鮑氏不動桿菌抗原 NcsP 與 TonB-R 之特性分析

莊斯凱

doi:10.6845/NCHU.2015.01417

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鮑氏不動桿菌抗原 NcsP 與 TonB-R 之特性分析

Characterization of the Acinetobacter baumannii antigens NcsP and TonB-R

莊斯凱 , Masters　　Advisor：楊秋英　　

繁體中文 DOI： 10.6845/NCHU.2015.01417

鮑氏不動桿菌；鐵離子；疫苗； Acinetobacter baumannii ； iron ； vaccine

Abstract │ Reference (48) │ Times Cited (4) │ Altmetrics

Abstract 〈TOP〉

鮑氏不動桿菌 ( Acinetobacter baumannii，AB ) 是革蘭氏陰性菌，為伺機性呼吸道病原菌，常造成院內感染。近年來抗生素的濫用，使得多重抗藥性 AB (multidrug-resistant AB，MRAB) 不斷地被發現。在 MRAB 的防疫中，除了尋找更強效的抗生素，研發有效的疫苗為另一可行之策略。先前本實驗室鑑定到兩個可被人類血清辨認的蛋白質 TonB-R (putative TonB-dependent siderophore receptor) 及 NcsP (non-classical secretory protein)。本研究主要探討 TonB-R 及 NcsP 之生理特性及評估作為 AB 菌疫苗之可行性。首先將重組蛋白質 TonB-R (rTonB-R) 及 NcsP (rNcsP) 純化並免疫小鼠製備具抗原專一性之抗血清，西方墨點法分析各分離菌株，其中 TonB-R 及 NcsP 於 AB 菌分別具有 89 % (65/73) 及 100 % (73/73) 之高度保守性。菌體分層分析結果顯示 TonB-R 及 NcsP皆存在於外膜。經 ELISA 分析經熱失活處理之 AB 菌菌體表面，以及利用免疫螢光染色經流式細胞儀及螢光顯微鏡分析活菌表面，結果皆顯示兩目標抗原外露於菌體表面。接著，本研究分析 AB 菌於缺鐵生長環境下的生長狀況及目標抗原表現情形，結果顯示提供鐵離子螯合劑 2, 2’- bipyridyl (BIP) 之 LB 於晚期 log phase 發現 AB 菌 TonB-R 表現量高於 LB 及添加 FeCl3 之 LB (LB/FeCl3)。相反的，相較於 LB 及 LB/FeCl3，NcsP 於 LB/BIP 具較高表現量於早期 log phase。這些結果指出 TonB-R 及 NcsP 的表現可能受鐵離子調控。為了評估目標抗原作為 AB 菌疫苗之可行性，將小鼠個別免疫含費氏佐劑 (Freund，s adjuvant，FA ) 的 rTonB-R、rNcsP 重組蛋白質及控制組 (僅施打 FA ) 後，以臨床分離之 AB 進行呼吸道感染。感染四小時後，取其肺葉計算菌數，中位數 (median) 計算結果 TonB-R、NcsP、控制組依序為 4176、3605、8513 CFU/g，這些具顯著差異 (TonB-R，P < 0.05；NcsP，P < 0.01) 之結果證明 TonB-R 及 NcsP 所引起的免疫反應可提供有效的保護力。進一步於in vitro 數據證實  anti-TonB-R 及 anti-NcsP 抗血清對 AB 菌具殺菌/抑菌效力。最後，利用 MTT assay 分析 rTonB-R 及 rNcsP 對人類細胞是否具細胞毒性，結果顯示對 A549 不具毒性。綜合上述結果，面對 AB 菌的感染，TonB-R 及 NcsP 適合作為疫苗研發之目標。

Parallel Abstract 〈TOP〉

Acinetobacter baumannii (AB), which is a Gram-negative opportunistic respiratory pathogen, often causes nosocomial infections. In recent years, many multidrug-resistant AB (MRAB) strains have been found because of antibiotic overuse. Besides searching of new antibiotics for control of the MRAB infections, one alternative strategy is to develop effective vaccines. TonB-R and NcsP are two human serum-reactive antigens previously identified in our laboratory. This study aims to investigate the characteristic of these two proteins and to evaluate their feasibility to be used as anti-AB vaccines. At first, mice were immunized with purified recombinant TonB-R (rTonB-R) and rNcsP, respectively, to prepare antigen-specific antisera. Western blot analysis revealed that TonB-R and NcsP were detected in 89 % (65/73) and 100 % (73/73) of the tested AB isolates suggesting that these two antigens are highly conserved in AB. The results of bacterial fractionation showed that both proteins were presented in the outer membrane. ELISA performed with the heat-killed AB and immunostaining the viable AB followed by flow cytometry and immunomicroscopy demonstrated that both antigens are surface exposed. Next, the levels of TonB-R and NcsP at different growth phases and under iron-limiting conditions were monitored. The results showed that in the presence of 2,2’- bipyridyl (BIP), an iron chelator, the levels of TonB-R were higher than those bacteria grown in LB and LB supplemented with FeCl3 (LB/FeCl3); particularly in the late log phase. In contrast, higher NcsP levels were observed only at the early log phase in the bacteria grown in LB/BIP compared to those in LB and LB/FeCl3. Nevertheless, the results indicate that the expression of TonB-R and NcsP may be iron-regulated. To evaluate whether these two proteins can be used as AB vaccines, mice immunized with rTonB-R and rNcsP in the presence of Freund，s adjuvant (FA) and the control group (immunized with FA only) were challenged via the respiratory tract with a clinical isolated AB. AB was recoverd from the lung four hours post challenged. The median bacterial burdens were 4176, 3605, and 8513 CFU/g for TonB-R, NcsP, and control mice, respectively. The differences between the immunized and the control group were significant (TonB-R, P < 0.05; NcsP, P < 0.01) indicating that both TonB-R and NcsP are able to elicit protective immune responses. The bactericidal/bacteriostatic activies of anti-TonB-R and anti-NcsP antisera were further confirmed in vitro. Finally, MTT assay was performed to evaluate whether rTonB-R and rNcsP are cytotoxic to human cells. The results showed that both proteins were not cytotoxic to human A549 cells. Collectively, these results indicate that TonB-R and NcsP are good vaccine candidates against AB infection.

Reference ( 48 ) 〈TOP〉

Antunes, L., Imperi, F., Towner, K. J. & Visca, P. (2011). Genome-assisted identification of putative iron-utilization genes in Acinetobacter baumannii and their distribution among a genotypically diverse collection of clinical isolates. Research in microbiology 162, 279-284.
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連結：

Times Cited (4) 〈TOP〉

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