力。另外，為了測試專一性抗體是否真能有效地偵測異檸檬酸裂解酶，我們利用液相層析串聯式質譜儀確認了抗體的專一性。在E. montiporae溫度逆境實驗中，首先發現E. montiporae在其適合生長的溫度下，具有分泌異檸檬酸裂解酶的能力；另外，當E. montiporae處於高溫之下能夠大量的分泌異檸檬酸裂解酶到細胞外。在探討珊瑚宿主在溫度逆境下對調節E. montiporae所產生之異檸檬酸裂解酶的研究中，為了找出最佳的實驗條件，我們首先測試了不同的培養時間以及不同宿主體積。根據西方墨點法之結果可見，細菌與珊瑚宿主的共培養時間以及珊瑚宿主的培養體積對於E. montiporae體內異檸檬酸裂解酶的產量影響不甚明顯。但在E. montiporae與逆境珊瑚共同培養的實驗中，隨著溫度增加，比起珊瑚組織以及控制組的海水，珊瑚黏液能夠明顯的改變E. montiporae體內異檸檬酸裂解酶的產量；而在高溫的狀況下，珊瑚組織也改變了異檸檬酸裂解酶的分泌量。根據本篇研究的結果可以得知，珊瑚宿主對於E. montiporae中異檸檬酸裂解酶的產量以及分泌是具有影響力的。總而言之，本篇論文為少數透過分子生物學的方式提供生化證據來探討珊瑚與細菌相互作用的研究，並且提供一個新的觀點來深入瞭解珊瑚與細菌的交互關係。

Endozoicomonas montiporae CL-33T is a potential coral symbiotic bacterium, isolated from the reef-building coral Montipora aequituberculata. Previous studies deduced that E. montiporae encodes type III secretion system (T3SS) effectors which could translocate into the coral cell to modulate metabolism or increase its fitness. This study is focused on one of the predicted T3SS effector proteins, isocitrate lyase (ICL), which is a key enzyme in glyoxylation pathway. Glyoxylation pathway genes were known to be highly expressed when the coral is under heat stress and functionally related to increasing coral’s fitness. In order to investigate whether ICL protein can be secreted out of the bacterium, we cultivated E. montiporae at different temperatures, mimicking heat stress conditions in the natural environment. We generated a specific antibody in order to detect the ICL protein. The antibody specificity was experimentally confirmed using polyacrylamide gel electrophoresis (PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We established and optimized western blotting technique for detection of the ICL using the specific antibody. According to western blotting results, the ICL protein was detected as a secreted protein in the supernatant, which proves the ICL can be secreted out of the bacterium. In the different temperature treatments, E. montiporae secretes more ICL proteins out of the bacterium under high temperature. Furthermore, we investigated changes in the ICL of E. montiporae incubating with coral cell lysates (i.e., mucus or tissue) under different temperatures. The result revealed that under heat stress, ICL expression change can be induced by coral mucus. Furthermore, the secretion of ICL also can affect by coral tissue lysate under higher temperature. This study provides more insight to understand how the expression of secretory ICL will modulate with heat stress and can be a potential metabolite to govern host response. Taken together, this thesis work is one of few studies to offer potential molecular and biochemical evidence for coral-bacteria interactions.