鮑氏不動桿菌是一種格蘭氏陰性的球桿菌，且為人類伺機感染病菌。在正常人體中共生存在，但在免疫能力低下的病人容易起而致病，嚴重則可能導致死亡。近年來全球出現許多不同的多重抗藥性鮑氏不動桿菌菌株，在臨床治療上造成了很大的困難。例如對抗鮑氏不動桿菌最後一道防線之藥物Tigecycline及Colistin，目前也已經有抗藥性菌株的出現，因此必需儘速尋找或研發新抗生素或其他有效抗菌物質，以克服鮑氏不動桿菌的感染問題。本研究中，我們發現人類抗菌胜肽LL-37能夠有效殺害鮑氏不動桿菌，並且在非致死劑量之下能抑制細菌貼附及移動能力。由鮑氏不動桿菌細胞萃取的脂多醣體(Lipopolysaccharide)能夠有效和LL-37競爭，使得細菌貼附能力得以恢復。除此之外、脂多醣體缺失的鮑氏不動桿菌變異株對LL-37有較高的抗性，推測LL-37有可能透過與脂多醣體之接合反應而造成細菌貼附能力之下降。除了脂多醣體，我們也發現LL-37會結合到鮑氏不動桿菌的外膜蛋白A (OmpA)。雖然外膜蛋白A和細菌貼附能力有很大相關，但和LL-37造成的貼附能力下降可能沒有直接的關聯。在實驗中也發現脂多醣體和外膜蛋白A的缺陷都會導致菌体對LL-37的敏感性。綜合上述結果，LL-37可能是一個具有潛力且未來能夠用於治療鮑氏不動桿菌感染的新試劑。

Acinetobacter baumannii is a Gram-negative coccobacillus and is a leading nosocomial pathogen worldwide. Recently, emergence of multidrug resistant A. baumannii has become a great threat to healthcare. Strains resist to the last line of anti-mocrobial agents, tigecycline and colistin, were appeared. Therefore, developing new drugs to treat A. baumannii infections is urgently needed. In this work, a human antimicrobial peptide LL-37 was evaluated for its effects on A. baumannii. We found that LL-37 kills A. baumannii efficiently and reduces cell adhesion and motility. Lipopolysaccharides extracted from A. baumannii cell surface can rescue LL37-mediated inhibition of cell adhesion. Moreover, far-western analysis indicated that LL-37 binds to outer membrane OmpA protein of A. baumannii (AbOmpA), but this binding seems not to correlate with the inhibitory effect on cell adhesion. However, both LPS and AbOmpA were related to sensitivity of A. baumannii to LL-37. Together, this study suggested that LL-37 may be a potential agent in the future treatment of A. baumannii infections.

Table of Contents
摘要 I
Abstract II
致謝 III
Table of Contents IV
List of Tables VII
List of Figures VIII
List of Supplemental Figure IX
1. Introduction 1
1.1 Acinetobacter baumannii: background and its significance in human health 1
1.2 Antimicrobial peptides 2
1.3 LL-37: an important human AMP 3
1.4 Drug resistance of A. baumannii 4
1.4.1 Decreasing cell permeability 4
1.4.2 Enzyme inactivation 4
1.4.3 Alteration of drug target sites 5
1.4.4 Efflux pumps 5
1.5 Virulence factors of A. baumannii 5
1.5.1 Surface hydrophobicity, acinetobactin and phospholipase D (PLD) 6
1.5.2 Penicillin-binding protein 7/8 (PBP-7/8), RecA and capsule 6
1.5.3 A. baumannii adhesion 7
1.5.4 A. baumannii OmpA (AbOmpA) porin protein 7
1.6 Aim of this study 7
2. Material and methods 9
2.1 Peptides and reagents 9
2.2 A. baumannii strains, media, and growth conditions 9
2.3 Assays for anti-Acinetobacter activity of LL-37 10
2.4 Assay for A. baumannii adhesion 10
2.5 Assay for A. baumannii motility 11
2.6 Assay for LL-37 and A. baumannii binding 11
2.6.1 Flow cytometric analysis 11
2.6.2 Immunofluorescence staining 12
2.7 OMPs extraction and western blot 12
2.7.1 A. baumannii OMPs extraction 12
2.7.2 Far-western analysis 13
2.7.3 Western analysis 14
2.8 AbOmpA/LL-37 binding assay 14
2.8.1 BA-LL37 pull-down assay 14
2.8.2 Coomassie Blue stain 15
2.9 Identification of LL37-binding protein(s) 15
2.9.1 MOLDI-TOF 15
2.9.2 LL37-AbOmpA binding prediction 17
2.10 Preparation of lipopolysaccharides (LPS) from A. baumannii 17
2.11 ELISA assay for LL-37/OmpA164-181 and LL-37/OmpA74-84 association 18
2.12 Genomic DNA extraction 19
2.13 Construction of ompA-deleted mutant of A. baumannii 20
2.14 RNA isolation and reverse transcription -PCR 21
2.15 Expression, purification, and refolding of recombinant AbOmpA 22
2.16 Cytotoxic activity of refolded AbOmpA 23
3. Results 25
3.1 LL-37 kills A. baumannii in a dosage-dependent manner 25
3.2 LL-37 inhibits A. baumannii adhesion and motility 25
3.3 LL-37 binds to A. baumannii cells 26
3.4 The effects of LL-37 on A. baumannii are specific 27
3.5 AbOmpA is a potential binding target of LL-37 28
3.6 LL-37 associated with certain regions of AbOmpA 29
3.7 OmpA is involved in A. baumannii adhesion 30
3.8 LL37-AbOmpA interaction 31
3.9 LPS represents another binding target of LL-37 32
3.10 Results of pull-down assay and MALDI-TOF 33
4. Discussion 34
5. References 39

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