以磷酸化蛋白質體學研究鮑氏不動桿菌臨床菌株SK17中磷酸化對乙內醯胺酶AmpC活性的調控機制

Comparative phosphoproteomics reveals the role of AmpC β-lactamase phosphorylation in the clinical imipenem-resistant strain Acinetobacter baumannii SK17

賴若馨(Juo-Hsin Lai)

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/博士(2015年)

https://doi.org/10.6342/NTU.2015.00514

摘要

鮑氏不動桿菌（Acinetobacter baumannii）為一伺機性致病菌，已知會感染住院、免疫力低下、及患病末期的病人。重要的是，具多重抗藥性的鮑氏不動桿菌（multidrug resistant Acinetobacter baumannii ，簡稱 MDRAB）的出現已導致此菌演變成高毒性的院內致病菌，且是引起院內感染大爆發的原因。全球散布的MDRAB已經對人類健康造成嚴重的威脅，尤其是近十年大量對抗生素碳青黴烯
（carbapenem）產生抗性的菌株出現。在許多的碳青黴烯衍生物之中，亞胺培南（imipenem）能有效的對抗鮑氏不動桿菌。在西元1990 年代初期，已有報導指出鮑氏不動桿菌已對亞胺培南產生抗性，然而參與其中的分子機制還不明朗。從細菌到人類，蛋白質的翻譯後修飾作用廣見於各類物種。其中，磷酸化是一種可逆的蛋白質的轉譯後修飾作用，且與多種的細胞、代謝、訊息傳導的過程相關。
研究病源菌的磷酸化蛋白質體學可提供一個系統性的觀察，以探討磷酸化蛋白在致病的調控中所扮演的角色。在本研究中，我們分析了兩株臨床鮑氏不動桿菌株；亞胺培南敏感性的菌株SK17-S，及對亞胺培南產生抗性的菌株SK17-R。從散彈式策略以及高解析度的質譜儀偵測實驗中，於SK17-S 樣本中找出248 個磷酸化蛋白及410 個磷酸化位置，其胺基酸分布為47.0:27.6:12.4:8.0:4.9%於絲胺酸/蘇胺酸/酪胺酸/天門冬胺酸/組胺酸。此外於SK17-R 樣本中找出211 個磷酸化蛋白及285 個磷酸化位置，其絲胺酸/蘇胺酸/酪胺酸/天門冬胺酸/組胺酸分布則為41.4:29.5:17.5:6.7:4.9%。其中首次在SK17-S 樣本裡發現，一種乙內醯胺酶AmpC的絲胺酸第九十號（Ser-90）位置上有被磷酸化修飾，且此第九十號絲胺酸就位在此酵素的催化結構域 (S88VS90K) 上。因此，我們假設AmpC 蛋白磷酸化可能調控乙內醯胺酶的活性，與中和抵銷亞胺培南抗生素的能力。透過點突變的實驗
方法，我們發現模擬Ser-90 去磷酸化的轉殖株 (S90A)對imipenem 產生較高的抗性，相反地，模擬Ser-90 磷酸化的轉殖株 (S90D) 則是對imipenem 敏感的。並且S90A 突變蛋白的乙內醯胺酶活性較強，藉由與imipene 更結合並賦予菌株SK17-S 更多的保護力去對抗imipenem，且保護力是優於原生(wild-type)的AmpC。總結本研究結果顯示，在鮑氏不動桿菌的乙內醯胺酶AmpC 上Ser-90 的磷酸化，可負向的調控鮑氏不動桿菌對imipenem 的感受性、乙內醯胺酶活性、
及中和抵銷imipenem 抗生素的效應。

關鍵字

多重抗藥性的鮑氏不動桿菌 ； 亞胺培南 ； 磷酸化蛋白質體學 ； 質譜儀分析儀 ； 二氧化鈦萃取磷酸化胜肽 ； 乙內醯胺酶AmpC

並列摘要

Acinetobacter baumannii is an opportunistic bacterial pathogen which is known to infect hospitalized, immunocompromised, or terminal patients. Importantly, this bacterium has become a high-virulent nosocomial pathogen due to the emergence of multidrug-resistant Acinetobacter baumannii (MDRAB) responsible for numerous outbreaks of infections. Global spread of MDRAB has emerged as a serious threat to human health; especially carbapenem resistance has greatly increased over recent decades. Among the many carbapenem derivatives, imipenem has been found to be effective against A. baumannii. It has been reported that imipenem resistance in A. baumannii threatens human health since the early 1990s; however, the underlying molecular mechanisms remain unclear. Accordingly, post-translational modifications (PTMs) of proteins have been found in a wide variety of species from bacteria to human. Among all, phosphorylation is a kind of reversible PTMs, and has been linked to a wide variety of cellular, metabolic, and signaling processes. Measuring the phosphoproteomics of pathogenic bacteria can provide a system view for investigating their roles in virulence regulation networks. In this study, we analyzed the phosphoproteomics of two clinical isolates of A. baumannii: imipenem-sensitive strain SK17-S and -resistant strain SK17-R. Using a shotgun strategy combined with high-accuracy mass spectrometry, we identified 410 phosphosites on 248 unique phosphoproteins from SK17-S and 285 phosphosites on 211 unique phosphoproteins from SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites from SK17-S and SK17-R are 47.0, 27.6, 12.4, 8.0, and 4.9% versus 41.4, 29.5, 17.5, 6.7, and 4.9%, respectively. Among all, the Ser-90 phosphosite, located on the catalytic motif S88VS90K91 of the AmpC β-lactamase, was first identified from SK17-S. We hypothesized that AmpC phosphorylation may control its enzyme activity and the ability of neutralizing imipenem due to the proximity of Ser-90 to the catalytic residue Ser-88. By site-directed mutagenesis, the nonphosphorylatable strain S90A was more resistant to imipenem, by contrast, the phosphorylation-simulated strain S90D was imipenem-sensitive. Also, S90A mutant protein exhibited higher β-lactamase activity and conferred more bacterial protection on SK17-S against imipenem than the wild-type. In summary, our results revealed that in A. baumannii Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem.

並列關鍵字

multidrug-resistant Acinetobacter baumannii ； imipenem ； phosphoproteome ； Mass spectrometry ； TiO2 phosphopeptide enrichment ； AmpC β-lactamase

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