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系統識別號 U0026-1908201416350800
論文名稱(中文) 創傷弧菌MARTX毒素抗吞噬機制之研究
論文名稱(英文) Studies on the mechanism of Vibrio vulnificus MARTX-mediated antiphagocytosis in macrophages
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 102
學期 2
出版年 103
研究生(中文) 陳俊良
研究生(英文) Chun-Liang Chen
電子信箱 muzhikmuzhik@yahoo.com.tw
學號 S58941418
學位類別 博士
語文別 英文
論文頁數 74頁
口試委員 指導教授-何漣漪
召集委員-鄧景浩
口試委員-吳俊忠
口試委員-呂增宏
口試委員-馬明琪
口試委員-陳念榮
中文關鍵字 創傷弧菌  MARTX毒素  抗吞噬作用  鈣離子 
英文關鍵字 V. vulnificus  MARTX toxin  antiphagocytosis  Ca2+ 
學科別分類
中文摘要 創傷弧菌生物一型(Vibrio vulnificus biotype 1)是一株能經食物或傷口感染人體且具高度致死率的伺機性革蘭氏陰性病原菌,感染後能快速造成敗血症及出血性皮膚壞死等病變。此菌具有多種毒力因子,包括能抑制吞噬作用的MARTX(multi-functional autoprocessing repeats in toxin)細胞毒素。為了進一步了解創傷弧菌生物一型MARTX(MARTXVv1)的抗吞噬機制,我一方面比較創傷弧菌生物一型野生型菌株YJ016及其rtxA1基因剔除株HL128對小鼠巨噬細胞株RAW264.7之吞噬作用相關特性的影響,另一方面,我將MARTXVv1序列中的特定區域剔除後測試其對於MARTXVv1所導致細胞毒性及抗吞噬作用的影響。我發現MARTXVv1能在YJ016感染7.5至15分鐘內造成RAW264.7細胞中參與吞噬作用之訊息傳遞分子特定位置的磷酸化程度明顯下降,如SFKs Y418 (Src family kinases,包括Lyn、Fgr與Hck)、FAK Y861、Pyk2 Y402、Syk Y525、PI3K p85 Y458及Akt S473等,卻不影響SFKs Y529及已知為toll like receptors (TLRs)下游p38 MAP kinase T180/Y182等位置的磷酸化程度;於15-30分鐘間造成RAW264.7細胞偽足消失、型態改變及F-actin分佈異常;並且在60-90分鐘間明顯導致其細胞硬度下降且導致細胞膜上lipid rafts無法聚集。預先將RAW264.7細胞處理SFKs抑制劑PP2、FAK/Pyk2抑制劑PF431396、PI3K抑制劑LY294002、Akt1/2抑制劑或F-actin抑制劑cytochalasin D後再感染HL128,這些抑制劑都能有效抑制HL128被吞噬,但處理PP2時所造成的結果與MARTXVv1的作用最為相似,顯示MARTXVv1可能透過抑制SFKs及其下游的訊息傳遞路徑來抑制RAW264.7細胞的吞噬作用。另外,我發現MARTXVv1能使RAW264.7細胞內的游離鈣離子濃度上升;加入2.0 mM 鈣離子螯合劑EGTA能抑制MARTXVv1的作用;同時再加入5 mM CaCl2能中和EGTA的作用;預先處理細胞內鈣離子螯合劑BAPTA-AM無法抑制MARTXVv1的作用,顯示細胞外的鈣離子為MARTXVv1功能所必需。我也發現在1.5 mM EGTA的情況下,YJ016的細胞毒性下降至與HL128相似,而抗吞噬作用則不受影響,顯示MARTXVv1可能透過不同機制造成細胞毒性與抗吞噬作用。將MARTXVv1中的ERM區域(ezrin/radixin/moesin domain)及GD -rich區域 (glycine asparagine-rich domain) 剔除能使MARTXVv1所導致的細胞毒性及抗吞噬作用顯著下降至與HL128相似,顯示這兩區域對MARTXVv1功能的重要性。最後,我顯示MARTXVv1對小鼠腹腔巨噬細胞(mPEM)的作用與其對RAW264.7細胞的作用相似。綜合以上,我認為MARTXVv1能在創傷弧菌YJ016感染後與宿主的巨噬細胞作用,抑制SFKs及其下游的訊息傳遞路徑,導致F-actin分佈異常,進而造成細胞變圓及吞噬細菌的能力下降,使該菌不易被清除。
英文摘要 Biotype 1 Vibrio vulnificus is an opportunistic gram-negative pathogen that causes serious wound infections and fulminant septicemia via ingestion of contaminated seafood, with a high mortality rate in humans. The virulence of biotype 1 V. vulnificus is multifactorial, and the virulence determinants include the MARTX (multi-functional autoprocessing repeats in toxin) cytotoxin that prevents the bacterial cell from engulfment by the phagocytes. To elucidate how the MARTX of biotype 1 V. vulnificus (MARTXVv1) inhibits phagocytosis of the macrophage, I first compared the MARTXVv1-deficient mutant (HL128) with the wild-type strain (YJ016) for a variety of phagocytosis-related properties in a mouse macrophage cell line, RAW264.7. I then deleted single domains in MARTXVv1 and examined their effects on MARTXVv1-mediated cytotoxicity and antiphagocytosis. I found that after 7.5-15 min of infection, MARTXVv1 caused dephosphorylation at specific amino acids of phagocytosis-related kinases, such as SFKs Y418 (Src family kinases that include Lyn, Fgr and Hck), FAK Y861, Pyk2 Y402, PI3 kinase p85 subunit Y458 and Akt S473, but not SFks Y529 and p38-T180/Y182, the downstream effector of the toll-like receptor (TLR). MARTXVv1 caused loss of pseudopodia, cell rounding and F-actin disorganization by 15-30 min of infection. At 60-90 min of YJ016 infection the cell rigidity decreased and coalesce of lipid rafts was impaired. Pretreatment of RAW264.7 cells with PP2 (SFKs inhibitor), PF431396 (FAK/Pyk2 inhibitor), LY294002 (PI3K inhibitor), Akt1/2 inhibitor and cytochalasin D (F-actin inhibitor) inhibited the engulfment of HL128 by RAW264.7 cells. The effects of PP2 were similar to MARTXVv1, which suggested that MARTXVv1 might inhibit the phagocytosis of RAW264.7 cells by causing dephosphorylation of the SFKs-mediated signaling pathway. In addition, MARTXVv1 caused the level of intracellular Ca2+ to increase; treatment with 2.0 mM EGTA (a Ca2+ chelator) inhibited the effects of MARTXVv1, and this inhibition was abolished in the presence of 5 mM CaCl2 at the same time. In contrast, pretreatment with BAPTA-AM (an intracellular Ca2+ chelator) did not inhibit the functions of MARTXVv1, indicating that the extracellular Ca2+ level was essential for the functions of MARTXVv1. Moreover, treatment with 1.5 mM EGTA caused MARTXVv1-mediated cytotoxicity to decrease, but the antiphagocytosis activity remained, suggesting that MARTXVv1 might exert cytotoxicity and antiphagocytosis by different mechanisms. The deletion of ezrin/radixin/moesin domain (ERM) and glycine asparagine (GD)-rich domain caused decrease of the MARTXVv1-mediated cytotoxicity and antiphagocytosis to the HL128 level, which indicated that both the ERM and GD-rich domains are important for the functions of MARTXVv1. Finally, I demonstrated that the effects of MARTXVv1 on RAW264.7 cells and mouse peritoneal exudate macrophages were similar. Taken together, I propose that the interaction of released MARTXVv1 with macrophage during V. vulnificus infection may cause dephosphorylation of SFKs-dependent signaling pathway, which in turn results in F-actin disorganization and, consequently, cell rounding as well as impaired engulfment of bacteria.
論文目次 中文摘要 i
Abstract ii
Acknowledgements iii
Table of contents iv
List of tables vii
List of figures viii
Abbreviations x
Introduction 1
Vibrio vulnificus 1
RTX toxins 2
MARTX toxin in V. vulnificus 3
Phagocytosis 4
Src family kinases 5
FAK/Pyk2 6
PI3K-Akt 6
TLRs-p38 MAP kinase 7
Antiphagocytosis mechanism 8
Objectives 10
Materials and methods 11
Cell lines, bacteria, antibiotics, medium and kinase inhibitors 11
Genomic DNA isolation 11
Plasmid DNA isolation 12
Polymerase chain reaction (PCR) 13
DNA clean up and gel extraction 13
Restriction enzyme digestion 14
Dephosphorylation of 5’-ends of DNA 14
DNA ligation 15
Heat-shock transformation and competent cells preparation 15
Conjugation 16
Isolation of MARTXVv1 domain deletion mutants 16
Isolation of mouse peritoneal exudate macrophages (mPEMs) 17
Cytotoxicity assay 18
Phagocytosis assay 18
Cell rigidity assay 19
Intracellular free calcium level analysis 20
Immunoblotting 20
Immunoprecipitation 21
Immunofluorescence microscopy 22
Expression and purification of His6-tagged fusion proteins 22
Antisera preparation 24
Statistical analyses 24
Ethics statement 25
Results 26
Effects of MARTXVv1 on phosphorylation levels of SFKs, FAK, Pyk2, PI3K, Akt and p38 kinase. 26
Effects of MARTXVv1 on coalesce of lipid raft in the macrophages. 28
Effects of inhibitors of SFKs, FAK, Pyk2, PI3K, Akt and F-actin on phagocytosis of MARTXVv1-deficient mutant. 28
Effects of calcium chelator on MARTXVv1-mediated cytotoxicity and antiphagocytosis. 29
Effect of MARTXVv1 on intracellular free Ca2+ level of macrophages. 31
Modular structures of the MARTXs of V. cholerae and V. vulnificus strains 32
Involvement of various MARTXVv1 domains in cytotoxicity and antiphagocytosis. 32
Effects of mutant MARTXVv1 proteins on the phosphorylation levels of SFKs, FAK, Pyk2, PI3K, and Akt. 33
Discussion 35
Perspectives 42
References 43
Publications 73
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