人體中存在著不同菌種，在人體健康時雖然不構成危險，可是一旦人體免疫力下降，這些伺機性病原便有可能攻擊人體。白色念珠菌是人體共生菌叢中一種能夠感染人體且產生極高致死率的真菌，然而因為抗菌藥物的廣泛使用，白色念珠菌抗藥性如今已經成為全球性的議題。且因現行的抗真菌藥物種類有限，因此除了市面上已有的臨床藥物之外，開發及尋找新形態的藥物是目前非常重要的課題。且因真菌在生物特性上與人類有許多的相似性，使得開發抗真菌藥物面臨更大的挑戰。
抗菌胜肽作為生物先天性免疫系統重要的一環，除了具有殺菌效果外，亦可影響免疫系統的作用，因此被認為具有開發成新藥物之潛力。本研究主要在探討人類唾液衍生的抗菌胜肽對白色念珠菌的殺菌效果及機理。這些抗菌胜肽展現了顯著的抗菌效果，且除了一般懸浮細胞，亦能有效對抗形成生物膜的白色念珠菌及臨床上分離的Candida屬菌株。並且發現這些抗菌胜肽在白色念珠菌中攻擊其粒線體，導致細胞中的活性氧自由基上升，並與粒線體中產生細胞所需能量的電子傳遞鏈活性高度相關。尤其我們實驗更進一步發現其攻擊目標為粒線體電子傳遞鏈起始的mitochondrial complex I，藉由攻擊complex I上的NADH脫氫酶，抑制complex I活性及細胞氧氣消耗。不同於以往的研究，這是第一次發現對mitochondrial complex I具攻擊性的胜肽。結合上述綜合而論，本研究提出一個抗真菌藥物開發可能的新方向，並顯示抗菌胜肽擁有比目前所知更為複雜的機制與功能。

Candida albicans is a fungal commensal of humans and is able to cause life-threatening systemic infections particularly in the immunocompromised patients. The emergence of C. albicans drug resistance is also becoming more and more serious due to the limited classes of antifungals available and widespread clinical use of antifungals. Therefore, developing new antifungals is an urgent need.
Antimicrobial peptides (AMPs) are important components of host innate immunity these peptides possess a broad-spectrum against different pathogens including fungi. AMPs are thus considered as a highly exploitable agents for new therapeutic strategy. In this work, we studied the activity and mechanisms of human saliva-derived AMPs against C. albicans. These AMPs show a potent candidacidal activity against both planktonic and biofilm cells of C. albicans, and also clinical isolates of various Candida spp. Moreover, we identified the mitochondrial electron transport chain complex I, particularly NADH dehydrogenase of complex I, is the main target of the AMPs. Together, the unique properties of the AMPs tested suggest the potential use of AMPs in therapeutic applications and also highlight complex mechanisms and functions of AMPs.

INTRODUCTION 1
• 1. Candida albicans 2
• 2. Biofilm and its formation in C. albicans 2
• 3. Antimicrobial peptides (AMPs), Histatins, P-113 and P-113 derivatives 3
• 4. Mitochondria and complex I of the electron transport chain 3
• 5. ROS and mitochondrial complex I 5
MATERIALS & METHODS 6
• 1. Peptides and reagents. 7
• 2. Strains, medium and growth condition. 7
• 3. Isolation of mitochondria. 7
• 4. Mitochondrial complex I activity assay. 8
• 5. Identification of mutants resistant to P-113. 8
• 6. Biofilm metabolic activity assay. 8
• 7. Determination of minimum inhibiting concentration (MIC) of AMPs. 9
• 8. Antifungal susceptibility assay. 9
• 9. Scanning electron microscopy to examine biofilm morphology. 10
• 10. Fluorescence colocalization of AMPs. 10
• 11. Measurement of intracellular ROS accumulation. 11
• 12. Measurement of C. albicans respiration. 11
• 13. Determination of malondialdehyde (MDA) in C. albicans. 12
• 14. Measurement of ATP content in C. albicans. 12
• 15. Statistical analysis. 12
RESULTS 13
• 1. P-113Du and P-113Tri exhibit superior candidacidal activity compared to P-113 14
• 2. ROS involve in biofilm killing of AMPs 14
• 3. All tested AMPs target mitochondria 15
• 4. AMPs activity is associated with mitochondrial respiration 15
• 5. AMP resistance arising from mutants defective in mitochondrial complex I 16
• 6. AMPs inhibit cell respiration 17
• 7. Complex I and NADH dehydrogenase inhibited by the AMPs 17
• 8. The candidacidal activity of the AMPs closely correlates with ROS generation 19
DISCUSSION 20
REFERENCES 24
TABLE 33
• Table 1 | Mutant library gene orthologs for complex I P-113 resistant strains. 33
FIGURES 34
• Figure 1 | P-113 series AMPs effectly extirpate the planktonic and biofilm cells of C. albicans. 34
• Figure 2 | AMPs induce vacuolar-like protuberances formation on C. albicans biofilm. 35
• Figure 3 | ROS scavenger abolished activity of P-113 series AMPs against biofilm cells. 36
• Figure 4 | P-113 series AMPs target to mitochondria. 37
• Figure 5 | Respiration influences AMPs activity. 38
• Figure 6 | Subnit mutations on complex I consider P-113 resistance. 39
• Figure 7 | P-113 series AMPs effectively inhibit reaspiration and NADH dehydrogenases. 40
• Figure 8 | AMPs kill cells throgh induce ROS accumulation. 42
SUPPLEMENTARY TABLE 43
• Supplementary Table 1 | The MIC of P-113 series AMPs against clinical isolates 43
SUPPLEMENTARY RESULTS 44
• Supplementary Fig. 1 44
• Supplementary Fig. 2 45
• Supplementary Fig. 3 46
• Supplementary Fig. 4 47
• Supplementary Fig. 5 48
• Supplementary Fig. 6 49

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