綠膿桿菌是臨床環境中常見的病原體之一。該病原體引起尿路，呼吸道，傷口，眼睛和系統性感染，並與多種抗生素之抗藥性相關，使治療複雜化。在對高細胞毒性、社區感染型、腹瀉型綠膿桿菌菌株的毒力進行分析時，我們注意到胺肽酶PaAP對培養的大腸直腸癌細胞表現出明顯的毒性。該酶是存在綠膿桿菌外膜囊泡中M28金屬肽酶家族的一員。到目前為止，尚未有PaAP與細胞毒性相關的報導。在這項研究中，我們探討細胞毒性B136-33菌株和標準菌株PAO1的上清液對大腸癌細胞的細胞毒性作用與其PaAP表現量的關聯。PaAP水平的升高與B13633菌株中綠膿桿菌細胞毒性的增加直接相關，而綠膿桿菌PAO1菌株的細胞毒性沒有觀察到差異。此外，為了近一步了解PaAP在細胞毒性作用的角色與貢獻，藉由大腸桿菌蛋白質表現系統建構重組PaAP蛋白，在純化後並用於測試其對哺乳動物細胞的影響。本論文發現單獨的重組PaAP不能誘導細胞死亡，也不能抑制細胞增殖。基於綠膿桿菌PAO1和純化的重組PaAP的細胞毒性測試結果均暗示PaAP非直接的細胞毒素。然而，當使用重組PaAP蛋白對細胞預先處理，發現在B13633和PAO1菌株下，綠膿桿菌的侵入與黏附的能力與PaAP的表現量成正相關，且綠膿菌素的量也與PaAP的表現量成正比。此外，發現綠膿桿菌的溶血、游動和表面移行(swarming)的能力隨著PaAP表現量的增加而增強。綜上所說，本論文發現PaAP不僅在細胞毒性中占有重要角色，也可提升綠膿桿菌的粘附性和侵入能力，甚至影響其他的毒力因子以及致病機制。

Pseudomonas aeruginosa is one of the most commonly encountered pathogens in clinical settings. The pathogen causes urinary tract, respiratory tract, wound, eye, and systematic infections and is frequently associated with multiple antibiotic resistance that complicates the treatment. During the analysis of the virulence of a highly cytotoxic, community-acquired, diarrheagenic P. aeruginosa strain, we noted that a mutant strain defective in aminopeptidase PaAP (PA2939) displayed a clear reduction in toxicity to cultured colorectal cancer cells. The enzyme is a metallo-enzyme of the M28 peptidase family that is present abundantly in the extracellular outer membrane vesicles of P. aeruginosa. So far, no report has associated PaAP with cytotoxicity. In this study, I investigated the cytotoxic effect of the culture supernatants of several derivatives of P. aeruginosa cytotoxic B136-33 and PAO1 on colorectal cancer cells in relation to their PaAP levels. The increased level of PaAP was directly related to the increased cytotoxicity of P. aeruginosa in B136-33 strain whereas little difference was observed in PAO1 strain. Further analysis using different truncated forms of PaAP produced in E. coli showed that PaAP alone could neither induce cell death nor inhibit cell proliferation. Nevertheless, the invasiveness and adhesion ability of P. aeruginosa were significantly enhanced by overexpression of PaAP in both B136-33 and PAO1. This finding was further supported by the fact that treating cultured cells with recombinant PaAP protein could significantly promote the bacterial invasion. Furthermore, higher PaAP expression seems to be proportional to higher pyocyanin production. Similarly, the hemolytic, swimming and swarming abilities of P. aeruginosa were found to be enhanced by the increased PaAP levels. In conclusion, we reported that PaAP plays an important role not only in cytotoxicity, adhesion and invasion ability of P. aeruginosa, but also influences other virulence factors and virulence associated mechanisms.

摘要 2
ABSTRACT 3
Table of Content 4
List of Tables 7
List of Figures 8
Abbreviations 10
1.1 Pseudomonas aeruginosa Prevalence 12
1.2 P. aeruginosa: Establishing and Maintaining an Infection 13
1.3 Proteases and their contribution in virulence 13
1.4 Introduction to Aminopeptidase (PaAP) 15
Materials and methods 17
1.5 Bacterial cultures and growth condition 17
1.5.1 Bacterial cultures preparation 17
1.5.2 Competent cell preparation 17
1.6 Cell culture and growth conditions 18
1.6.1 Medium preparation 18
1.6.2 Culture conditions 18
1.7 Overexpression of PaAP in P. aeruginosa 19
1.7.1 Construction of pMMB66EH-PaAP 19
1.8 Construction of recombinant PaAP variants 20
1.8.1 Construction of rPaAP 20
1.8.2 Construction of rPaAP_273-536 20
1.8.3 Construction of rPaAP_37-517 21
1.8.4 Genomic DNA extraction and purification 22
1.8.5 Primers 22
1.8.6 Polymerase Chain Reaction (PCR) 22
1.8.7 DNA purification 23
1.8.8 Isolation of plasmids 23
1.9 Purification of His-tagged rPaAP, rPaAP_273-536 and rPaAP_37-517 24
1.9.1 Solubilization of inclusion bodies 24
1.9.2 Refolding of rPaAP proteins 24
1.9.3 Purification of His-tagged protein 25
1.9.4 Activation of recombinant rPaAP 25
2.0 Enzyme activity assay 25
2.1 Cytotoxicity assay 26
2.2 Cell viability assay 26
2.3 Cell invasion assay and adherence assay 26
2.3.1 Preparation of bacteria 26
2.3.2 Preparation of cell culture 27
2.3.3 Cell invasion assay 27
2.3.4 Cell adherence assay 27
2.5 Cell proliferation inhibition assay 28
2.6 Hemolysis test 28
2.7 Swimming and swarming motility assay 28
2.8 Pyocyanin quantitation assay 29
2.9 SDS-PAGE and immunoblotting analysis 29
Results 31
3.0 Construction of a P. aeruginosa △PaAP mutant, complementation strains and recombinant PaAP 31
3.1 Recombinant PaAPs enzymatic activity and activation of rPaAP 32
3.2 Recombinant PaAP size conversion from 56 kDa to 28 kDa upon heating 33
3.3 Increased cytotoxicity of P. aeruginosa against mammalian cells is observed in overexpression PaAP strains 34
3.4 rPaAP does not have the same cytotoxicity on HCT8 cells 34
3.5 rPaAP does not inhibit HCT8 cell proliferation 35
3.6 Effects of PaAP expression on cell adhesion and cell invasion ability of P. aeruginosa 35
3.7 rPaAP_37-517 promotes P. aeruginosa adhesion and invasion of PaAP producing cells 36
3.8 Overexpression of PaAP showed increasing hemolytic activity 37
3.9 Influence of overexpression PaAP on pyocyanin production 38
3.10 Effects of PaAP overexpression in swarming and swimming ability of P. aeruginosa 38
Discussion 39
Tables 43
Figures 46

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