白色念珠菌是人類最重要的真菌病原體之一，共生在健康人體中但是會伺機性造成局部的黏膜感染，甚至致命的全身侵入性感染，特別是在免疫功能低下的患者中。抗菌胜肽在宿主先天性免疫系統中扮演重要角色，在我們實驗室先前的研究顯示人類抗菌胜肽LL-37會和白色念珠菌的細胞壁結合並改變細胞壁完整性，進而影響白色念珠菌的貼附能力。另外，我們的初步實驗也發現白色念珠菌的轉錄因子Sfp1參與調控生物膜的形成及細胞壁完整性。依據上述結果Sfp1是否有參與白色念珠菌對於LL-37的壓力反應，並且其分子機制為何，是本研究的主要目標。首先，與野生型及SFP1基因回復型菌株相比，我們發現SFP1基因剔除菌株具有較高的細胞壁醣類含量，並且對細胞壁干擾劑具有較高的抗性。另外， 經LL-37處理後，雖然SFP1基因剔除菌株的細胞壁完整性和內質網壓力反應相關基因沒有顯著改變，然而與野生型及SFP1基因回復型菌株相比，此SFP1基因剔除菌株表現出比較低的未折疊蛋白反應（此反應已知與ER壓力相關），並且累積較低的細胞內ROS及有較高的存活率等特性。總結來說，本研究結果揭示白色念珠菌對抗菌胜肽LL-37反應的新機制，並說明了抗菌胜肽在未來發展新抗真菌藥物的潛力。

Candida albicans is a commensal in healthy individuals but can become pathogenic particularly in the immunocompromised patients. C. albicans is responsible for a wide range of infections, including superficial and life-threatening systemic infections. Antimicrobial peptides (AMPs) play an important role in the host innate immune system. Our previous studies showed that the AMP LL-37 binds to the cell wall and alters the cell wall integrity (CWI) of C. albicans. Moreover, our preliminary data indicated that the transcription factor Sfp1 involves in maintaining CWI. These results raise a possibility that Sfp1 may participate in cell response to LL-37. In this study, therefore, we aimed to determine the role of Sfp1 and to reveal the possible mechanisms in cell response to LL-37. Our results showed that the sfp1-deleted mutant had higher cell wall glycan contents and was more resistant to cell wall-disrupting agents compared to the control cells with LL-37 treatment. In addition, although CWI and endoplasmic reticulum (ER) stress-responsive genes did not show significant difference in the sfp1-deleted mutant, this mutant did exhibit a lower unfolded protein response (related to the ER stress response), intracellular ROS accumulation and higher viability with the treatment of LL-37, compared to the controls. Together, our findings reveal a novel mechanism for C. albicans response to LL-37 and demonstrate the potential use of AMPs in the future development of new antifungal agents.

中文摘要 I
Abstract II
致謝辭 III
Table of contents IV
1. Introduction 1
1.1 Candida albicans infection and its risk factors 2
1.2 Virulence factors and pathogenesis of C. albicans 3
1.3 Antimicrobial peptides and LL-37 4
1.4 Cell wall component and cell wall integrity in C. albicans 7
1.5 Endoplasmic reticulum stress, unfolded protein response and cell wall 8
1.6 Transcription factor Sfp1 in C. albicans 10
1.7 Aim of this study 11
2. Materials and Methods 12
2.1 Peptide synthesis 13
2.2 Strains and growth condition 13
2.3 Construction of ERO1-FLAG tagging strains 13
2.4 Protein extraction and quantification 14
2.5 Cell susceptibility to LL-37 15
2.6 Intracellular reactive oxygen species (ROS) accumulation 16
2.7 Lipid peroxidation assay 16
2.8 Total RNA isolation and reverse transcription (RT) real-time quantitative PCR (qPCR) 16
2.9 Cell susceptibility to cell wall-perturbing agents 17
2.10 Determination of the carbohydrate content of the cell wall 18
2.11 Assay for Mkc1 phosphorylation 19
2.12 Protein secretion assay 20
2.13 HAC1 mRNA splicing assay 20
2.14 Detection of the Ero1 oxidation state 21
2.15 Statistical analysis 21
3. Results 22
3.1 Sfp1 is involved in cell susceptibility to LL-37 23
3.2 Deletion of SFP1 alters cell wall composition and affects cell tolerance to LL-37 23
3.3 Deletion of SFP1 enhances clearance of LL-37-induced reactive oxygen species (ROS) 25
3.4 LL-37-induced endoplasmic reticulum (ER) dysfunction 27
3.5 Activation of the unfolded protein response (UPR) pathway by LL-37 28
3.6 LL-37 impairs ER redox homeostasis 29
3.7 LL-37 and the calcineurin inhibitor FK506 have a combinatorial effect against C. albicans 30
4. Discussion 32
5. References 37
Tables 49
Table 1. Strains used in this study 49
Table 2. Oligonucleotides used in this study 50
Table 3. MICs of LL-37 against C. albicans 51
Figures 52
Figure 1. The sfp1-deleted mutant has a higher tolerance to LL-37 comparing to the wild-type and the SFP1-reintegrated strains 52
Figure 2. The sfp1-deleted mutant with LL-37 treatment is more tolerant to cell wall-disrupting agents than the control strains 53
Figure 3. The levels of individual cell wall carbohydrate in cells treated with or without LL-37. Cells were treated with or without LL-37 (8 μg/ml) for 30 min, and cell wall glucan (A), mannan (B) and chitin (C) were measured using a high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The results are expressed as the mean ± standard deviation of four independent assays. *, p<0.05. 54
Figure 4. The level of Mkc1 phosphorylation is detected in cells treated with or without LL-37 55
Figure 5. Detection of intracellular ROS accumulation and lipid peroxidation in various strains treated with or without LL-37 56
Figure 6. Investigation of ER functions possibly affected by LL-37 and deletion of SFP1 58
Figure 7. LL-37 treatment induces splicing of the transcription factor gene HAC1 mRNA 59
Figure 8. LL-37 induces more UPR activation in the wild-type than the sfp1-deleted mutant 60
Figure 9. LL-37 enhances more Ero1 oxidation in the wild-type than the sfp1-deleted mutant 61
Figure 10. LL-37 and FK506 have a combinatorial effect against C. albicans 62
Figure 11. A simple model for C. albicans to maintain CWI & response to LL-37 63
Supplement Figures 64
Figure S1. The wild-type and the SFP1-reintegrated strains show an increased chitin content of the cell wall after LL-37 treatment 64
Figure S2. The sfp1-deleted mutant exhibits a higher expression changes for the antioxidant genes in cells treated with LL-37 65

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