利用定點突變分析豬霍亂沙門氏桿菌細胞毒素flagellin的毒性可能區域

Abstract

P56是從Salmonella choleraesuis CH12440培養上清液纯化出來，分子量為56kDa的蛋白質，以此蛋白質處理小鼠巨噬細胞(mouse marcophage)，會導致細胞變性及壞死，故P56可視為一種細胞毒素(cytotoxin)。經由胺基酸序列比對發現P56為構成細菌鞭毛的次單位-flagellin，且將P56基因命名為sal15，但現今仍未有文獻報導flagellin具有細胞毒性。與其他S. choleraesuis菌株之flagellin DNA序列 (Genbank number X03394 )做比較，發現S. choleraesuis CH12440之flagellin基因多了36 bp的序列，而初步研究發現如將此段序列刪除則其毒性消失。本論文的主題是對這一區域做更進一步的探討。首先，將可能毒性區域的36 bp DNA序列插入不具毒性的E. coli之 fliC基因之中，測驗是否能賦予E. coli的FliC毒性，結果發現無論DNA序列是否多出了36個bp，FliC都具有毒殺小鼠巨噬細胞株RAW264.7及P388D1的能力。另外，將Sal15此區域中的胺基酸序列K 247、A 252、D 253利用PCR進行定點突變改變為T 247、T 252、G 253，但是此三個重組蛋白質的毒性均無顯著的下降，故此三個胺基酸與Sal15的毒性無明顯的關係。另一方面，探討Sal15是否是導致巨噬細胞的凋亡(apoptosis)，目前已知當細胞凋亡時PARP-1 [poly(ADP-ribose) polymerase-1]會被分解、抑制，故利用PARP-1作為細胞凋亡標記，進行西方墨點法，但結果得知PARP-1並沒有被分解、抑制，故推測Sal15導致巨噬細胞的死亡並非經由凋亡的途徑。

P56, a protein with a molecular mass of 56 kDa was isolated form the culture supernatant of Salmonella choleraesuis CH12440. It caused the degeneration and necrosis of mouse macrophage cells, and was considered as a cytotoxin. It was demonstrated to be a flagellin, a filament subunit of bacterial flagellum. The gene encoding P56 was cloned, named sal15, and discovered that it had extra region of 36 nucleotide absent on other flagellin genes. The primary data showed that P56 without the extra region had a decreased cytotoxicity on RAW264.7 cell. In this study, the role of the extra region was further addressed. Firstly, the 36 nucleotide region was inserted into the corresponding site of Escherichia coli flagellin gene. Next, several site-directed mutants within this region of sal15 including K247T, A252T, and D253G were constructed. MTT analysis showed that Escherichia coli flagellin gene with or without the extra region had toxicity to RAW264.7 and P388D1 cells. The three site-directedly mutated amino acid had no effect on the cytotoxicity of P56. In this study the mechanisms of cell death was also examined. The results suggested that the death of cells was caused by necrosis or apoptotic pathways other than PARP-1.