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The molecular factors derived from potent bactericidal activity of reduced cryptdin-4

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Title: The molecular factors derived from potent bactericidal activity of reduced cryptdin-4
Other Titles: 還元型cryptdin-4の強力な殺菌活性の分子機構に関する研究
Authors: 佐藤, 優次1 Browse this author
Authors(alt): Sato, Yuji1
Issue Date: 24-Mar-2022
Publisher: Hokkaido University
Abstract: Defensins, a major family of antimicrobial peptides (AMPs), are cysteine-rich AMPs with three disulphide bonds under normal oxidative conditions. For a long time, studies concerning defensins have focused on oxidised forms consisting of three disulphide bridges. However, it has been reported that reduced defensin exhibits bactericidal activity equal to or greater than that of its oxidised form. After that, it has been focused on the reduced defensins studies. α-Defensins secreted by small intestinal Paneth cells are known as cryptdins. Cryptdin-4 (Crp4) is the most widely studied cryptdin. Reduced Crp4 (Crp4red) has been reported to exert potent bactericidal activity against enteric commensal and non-commensal bacteria, whereas oxidised Crp4 (Crp4ox) is only active against noncommensal bacteria. An increase in reduced cryptdins is correlated with dysbiosis in a mouse model of Crohn’s disease. This study suggests that alternative defensin activity is associated with disease mechanisms and progression. However, the precise bactericidal mechanism of both Crp4ox and Crp4red has yet to be fully elucidated, considering that Crp4ox permeabilises bacterial membranes, and that such permeabilisation is correlated with bactericidal activity. On the other hand, some studies have demonstrated that certain defensins do not disrupt cell membranes but exhibit other modes of action, defined as 'molecular targeting mechanisms', and have reported a fully new activity mechanism termed nanonets. In this study, I aimed to elucidate the molecular factors that affect the potent bactericidal activity of Crp4red. I tested free cysteine, structure, and hydrophobicity as molecular factors, and determined whether bacterial membrane interactions are critical for the mode of action of Crp4red. In Chapter 1, I describe the preparation of peptides to study the bactericidal activity of Crp4red. Crp4 was co-expressed in E. coli to generate Crp4ox, and Crp4red was obtained by completely reducing Crp4ox. To study the function of the thiol group of cysteine residues, Crp4red was chemically modified with N-ethylmaleimide (NEM). In Chapter 2, I sought to elucidate the bactericidal mechanism of Crp4red at the molecular level using the peptides described in the previous chapter; Crp4ox, Crp4red, NEM-Crp4, and 6C/S-Crp4, in which all Cys residues were substituted with Ser residues. The bactericidal activities of these peptides against non-commensal and commensal bacteria were assessed. All peptides showed bactericidal activity against non-commensal bacteria. Crp4red and NEM-Crp4 exhibited bactericidal activity against one commensal, Lactobacillus johnsonii (L. johnsonii) whereas 6C/S-Crp4 was not effective against L. johnsonii but killed another commensal, Bifidobacterium breve (B. breve). On the other hand, Crp4ox did not exert any activity against either commensal bacteria. These results suggested that it is not important for the thiol group of cysteine residues in Crp4red to exhibit potent bactericidal activity. The secondary structure of the peptides showed no significant changes. Hydrophobicity was strongly correlated with bactericidal activity. These results suggested that the potent Crp4red bactericidal activity is derived from its hydrophobicity. Moreover, I attempted to determine whether bacterial membrane interactions are critical for the Crp4red mode of action. A liposome leakage assay using lipids extracted from L. johnsonii suggested a degree of correlation with bactericidal activity These results suggested that membrane interactions are crucial for potent Crp4red activity and that the potent bactericidal capacity of Crp4red is derived from its hydrophobicity and involves disruption of he bacterial membrane.
Conffering University: 北海道大学
Degree Report Number: 甲第14846号
Degree Level: 博士
Degree Discipline: ソフトマター科学
Examination Committee Members: (主査) 教授 相沢 智康, 教授 綾部 時芳, 教授 出村 誠, 教授 芳賀 永, 准教授 菊川 峰志
Degree Affiliation: 生命科学院(ソフトマター専攻)
Type: theses (doctoral)
Appears in Collections:課程博士 (Doctorate by way of Advanced Course) > 生命科学院(Graduate School of Life Science)
学位論文 (Theses) > 博士 (ソフトマター科学)

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