可用於對抗人類病原菌的抗菌胜肽之設計及應用

Design and application of antimicrobial peptides against human pathogens

陳頌邦(Sung-Pang Chen)

指導教授 ： 陳佩燁

國立臺灣大學/生命科學院/生化科學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202004350

摘要

近年來，由於抗生素藥物的過度使用造成抗藥性菌株的大量出現，且新的抗生素發展速度緩慢，發展新型態的抗生素已成為當務之急。抗微生物胜肽(Antimicrobial peptides，AMPs) 或稱為抗菌胜肽，被認為極具潛力發展成為取代傳統抗生素的新一類抗生素。
在實驗室先前的研究中，我們設計了五條陽離子抗菌胜肽，均由18個胺基酸組成，擁有不同的疏水性/親水性胺基酸分佈。在經過初步的抗菌活性測試(Minimal Inhibitory Concentration/ Minimal Bactericidal Concentration ; MIC/MBC測試) 後，我們挑選了抗菌活性最好的胜肽，並在本研究中，以此胜肽為模板進行不同的修飾，例如縮短胜肽長度、替換相同性質的胺基酸、改變N端的保護基、使用D-form胺基酸或在序列中加入類肽結構。我們測試這些修飾後的胜肽對於4種醫院內病原菌 (Acinetobacter baumannii、Klebsiella pneumoniae、Staphylococcus aureus及Staphylococcus epidermidis) 的MIC/MBC，以及這些胜肽對於大鼠或狗紅血球細胞的溶血活性。接著，我們挑選出展現最佳選擇性 (低MIC/MBC與低溶血活性) 的胜肽，測試它們對人類胚胎腎細胞 (HEK293) 的細胞毒性，並測試這些胜肽在大鼠血漿中的穩定性，以判斷胜肽是否易被血漿中的蛋白酶降解，同時也利用大鼠的全血測試胜肽在血液中的殺菌能力與速度。最終，我們以薄層層析分析細菌細胞膜的磷脂質組成，藉此實驗幫助找出細菌細胞膜磷脂質成分與抗菌活性之間的關係。
根據本研究的實驗結果，我們總結出將胜肽的N端以較長鏈的醯基修飾會增加溶血活性並且降低抗菌效果。在本研究所有設計的胜肽中，將序列修短的pepD3、換成D-form胺基酸的pepdD2以及將leucine換成isoleucine的pepI2表現出較好的抗菌活性和較低的溶血活性。pepdD2在血漿中最穩定，pepI2對HEK293的細胞毒性最低。在未來的研究中，我們將著重於pepD3、pepdD2、pepI2，評估此三條胜肽在活體內的功效和毒性，並測試它們是否同樣對抗藥性細菌展現出抗菌活性。

關鍵字

抗菌胜肽 ； 院內感染性細菌 ； 鮑氏不動桿菌 ； 克雷伯氏肺炎菌 ； 金黃色葡萄球菌 ； 表皮葡萄球菌 ； 抗生素抗藥性細菌

並列摘要

Recently, the overuse of traditional antibiotics makes the emergence of antibiotic-resistant bacterial strains. Unfortunately, there are not enough new drugs in the pharmaceutical pipeline to keep pace with drug-resistant bacterial development. Thus, the development of an effective novel class of antibiotics is an urgent need. Antimicrobial peptides (AMP) is considered as one of the most promising candidates for a novel class of antibiotics.
In our previous investigation, we designed five cationic AMPs. All of them composed of 18 amino acids with different hydrophobic/hydrophilic amino acid distribution. After a preliminary antimicrobial test (Minimal Inhibitory Concentration/ Minimal Bactericidal Concentration test, MIC/MBC test), we chose the one which showed the best antimicrobial activity and then modified this peptide with different methods, such as decreasing peptide length, replacing amino acids with the ones with similar property, changing the N-terminal capping group, using D-form amino acids or inserting peptoid structure. We tested the MIC/MBC of these modified peptides against four nosocomial pathogens (Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus and Staphylococcus epidermidis) and the hemolysis of these peptides against rat or dog red blood cells. After that, we chose the ones that showed the best selectivity (low MIC/MBC and hemolysis) and following test their cytotoxicity to human embryonic kidney cells (HEK293). We also tested their stability in rat plasma to evaluate whether these peptides easily degraded by plasma protease. Besides, we also used rat whole blood to evaluate bactericidal ability and time-kill kinetics of peptides in blood environment. Finally, we used thin-layer chromatography to analyze the lipid composition of the bacterial cytoplasmic membrane. From this test, we can find out the relationship between bacterial membrane lipid composition and antimicrobial activity.
Based on our data, we conclude that longer chain-length of the acyl group at N-terminus increased hemolysis activity and decreased antimicrobial activity. Among all the designed peptides, pepD3 (shorter length), pepdD2 (D-form peptide), and pepI2 (replace Leu with Ile) displayed better antimicrobial activity and lower hemolysis. pepdD2 is the most stable peptide in plasma. pepI2 has the lowest cytotoxicity to HEK293 cells. In the future, we will focus on pepD3, pepdD2, and pepI2 to evaluate their in vivo efficacy, in vivo toxicity, and test whether they are effective against antimicrobial-resistant bacteria.

並列關鍵字

Antimicrobial peptides (AMPs) ； nosocomial pathogen ； A. baumannii ； K. pneumoniae ； S. aureus ； S. epidermidis ； antimicrobial-resistant bacteria

參考文獻

[1] Haney EF, Hancock REW (2013). Peptide design for antimicrobial and immunomodulatory applications. Biopolymers. 100:572-583.

[2] Schneider JJ, Unholzer A, Schaller M, Schäfer-Korting M, Korting HC (2005). Human defensins. Journal of Molecular Medicine. 83:587-595.

[3] Dubos RJ (1939). Studies on a bactericidal agent extracted from a soil bacillus: I. Preparation of the agent. Its activity in vitro. Journal of Experimental Medicine. 70:1-10.

[4] Hotchkiss RD, Dubos RJ (1940). Fractionation of the bactericidal agent from cultures of a soil bacillus. Journal of Biological Chemistry. 132:791-792.

[5] Kamysz W (2005). Are antimicrobial peptides an alternative for conventional antibiotics? Nuclear Medicine Review Central Eastern Europe. 8:78-86.