Analysis of a putative toxin region in cytotoxin flagellin from Salmonella choleraesuis by site-directed mutagenesis
Date Issued
2005
Date
2005
Author(s)
Huang, Shin-Tsung
DOI
zh-TW
Abstract
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.
Subjects
豬霍亂沙門氏桿菌
鞭毛
細胞毒素
cytotoxin
Salmonella choleraesuis
flagellin
SDGs
Type
other
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