鮑氏不動桿菌BaeSR二元調節系統對輸出幫浦調節之探討

Regulation of Efflux Pumps by BaeSR Two-Component Regulatory System in Acinetobacter baumannii

林明鋒(Ming-Feng Lin)

指導教授 ： 藍忠昱

國立清華大學/生命科學院/分子與細胞生物研究所/博士(2015年)

https://doi.org/10.6843/NTHU.2015.00445

摘要

二元調節系統在細菌對環境刺激適應的調節與訊號的傳遞上扮演重要角色。二元調節系統BseSR為五大胞外反應路徑之一，能偵測環境訊號並藉由改變細菌外套而反應。在此研究中，我們發現鮑氏不動桿菌的二元調節系統基因baeR 及baeS被共同轉錄，且於高滲透壓的情形下可對此壓力做出反應。相較於標準株，具老虎黴素抗藥性之誘導株或臨床株，其baeSR與幫浦基因adeAB之轉錄量均增加。若將標準株 ATCC 17978之baeR去除，將分別導致對adeA與adeB基因表達量下降67-73%與68%，並使老虎黴素的最低抑菌濃度下降。去baeR對adeAB基因表達量的影響亦可在經實驗誘導之具老虎黴素抗藥性菌株中發現。殺菌效力評估試驗顯示，當老虎黴素的濃度為0.25 μg/mL時，去baeR變種相較於標準株，其整個生長期間的菌落形成單位約減少1-log10。當老虎黴素的濃度增加至0.5 μg/mL時，去baeR變種的菌落形成單位更顯著減少4.7-log10。輸出幫浦AdeIJK 及MacAB-TolC 也被證明為BaeSR所調控。其中，MacAB-TolC於具老虎黴素抗藥性鮑氏不動桿菌之誘導株與臨床株均有較高的基因表達量，意味除AdeABC外其對老虎黴素抗藥性的重要性。表現型微陣列的結果顯示，鮑氏不動桿菌於去baeR後對某些化學物質呈易感性, 特別是鞣酸。鮑氏不動桿菌標準株於含有100 µg/mL鞣酸的培養基中，其adeJ 與macB 的基因表達量均增加，此增加可被去baeR所完全阻止。His-BaeR與adeA、adeJ與macA啟動區的交互作用之凝膠遷移滯後實驗並未證明其直接結合，意指其交互作用為間接性。綜言之，鮑氏不動桿菌可利用二元調節系統BaeSR可以間接方式透過輸出幫浦的調控來感應與反應處理化學物質，如鞣酸與老虎黴素。我們的發現提供一有潛力的標的可用以發展抗微生物製劑對抗具抗藥性鮑氏不動桿菌。

關鍵字

鮑氏不動桿菌 ； 老虎黴素 ； 二元調節系統 ； 輸出幫浦 ； 鞣酸

並列摘要

Bacterial two-component regulatory system (TCS) plays an important role in the regulation of adaptation to and signal transduction of environmental stimuli. BaeSR, a TCS, which detects environmental signals and responds by altering the bacterial envelope, is one of the five extracytoplasmic response pathways. In this study, we found that the TCS genes baeR and baeS in Acinetobacter baumannii are co-transcribed and function as stress responders under high osmotic conditions. The baeSR and adeAB genes showed increased transcription in both the laboratory-induced and clinical tigecycline-resistant strains compared with the wild-type strain. The deletion of baeR in the ATCC 17978 strain led to 67–73% and 68% reduction in adeA and adeB expression, respectively, with a decrease in the tigecycline minimal inhibition concentration (MIC). The influence of baeR knockout on adeAB gene expression can also be observed in the laboratory-induced tigecycline-resistant strains. The time-kill assay showed that the baeR deletion mutant showed an approximate 1-log10 reduction in colony forming units (CFUs) relative to the wild-type strain when the tigecycline concentration was 0.25 μg/mL throughout the assay period. Increasing the tigecycline concentration to 0.5 μg/mL produced an even more marked 4.7-log10 reduction in CFUs of the baeR deletion mutant. Efflux pump AdeIJK and MacAB-TolC are also demonstrated under the regulation of BaeSR. Of them, MacAB-TolC had higher gene expression in both the laboratory induced and clinical tigecycline-resistant A. baumannii strains, implicating its importance to tigecycline resistance in addition to AdeABC. Phenotypic microarray results showed that A. baumannii is vulnerable to certain chemicals, especially tannic acid, after deleting baeR, which was confirmed using the spot assay. The reference strain A. baumannii also exhibited increased gene expression of adeJ and macB in the medium with 100 µg/mL tannic acid, but the increase was fully inhibited by baeR deletion. An electrophoretic motility shift assay based on an interaction between His-BaeR and the adeA, adeI and macA promoter regions did not demonstrate direct binding, which implies that their mutual interaction was indirect. In conclusion, A. baumannii can make use of TCS BaeSR system to sense and respond in disposing chemicals, such as tannic acid and tigecycline, through regulating indirectly the efflux pumps. Our finding provides a promising target to develop future antimicrobials against drug- resistant A. baumannii.

並列關鍵字

Acinetobacter baumannii ； Tigecycline ； Two-component regulatory system ； Efflux pumps ； Tannic acid

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