探討經由 D-胺基酸取代之新型抗菌胜肽 Tilapia piscidin 4 衍生物的殺菌活性和穩定性

Investigation of Bactericidal Activity and Stability of Novel D-Amino Acid Substituted Antimicrobial Peptides Derived from Tilapia piscidin 4

許博咸(Po-Hsien Hsu)

指導教授 ： 陳志毅

共同指導教授 ： 李宗徽(Tzong-Huei Lee)

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202202839

摘要

抗菌胜肽 (Antimicrobial peptides, AMPs) 對細菌外膜的物理性破壞機制，被認為是可以當作多重抗藥性細菌的替代藥物候選物。但天然AMPs在臨床應用上，卻因其對生理環境的不穩定而失活。所幸經許多研究的累積得知，AMPs的螺旋性、陽離子和疏水面的兩性結構為其活性的主因之一。因此我們利用從Oreochromis niloticus分離出的Tilapia piscidin 4 (TP4)，經過胺基酸的替換修飾，透過Lysine (K) 提高其正電荷的同時也調整親疏水性的平衡，並將第13個胺基酸替換成Proline (P)，再分別把不同位置上的L-胺基酸替換成鏡像異構物 (D-胺基酸) 以提升穩定性，進而合成出三種不同的TP4異手性 (Heterochiral) 衍生物TP4-α、TP4-β和TP4-γ。三種AMPs皆對多數細菌有良好的抗菌活性，特別在對抗NDM-1 Klebsiella pneumoniae時展現出最好的活性，並大大的改善了原本TP4極差的細胞選擇性和在生理條件下的不穩定，像是獲得極低的溶血性，或是在人類血清、不同的生理鹽類濃度、pH 7.4-10的環境下都能維持抗菌活性的穩定，並且不受不同溫度和蛋白酶的影響，同時也能從Circular dichroism (CD) 的二級結構光譜圖證實其結構的穩定。除此之外，利用不同螢光探針的殺菌機制研究可知，TP4-α、TP4-β和TP4-γ皆會和Lipopolysaccharide (LPS) 做結合，接著造成細菌外膜破裂、細胞膜去極化和增加內膜的滲透性，最終導致細菌死亡，同時也可以透過電子顯微鏡證實細菌表面的破洞和膜的破損。雖然在細胞毒性方面沒有得到改善，但可以利用和不同種類的抗生素做搭配形成優秀的協同作用，除了可以降低劑量的使用和成本外，還可以增強兩種藥物的藥效，甚至擴大抗菌的範圍。總體來說，或許TP4的異手性衍生物可利用和其他藥物做搭配的方式，更進一步的應用於抗NDM-1 K. pneumoniae或廣譜藥物的發展。

關鍵字

抗菌胜肽 ； Tilapia piscidin 4 (TP4) ； 鏡像異構物 ； 穩定性 ； 多重抗藥性 ； NDM-1克雷白氏肺炎菌 ； 協同作用

並列摘要

Antimicrobial peptides (AMPs) are physiological defense molecules that counter pathogenic consequences. AMPs act through physical interaction followed by bacterial membrane lysis. They are thought to be potential alternatives of clinical antibiotics due to reported activity against multidrug resistant (MDR) pathogens. However, the clinical application of natural AMPs are limited due to their instability in physiological conditions. Therefore, we modified a natural AMP known as Tilapia piscidin 4 (TP4) to develop a series of novel molecules with enhanced stability and notable activity, while maintaining minimal toxicity in in Vivo condition. We performed amino acid substitution, followed by chiral alteration, that resulted in development of heterochiral derivatives of TP4 (TP4-α, TP4-β and TP4-γ). All three AMPs displayed notable antibacterial activity. These peptides were significantly potential against MDR species like NDM-1 Klebsiella pneumoniae. The modified peptides showed negligible hemolysis and enhanced stability when compared with the TP4, which is known to be toxic and unstable in physiological conditions. The hybrid peptides showed notable activity in presence of physiological ions, varying pH and temperature and even in presence of physiological enzymes. The stability of its structure was confirmed from the secondary structure spectrum of circular dichroism (CD). The modified peptides act through lipopolysaccharide (LPS) binding, cause the rupture of the bacterial outer membrane, the depolarization of the cell membrane, and increase the permeability of the inner membrane. The holes on the bacterial surface and membrane were confirmed under the electron microscope. Although the cytotoxicity has not been improved, these peptides show synergistic activity with clinical antibiotics. This could be an innovative way to repurpose old antibiotics against MDR species and reduce the dosage and cost. Hence, we conclude that informed amino acid substitution with heterochiral construct can be employed to develop potential AMPs that may be useful for clinical applications in near future.

並列關鍵字

Antimicrobial peptides ； Tilapia piscidin 4 (TP4) ； D-form enantiomer ； Stability ； Multidrug resistance ； NDM-1 K. pneumoniae ； Synergistic effect

參考文獻

(1) Verma, T.; Aggarwal, A.; Singh, S.; Sharma, S.; Sarma, S. J. Current challenges and advancements towards discovery and resistance of antibiotics. Journal of Molecular Structure 2022, 1248, 131380.

(2) Michael, C. A.; Dominey-Howes, D.; Labbate, M. The Antimicrobial Resistance Crisis: Causes, Consequences, and Management. Frontiers in Public Health 2014, 2, Review.

(3) Spellberg, B.; Gilbert, D. N. The Future of Antibiotics and Resistance: A Tribute to a Career of Leadership by John Bartlett. Clinical Infectious Diseases 2014, 59 (suppl_2), S71-S75.

(4) Hossain, A.; Habibullah-Al-Mamun, M.; Nagano, I.; Masunaga, S.; Kitazawa, D.; Matsuda, H. Antibiotics, antibiotic-resistant bacteria, and resistance genes in aquaculture: risks, current concern, and future thinking. Environmental Science and Pollution Research 2022, 29 (8), 11054-11075.

(5) Ventola, C. L. The antibiotic resistance crisis: part 1: causes and threats. P t 2015, 40 (4), 277-283.