針對鮑氏不動桿菌17978 的亞胺培南抗藥性機制進行蛋白質體與磷酸化蛋白質體分析

Investigation of Imipenem-Resistant Mechanism in Acinetobacter baumannii 17978 by Proteomic and Phosphoproteomic Analysis

林孟筑(Meng-Chu Lin)

指導教授 ： 吳世雄

國立臺灣大學/生命科學院/生化科學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902499

摘要

介紹：鮑氏不動桿菌是眾所週知的重要院內感染菌，能夠呈現抗藥性並能夠在乾燥或消毒的醫院環境以生物薄膜的形式生存在醫療器材上。這樣爆發性感染的鮑氏不動桿菌以能夠獲得多重抗藥機制為勝，已造成公共衛生重大的議題。Carbapenem類抗生素包括亞胺培南imipenem（一種院內常用的Carbapenem類抗生素）和美羅培南meropenem已經被普遍用在對付這種多重抗藥性的鮑氏不動桿菌。然而隨著使用頻率增加，這些對於Carbapenem有抗藥性的鮑氏不動桿菌感染事件越來越來多，並獲得關切。
目的：為了以評估imipenem在細菌細胞間溝通與釋放抗藥因子的影響力，我們使用磷酸化蛋白質體分析去了解鮑氏不動桿菌在接受imipenem抗生素後的早期（形成基因突變的抗藥性菌株之前）如何去作反應因應壓力變化。這篇研究使用對imipenem具有耐受性的菌株鮑氏不動桿菌17978以驗證過往使用低於最小抑菌濃度抗生素的方式做表現型篩選來誘導異質性抗藥性的產生。我們使用細菌指數生長期的細菌培養加藥後，並收取細胞作蛋白質體和磷酸化蛋白體的分析。我們根據生物資訊分析的結果，針對對於imipenem有表現變化的蛋白質和經過基因功能註解的磷酸化蛋白質去做討論和比較。而在這其中我們集中探討一個磷酸化蛋白（磷酸化的NorM蛋白質，是一種MATE家族的藥物排出幫浦）。經過蛋白模擬結構與計算，這個藥物幫浦蛋白上的酪氨酸磷酸化修飾剛好位於幫浦蛋白通道的中間位置。以這樣的情況來說，我們推測這個磷酸化酪氨酸基團在這結構中可能造成立體阻礙而導致幫浦功能效率減弱。更近一步推論在施加imipenem後，這個位置的磷酸根被移去，使立體障礙同時減去，以方便藥物輸出幫浦能夠正常地作構型互變來將藥物打出。我們在未來會提供更多有效證據來支持這樣的假說和模型，以證明磷酸化在這個幫浦蛋白對於藥物抗藥性的重要性。

關鍵字

亞胺培南 ； 磷酸化蛋白質體 ； 鮑氏不動桿菌 ； 藥物抗藥性 ； 藥物輸出幫浦

並列摘要

Introduction: Acinetobacter baumannii has emerged as an important nosocomial and opportunistic pathogen worldwide, reflecting antimicrobial resistance, tolerance to desiccation and disinfection and biofilm formation on common abiotic surfaces in healthcare settings. Institutional outbreaks caused by Acinetobacter baumannii strains that have acquired multiple mechanisms of antimicrobial drug resistance constitute a growing public-health problem. Carbapenems, primarily imipenem (a frequently used carbapenem in clinical practices) and meropenem, have been used to treat multidrug-resistant (MDR) A. baumannii infections. However, the increasing incidence of carbapenem-resistant A. baumannii (CRAB) infections has become of major concern.
Objectives: In order to assess the influence of imipenem on bacterial cell communication and the release of different factors of drug resistance, we utilize phosphoproteomics analysis to understand how A. baumannii responds to carbapenem at the early stage before transforming to a genotypic variation-derived drug-resistant strain. In order to verify the selective effect using sub-MIC antibiotic and the commonly seen heteroresistance induced by environmental stress, a drug-sensitive A. baumannii 17978 was chosen as study. By growth phase-dependent sampling, the proteomes and phosphoproteomes of A. baumannii 17978 in the presence of imipenem were identified. Based on bioinformatics analysis, differentially expressed proteins and functional annotations of phosphoproteins in imipenem-treated A. baumannii 17978 were discussed. The phosphoprotein (a phosphorylated NorM), a MATE drug efflux antiporter, was identified. Consistent with our modeling calculation, the phosphorylation of Tyrosine (pY) of MATE drug efflux antiporter was shown to be in the middle of the efflux channel. In this scenario, the phospho-Tyrosine residue in the channel might cause a steric hindrance to reduce the pumping efficiency of MATE drug efflux antiporter. Therefore, upon the addition of imipenem, the phosphate group is suggested to be removed in order to facilitate the progression of the structural switching in the topology of the transporter. In the future, we will perform more evidence-based studies about this protein to better clarify the role of phosphorylation in this protein in relation of drug resistance.

並列關鍵字

Imipenem ； phosphoproteome ； Acinetobacter baumannii ； drug resistance ； efflux pump

參考文獻

1. Control, C.f.D. and Prevention, Acinetobacter baumannii infections among patients at military medical facilities treating injured US service members, 2002-2004. MMWR. Morbidity and mortality weekly report, 2004. 53(45): p. 1063.

2. Davis, K.A., et al., Multidrug-resistant Acinetobacter extremity infections in soldiers. Emerging infectious diseases, 2005. 11(8): p. 1218.

3. Roca, I., et al., The Acinetobacter baumannii Oxymoron: Commensal Hospital Dweller Turned Pan-Drug-Resistant Menace. Frontiers in Microbiology 2012. 3: p. 148.

4. Espinal, P., S. Marti, and J. Vila, Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. Journal of Hospital Infection, 2012. 80(1): p. 56-60.

5. Navon-Venezia, S., R. Ben-Ami, and Y. Carmeli, Update on Pseudomonas aeruginosa and Acinetobacter baumannii infections in the healthcare setting. Current opinion in infectious diseases, 2005. 18(4): p. 306-313.