綠膿桿菌PAO1中雜合感應子的特性分析

Abstract

中文摘要 綠膿桿菌一種伺機性感染的細菌，常在免疫系統不全的個體，例如燒傷患者及肺纖維囊腫病患身上引發院內感染。目前綠膿桿菌的基因序列已全數定序完成（http://www.psuedomonas.com/），總數達6.3 Mbp，其中包含5,570個開放讀架，預估能製造多達6,200個蛋白質。經專家們依其序列相似性以及相關研究統合的結果，大致將這些基因分為26個不同功能的群組，其中的雙分子訊息傳遞系統群組又可細分為三類：感應子類、調控子類、以及感應/調控雜合子類。感應子是用來感應外界環境並將訊息傳至細胞內的調控子；調控子則在接收感應子所傳來的訊息後，依生存所需去調控基因表現；目前已有文獻指出感應/調控雜合子其實為一種感應子，其訊息傳遞需藉由游離的組胺酸磷酸轉移分子作為橋樑，才能順利將訊息傳到調控子。 因此，我們設計了三個實驗：其一，是挑選兩個感應/調控雜合子基因PA0928及PA1611構築其缺損株，觀察此兩者基因的缺損會為綠膿桿菌帶來什麼影響，結果發現PA1611基因缺損株無論以生長曲線或游動分析，皆與野生株無異，但缺損株的pyocyanin生成量則比野生株為高，因此其確實的功能還須進一步實驗探知，而PA0928缺損株則在挑選過程中不斷出現高濃度卡那黴素耐受菌株，因此目前尚未順利取得。其二，我們表現綠膿桿菌中被提出可能的游離組胺酸磷酸轉移分子蛋白質HptA, B, 及C，以及PA0928、PA1611部分蛋白質，藉由體外磷酸化實驗發現重組蛋白0928H, 0928HD, 1611H, 1611HD皆具有自我磷酸化能力，HptA, B, 及C則沒有，且1611HD只會將其磷酸根轉移至HptB。其三，我們挑選不同的生長環境，以北方墨漬法偵測PA0928及PA1611所轉錄轉譯出的雜合感應子感應的訊息為何，進而推測此二者所扮演的角色及調控的下游基因，結果發現鐵離子的濃度以及H2O2所造成的氧化壓力皆能使PA0928的基因表現量增加，此外，我們所挑選的生長條件皆不會影響PA1611的表現量。

Abstract Pseudomonas aeruginosa is a typical opportunistic pathogen which colonizes the lungs of cystic fibrosis patients and causes severe infections in immunocompromised host. To modulate adaptive responses to the environment, this bacterium utilizes two-component systems, in which the signals from environmental stimuli are transducted by means of phosphorylation from sensor kinases to corresponding regulators for appropriate gene expression. 10 hybrid sensors that contain the kinase domain of sensor and the receiver domain of the response regulator were found in the genome of P. aeruginosa PAO1. Comparing with the unorthodox sensors, these hybrid sensors do not have a histidine-containing phosphotransfer (Hpt) domain. In consideration of the three steps phosphorylation model of the unorthodox systems, it has been proposed that the hybrid sensors possibly also carry out three-step signal transduction by transmitting their signal to the down stream response regulators by the three Hpt-like ORFs (HptA, B, and C) which were found elsewhere in the genome. To demonstrate the functional roles of the hybrid sensors, PA0928 and PA1611 were selected for further investigation. The mutants were firstly constructed and their properties including changes of growth curve and swarming assay were characterized. However, there was no difference between wild-type PAO1 and the PA1611 mutants. PA1611 mutants appear to produce more pyocyanin, which indicate that PA1611 may involve in the control of pyocyanin production. PA0928 mutant was failed to isolate because of the tolerance of P. aeruginosa. The phosphorelay specificity between the hybrid sensors and the Hpt molecules was also determined. The C-terminal DNA fragments of PA0928 and PA1611 containing both histidine kinase domain (H) and receiver domain (D), or only the H domain were respectively cloned. The recombinant proteins including 0928HD, 1611HD, 0928H, 1611H, HptA, HptB and HptC, were respectively overexpressed in E. coli expression system, and then purified by affinity chromatography. These purified proteins were then used in the in-vitro phosphorelay assay with [γ-32P]ATP as a phosphodonor. The two recombinant proteins PA0928 and PA1611 appeared to have autokinase activities, and the phosphoryl group from the phosphorylated 1611HD was able to be transferred specifically to HptB, but not HptA nor HptC. In addition, northern blotting was used to detect the expressions of PA0928 and PA1611 under various growth conditions, including LB and M9 medium, M9 medium with 1.5 mM NaCl, 200 mM FeCl3, or 19.5 mM H2O2, M9 medium with 25 mM glycerol, acetate, or succinate as carbon source instead of glucose. The results indicate that the concentration of Fe3+ and the oxidative stress stimulate the expression of PA0928.