白色念珠菌是人類最常見的真菌病原，存在於絕大多數人類的正常菌叢。在免疫力低下像是化療、器官移植、愛滋等病人可造成感染，其中包含表淺型與具有高致死率的念珠菌血症。抗菌胜肽(例如: defensins、cathelicidinsm與histatins)在人體先天性免疫系統中，扮演對抗病原菌入侵之重要角色。LL-37是目前在人體內唯一找到屬於cathelicidin抗菌蛋白家族的抗菌胜肽。在我們實驗室先前的研究中顯示低濃度的LL-37會改變白色念珠菌的細胞壁組成，並影響其貼附至聚苯乙烯塑膠材質、口腔表皮細胞OECM-1及BALB/c 小鼠的膀胱的能力。而在本研究中，則在探討高濃度LL-37對白色念珠菌的殺菌機制。首先，利用觀察程序性細胞凋亡(programmed cell death；PCD)的標示性特徵證明LL-37不會使白色念珠菌走向PCD。再者，先前的研究中多數指出LL-37擁有干擾細胞膜的能力，推論LL-37可能在細胞中造成脂質毒性(lipotoxicity)，近期的研究也顯示轉錄因子Rim101的相關途徑與脂質毒性有關。於是利用剃除Rim101相關基因(其中包含rim9、rim13、rim101) 的突變株，我們發現LL-37會引起在白色念珠菌的脂質毒性並促使其走向調節性細胞凋亡(Regulated cell death)。

Candida albicans is the most prevalent fungal pathogen in human. In immunocompromised patients, C. albicans causes a range of infections including superficial disease and systemic bloodstream infection with a high mortality. Antimicrobial peptides (AMPs) such as defensins, cathelicidins, and histatins play an important role in the innate immunity to against microbial invasion. LL-37 is the only member of the human cathelicidin family of AMPs and is commonly expressed in epithelial cells of the oral cavity and urogenital tract. In our previous studies, sub-lethal doses of LL-37 inhibit C. albicans adhesion to polystyrene surface, oral epidermoid OECM-1 cells and urinary bladders of BALB/c mice. In this study, we further investigate the killing mechanisms of high doses of LL-37 against C. albicans. We found that LL-37 induces reactive oxygen species (ROS) production, caspase activation, and DNA fragmentation, suggesting the occurrence of programmed cell death (PCD). However, because treating with ROS scavenger and caspase inhibitor fail to rescue C. albicans, we further discriminated cell death induced by LL-37 from regulated cell death. Moreover, the C. albicans RIM13 and RIM101 gene encodes a protease and a transcription factor, respectively. The Rim101-dependent pathway is related to lipotoxicity in yeast. Interestingly, we found that C. albicans rim9-, rim13- and rim101-deleted mutants are resistant to LL-37. These results suggested that the Rim101-dependent pathway and lipotoxicity is related to C. albicans PCD induced by LL-37. Together, this study provides new insights into cadidacial activity of the human AMP LL-37.

A. 中文摘要-I
B. Abstract-II
C. 致謝-I
D. CONTENTS-IV
E. INTRODUCTION-1
F. MATERIALS & METHODS-8
G. RESULTS-15
H. DISCUSSION-23
I. REFERENCES-28
J. TABLES & FIGURES-40

1. Matthew B. Lohse, et al. Development and regulation of single- and multi-species Candida albicans biofilms. Nat Rev Microbiol 16(1), 19-31 (2018).
2. Achkar J. M. & Fries B. C. Candida infections of the genitourinary tract. Clin Microbiol Rev 23, 253–273 (2010).
3. Ganguly S. & Mitchell A. P. Mucosal biofilms of Candida albicans. Curr Opin Microbiol 14, 380–385 (2011).
4. Kumamoto C. A. Inflammation and gastrointestinal Candida colonization. Curr Opin Microbiol 14, 386–391 (2011).
5. Brown G. D., et al. Hidden killers: human fungal infections. Sci Transl Med 4 (165), 165rv13 (2012).
6. Grosset M., et al. Recurrent episodes of Candidemia due to Candida glabrata, Candida tropicalis and Candida albicans with acquired echinocandin resistance. Med Mycol Case Rep 14, 20-23 (2016)
7. Guinea J., Global trends in the distribution of Candida species causing candidemia. Clin Microbiol and Infect 20 (Suppl 6), 5-10 (2014).
8. Fekkar A., et al. Emergence of echinocandin-resistant Candida spp. in a hospital setting: a consequence of 10 years of increasing use of antifungal therapy? Eur J Clin Microbiol Infect Dis 33 (9), 1489-1496 (2014).
9. Matsumoto E., et al. Candidemia surveillance in Iowa: emergence of echinocandin resistance. Diagn Microbiol Infect Dis. 79 (2), 205-208 (2014).
10. Pfaller M. A., et al. Clinical breakpoints for the echinocandins and Candida revisited: integration of molecular, clinical, and microbiological data to arrive at species-specific interpretive criteria. Drug Resist Updat. 14 (3), 164-176 (2011).
11. Fisher M. C., et al. Worldwide emergence of resistance to antifungal drugs challenges human health and food security. Science 360 (6390), 739-742 (2018).
12. Fisher M. C., et al. Emerging fungal threats to animal, plant and ecosystem health. Nature 484(7393), 186-94 (2012).
13. Mahlapuu M., et al. Antimicrobial Peptides: An Emerging Category of Therapeutic Agents. Front Cell Infect Microbiol 6, 194 (2016).
14. Fjell C. D., et al. Designing antimicrobial peptides: form follows function. Nat Rev Drug Discov 11 (1), 37-51 (2011).
15. Lai Y., Gallo R. L. AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol 30 (3), 131-41 (2009).
16. Gordon Y. J., et al. A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs. Curr Eye Res 30 (7), 505-515 (2005).
17. Yeaman M. R. & Yount N. Y. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55 (1), 27-55 (2003).
18. Bradshaw J. Cationic antimicrobial peptides : issues for potential clinical use. BioDrugs 17 (4), 233-40 (2003).
19. Koczulla A. R. & Bals R. Antimicrobial peptides: current status and therapeutic potential. Drugs 63 (4), 389-406 (2003).
20. Fox J. L. Antimicrobial peptides stage a comeback. Nat Biotechnol 31, 379–382 (2013).
21. Lázár V., et al. Antibiotic-resistant bacteria show widespread collateral sensitivity to antimicrobial peptides. Nat Microbiol 3 (6),718-731 (2018).
22. Dürr U.H., et al. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim Biophys Acta 1758 (9), 1408-25 (2006).
23. Jeffrey T., et al. Activities of LL-37, a Cathelin-Associated Antimicrobial Peptide of Human Neutrophils. Antimicrob Agents Chemother 42 (9), 2206–2214 (1998).
24. Nijnik A., et al. The roles of cathelicidin LL-37 in immune defences and novel clinical applications. Curr Opin Hematol 16 (1), 41-47 (2009).
25. Tsai P. W., et al. Characterizing the role of cell-wall β-1,3-exoglucanase Xog1p in Candida albicans adhesion by the human antimicrobial peptide LL-37. PLoS One 6 (6), e21394 (2011).
26. Tsai P. W., et al. Human antimicrobial peptide LL-37 inhibits adhesion of Candida albicans by interacting with yeast cell-wall carbohydrates. PLoS One 6 (3), e17755 (2011).
27. Den Hertog A.L., et al. Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane. Biochem J 388 (Pt 2), 689-95 (2005).
28. Kornelius Zeth and Enea Sancho-Vaello. The Human Antimicrobial Peptides Dermcidin and LL-37 Show Novel Distinct Pathways in Membrane Interactions. Front Chem 5 (86), (2017).
29. Galluzzi L., et al. Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ 22 (1), 58-73 (2015).
30. Büttner S., et al. Why yeast cells can undergo apoptosis: death in times of peace, love, and war. J Cell Biol 175 (4), 521-525 (2006).
31. Petranovic D., et al. Prospects of yeast systems biology for human health: integrating lipid, protein and energy metabolism. FEMS Yeast Res 10 (8), 1046-1059 (2010).
32. Carmona-Gutierrez D, et al. Guidelines and recommendations on yeast cell death nomenclature. Microb Cell 5 (1), 4-31 (2018).
33. Chen Y. J. Study of Molecular Mechanisms of Candida albicans Response to Human Antimicrobial Peptide LL-37. Master Thesis of National Tsing Hua University (2009).
34. Cui W., et al. Free Fatty Acid Induces Endoplasmic Reticulum Stress and Apoptosis of β-cells by Ca2+/Calpain-2 Pathways. PLoS One 8 (3), e59921 (2013).
35. M. Li and S. J. Martin, et al. Candida albicans Rim13p, a Protease Required for Rim101p Processing at Acidic and Alkaline pHs. Eukaryot Cell 3 (3), 741–751 (2004).
36. Schaffer J. E. Lipotoxicity: when tissues overeat. Curr Opin Lipidol 14 (3), 281-287 (2003).
37. Unger R. H., et al. Lipid homeostasis, lipotoxicity and the metabolic syndrome. Biochim Biophys Acta 1801 (3), 209-214 (2010).
38. Rockenfeller P., et al. Diacylglycerol triggers Rim101 pathway-dependent necrosis in yeast: a model for lipotoxicity. Cell Death Differ 25 (4), 765-781 (2018).
39. Gillum A. M., et al. Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198 (2), 179-182 (1984).
40. S. M. Noble, et al. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat Genet 42 (7), 590–598 (2010).
41. Lee S. Y., et al. The Transcription Factor Sfp1 Regulates the Oxidative Stress Response in Candida albicans. Microorganisms 7 (5), (2019).
42. Poljsak B., Pro-oxidative vs antioxidative properties of ascorbic acid in chromium(VI)-induced damage: an in vivo and in vitro approach. J Appl Toxicol 25 (6), 535-548 (2005).
43. María T. A., et al. Human Lactoferrin Induces Apoptosis-Like Cell Death in Candida albicans: Critical Role of K+-Channel-Mediated K+ Efflux. Antimicrob Agents Chemother 52 (11), 4081–4088 (2008).
44. Shuyuan L., et al. Components of the Calcium-Calcineurin Signaling Pathway in Fungal Cells and Their Potential as Antifungal Targets. Eukaryot Cell 14 (4), 324–334 (2015).
45. K. De Cremer, et al. Stimulation of superoxide production increases fungicidal action of miconazole against Candida albicans biofilms. Sci Rep 6, 27463 (2016).
46. Kwon Y. Y., et al. Caloric Restriction-Induced Extension of Chronological Lifespan Requires Intact Respiration in Budding Yeast. Mol Cells 40 (4), 307–313 (2017).
47. Wang H. I, et al. Resveratrol Modulates Mitochondria Dynamics in Replicative Senescent Yeast Cells. PLoS One 9 (8), e104345 (2014).
48. B. Hao, et al. Caspofungin Kills Candida albicans by Causing both Cellular Apoptosis and Necrosis. Antimicrob Agents Chemother 57 (1), 326–332 (2013).
49. Wu X. Z., et al. Plagiochin E, an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway. Biochim Biophys Acta 1800 (4), 439-447 (2010).
50. Giannattasio S., et al. Cytochrome c is released from coupled mitochondria of yeast en route to acetic acid-induced programmed cell death and can work as an electron donor and a ROS scavenger. FEBS Lett 582 (10), 1519-1525 (2008).
51. Lee J., et al. Melittin triggers apoptosis in Candida albicans through the reactive oxygen species-mediated mitochondria/caspase-dependent pathway. FEMS Microbiol Lett 355 (1), 36-42 (2014).
52. Du H., N-Acetylglucosamine-Induced Cell Death in Candida albicans and Its Implications for Adaptive Mechanisms of Nutrient Sensing in Yeasts. MBio 6 (5), e01376-15 (2015).
53. Rothstein D. M., et al. Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5. Antimicrob Agents Chemother 45 (5), 1367-1373 (2001).
54. Schaller-Bals S., et al. Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection. Am J Respir Crit Care Med 165 (7), 992-995 (2002).
55. S. G. Lapierre, et al. Cystic fibrosis respiratory tract salt concentration: An Exploratory Cohort Study. Medicine (Baltimore) 96 (47), e8423 (2017).
56. Crowley L. C., Scott A.P., and Marfell B.J., et al. Measuring Cell Death by Propidium Iodide Uptake and Flow Cytometry. Cold Spring Harb Protoc, (2016).
57. Fang F. C. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat Rev Microbiol 2 (10), 820-832 (2004).
58. Leadsham J. E., et al. Loss of cytochrome c oxidase promotes RAS-dependent ROS production from the ER resident NADPH oxidase, Yno1p, in yeast. Cell Metab 18 (2), 279-86 (2013).
59. X. Li, et al. Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases and cancers. J Hematol Oncol 6 (19), (2013).
60. Kujoth G. C., et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science 309 (5733), 481-484 (2005).
61. L. D. Zorova, et al. Mitochondrial membrane potential. Anal Biochem 552, 50–59 (2018).
62. Plenchette S., et al. Analyzing markers of apoptosis in vitro. Methods Mol Biol 281, 313-331 (2004).
63. M. Brini, et al. Calcium in Health and Disease. Interrelations between Essential Metal Ions and Human Diseases, 81-137 (2013).
64. Nicotera P., et al. The role of calcium in apoptosis. Cell Calcium 23 (2-3), 173-180 (1998).
65. Bravo-Sagua R., et al. Calcium Transport and Signaling in Mitochondria. Compr Physiol 7(2), 623-634 (2017).
66. Rizzuto R., et al. Flirting in little space: the ER/mitochondria Ca2+ liaison. Sci STKE 215, re1 (2004).
67. Garrido C., et al. Mechanisms of cytochrome c release from mitochondria. Cell Death Differ 13 (9), 1423-1433 (2006).
68. Mazzoni C. and Falcone C. Caspase-dependent apoptosis in yeast. Biochim Biophys Acta 1783 (7), 1320-1327 (2008).
69. Madeo F., et al. A caspase-related protease regulates apoptosis in yeast. Mol Cell 9 (4), 911-917 (2002).
70. Wu X. Z., et al. Plagiochin E, an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway. Biochim Biophys Acta 1800 (4), 439-447 (2010).
71. Guaragnella N, et al. Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1. FEBS Lett 584 (1), 224-228 (2010).
72. Y. R. Saadat and N. Saeidi, et al. An update to DNA ladder assay for apoptosis detection. Bioimpacts 5 (1), 25–28 (2015).
73. Bortner C. D., et al. The role of DNA fragmentation in apoptosis. Trends Cell Biol 5 (1), 21-26 (1995).
74. R. V. Rao, et al. Misfolded proteins, endoplasmic reticulum stress and neurodegeneration. Curr Opin Cell Biol 16 (6), 653–662 (2004).
75. Kaufman R. J. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev 13 (10), 1211-1233 (1999).
76. Henzler-Wildman K. A., et al. Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37. Biochemistry 43 (26), 8459-8469 (2004).
77. Brogden K. A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3 (3), 238-250 (2005).
78. Senyürek I., et al. Dermcidin-derived peptides show a different mode of action than the cathelicidin LL-37 against Staphylococcus aureus. Antimicrob Agents Chemother 53 (6), 2499-2509 (2009).
79. Yeaman M. R., et al. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55 (1), 27-55 (2003).
80. den Hertog A. L., et al. The human cathelicidin peptide LL-37 and truncated variants induce segregation of lipids and proteins in the plasma membrane of Candida albicans. Biol Chem 387 (10-11), 1495-1502 (2006).
81. Xavier P-S. The Medical Risks of Obesity. Postgrad Med 121 (6), 21–33 (2009).
82. Davis D., et al. Candida albicans RIM101 pH response pathway is required for host-pathogen interactions. Infect Immun 68 (10), 5953-5959 (2000).
83. Albert S-C. Coordinate responses to alkaline pH stress in budding yeast. Microb Cell 2 (6), 182–196 (2015).
84. Nishino K, et al. The C-terminal cytosolic region of Rim21 senses alterations in plasma membrane lipid composition: insights into sensing mechanisms for plasma membrane lipid asymmetry. J Biol Chem. 290 (52), 30797–30805 (2015).
85. Åse B., et al. The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes. Antimicrob Agents Chemother. 2009 Mar; 53(3): 1027–1038.
86. Han J., et al. The role of ER stress in lipid metabolism and lipotoxicity. J Lipid Res 57 (8), 1329-1338 (2016).
87. Fagone P., et al. Membrane phospholipid synthesis and endoplasmic reticulum function. J Lipid Res 50 (Suppl), S311-S316 (2009).
88. Rockenfeller P., et al. Lipotoxicty in yeast: a focus on plasma membrane signalling and membrane contact sites. FEMS Yeast Res 18 (4), (2018).
89. Aerts A. M., et al. The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans. FEBS Lett 583 (15), 2513-2516 (2009).
90. Sinha K., et al. Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis. Arch Toxicol 87 (7), 1157-1180 (2013).
91. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol 35 (4), 495–516 (2007).
92. Nakagawa T., et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403 (6765), 98-103 (2000).
93. Madeo F., et al. Caspase-dependent and caspase-independent cell death pathways in yeast. Biochem Biophys Res Commun 382 (2), 227-231 (2009).
94. Hauptmann P., et al. Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging. J Biol Chem 283 (27), 19151-19163 (2008).
95. Silke W., et al. An AIF orthologue regulates apoptosis in yeast. J Cell Biol 166 (7), 969–974 (2004).
96. Palermo V., et al. p53 death signal is mainly mediated by Nuc1(EndoG) in the yeast Saccharomyces cerevisiae. FEMS Yeast Res 13 (7), 682-688 (2013).
97. Büttner S., et al. Endonuclease G regulates budding yeast life and death. Mol Cell 25 (2), 233-46 (2007).