清大生醫學院學士後醫學系黃建銘助理教授（右二）與研究團隊成員

Assistant Professor  Jian-Ming Huang of School of Medicine, College of Life Sciences and Medicine, National Tsing Hua University, with research team member.

論文標題Paper Title

Inhibition of GABA metabolism by β-lactam antibiotics affects encystation in Acanthamoeba

學術期刊刊名Journal Name

biomedicine & pharmacotherapy

研究團隊 Research Team

主持人Principle Investigator：

黃建銘 助理教授  Jian-Ming Huang, Assistant Professor

國立清華大學學士後醫學系

School of Medicine, College of Life Sciences and Medicine, National Tsing Hua University(NTHU).

參與者Participants:：

摘要

Abstract

棘阿米巴囊化是一種休眠的過程，推測其代謝會在此過程中有重大變化。為探索這些變化，我們採用標記代謝體分析（targeted metabolomics），比較營養體與囊體在代謝物組成上的差異。結果顯示，囊化前的營養體以糖解作用與三羧酸循環活性為主，具高能量代謝特徵；而囊化後，能量產生相關代謝明顯下降。特別的是，我們觀察到GABA、谷胺醯胺等神經傳導相關代謝物在囊化初期顯著上升，推測其可能參與訊號傳遞或囊化誘導。此外，NAD+/NADH比例改變也暗示著棘阿米巴在囊化過程中發生氧化還原平衡調整。這些代謝變化提供了潛在的囊化標誌物，也為未來干擾囊化的藥物開發提供目標。

Encystation of Acanthamoeba is a dormant process characterized by presumed significant metabolic shifts. To explore these changes, we employed targeted metabolomics to compare the metabolite compositions between trophozoites and cysts. Our results indicate that pre-encystment trophozoites are primarily driven by glycolysis and the TCA cycle, exhibiting features of high energy metabolism. In contrast, energy production-related metabolism significantly declines following encystation. Notably, we observed a marked increase in neurotransmitter-related metabolites, such as GABA and glutamine, during the early stages of encystation, suggesting their potential involvement in signal transduction or the induction of the encystation process. Furthermore, changes in the NAD+/NADH ratio imply a recalibration of redox balance during Acanthamoeba encystation. These metabolic shifts not only provide potential biomarkers for encystation but also identify targets for the future development of drugs aimed at disrupting the encystation process.

研究成果Result/Contributions

棘阿米巴角膜炎（Acanthamoeba keratitis, AK） 是一種由致病性自由營生性棘阿米巴原蟲引起的嚴重疼痛性角膜感染，主要影響衛生習慣不佳的隱形眼鏡使用者。其臨床表現包括角膜浸潤、上皮與基質損傷以及劇烈的眼部疼痛，在晚期病例中可能導致視力喪失或必須進行眼球摘除。現行治療方法（如聚六亞甲基雙胍, PHMB）常因該寄生蟲能形成具抗藥性的囊體（cysts）而宣告失敗。在本研究中，代謝體分析顯示γ-胺基丁酸（GABA）在囊化過程中會大量累積。針對 GABA 支路（GABA shunt pathway）關鍵基因的表達分析顯示，麩胺酸脫氫酶（GDH）與麩胺酸脫羧酶（GAD）顯著上調。實驗發現，補充外源性 GABA 會提升囊化率，並上調囊化相關基因（包括纖維素合成酶 I、自噬相關蛋白 8 以及囊化媒介絲胺酸蛋白酶），這暗示 GABA 可能作為調節囊化過程的訊息分子。為了探索潛在的抗囊化策略，本研究測試了作為已知GABA_A 受體拮抗劑的 beta-內醯胺類抗生素。結果顯示，頭孢噻肟（Cefotaxime）、卡苯西林（Carbenicillin）及青黴素 G（Penicillin G）能在不產生細胞毒性的情況下，顯著抑制囊體形成；而非beta-內醯胺類抗生素則無此效果。這些發現顯示，此抑制作用與 beta-內醯胺環結構有關，且可能涉及對 GABA 媒介訊息傳導路徑的干擾。總結而言，本研究揭示了 GABA 代謝在棘阿米巴囊化過程中的關鍵作用，並指出beta-內醯胺類抗生素有望成為輔助藥物，用以克服 AK 治療中因囊體引起的抗藥性問題。

Acanthamoeba keratitis (AK) is a painful corneal infection caused by pathogenic free-living amoebae of the genus Acanthamoeba, primarily affecting contact lens users with inadequate hygiene practices. Clinical manifestations include corneal infiltration, epithelial and stromal damage, and severe ocular pain, which may lead to vision loss or enucleation in advanced cases. Current treatments, such as polyhexamethylene biguanide (PHMB), often fail due to the parasite’s ability to form drug-resistant cysts. In this study, metabolomic profiling revealed a significant accumulation of γ-aminobutyric acid (GABA) during encystation. Expression analysis of key genes associated with the GABA shunt pathway showed marked upregulation of glutamate dehydrogenase (GDH) and glutamate decarboxylase (GAD). Supplementation with exogenous GABA enhanced encystation rates and upregulated encystation-associated genes, including cellulose synthase I, autophagy-related protein 8, and encystation-mediating serine proteinase, suggesting that GABA may function as a signaling molecule regulating encystation. To explore potential anti-encystation strategies, β-lactam antibiotics, well-known GABAA receptor antagonists, were tested. Cefotaxime, carbenicillin, and penicillin G significantly inhibited cyst formation without inducing cytotoxicity, whereas non-β-lactam antibiotics showed no such effect. These findings suggest that the inhibitory effect is associated with the β-lactam ring structure and may involve interference with GABA-mediated signaling pathways. Collectively, our study reveals a critical role of GABA metabolism in Acanthamoeba encystation and highlights β-lactam antibiotics as potential adjunctive agents for overcoming cyst-associated drug resistance in AK treatment.

論文連結 Paper Link

https://doi.org/10.1016/j.biopha.2025.118841