福氏志賀菌 (Shigella flexneri)侵入人類的腸道細胞，引發急性發炎反應，導致腹瀉、腹痛和血便等。福氏志賀菌帶有一毒性質體，含許多致病基因，其中ipaB基因負責產生Invasion plasmid antigen B (IpaB) 蛋白，當S. flexneri進入人體腸道時，M細胞及其下之巨噬細胞會先吞噬S. flexneri，但S. flexneri分泌之 IpaB蛋白可使菌從phagosome逃脫，並活化巨噬細胞的死亡徑路，使巨噬細胞死亡。本研究探討福氏志賀菌與其分泌的IpaB蛋白對人類直腸癌HCT116細胞株的影響。 首先將福氏志賀菌野生株SH2308與ipaB缺失株SHB2308，分別與HCT116細胞共同培養2與4小時，以原位免疫抗體染色後以共軛焦顯微鏡觀察，發現SH2308會進入細胞質與細胞核，SHB2308只在細胞膜外。IpaB蛋白的C 端具有一段疏水區，接著將可表現出EGFP與IpaB融合蛋白，及EGFP與IpaB去除疏水區的IpaB803蛋白的融合蛋白的表現載體，轉殖到HCT116中，培養24小時後進行原位免疫抗體染色及共軛焦顯微鏡觀察。發現IpaB-EGFP與IpaB803-EGFP融合蛋白會在HCT116的細胞質，或同時在細胞質與細胞核中表現。培養盤的上清液中，有表現IpaB-EGFP與IpaB803-EGFP融合蛋白的細胞百分比分別為10% 與53%，而培養盤的貼附細胞中，有表現IpaB-EGFP與IpaB803-EGFP融合蛋白的細胞百分比分別為少於1% 與38%，顯示IpaB及IpaB803會使細胞失去貼附性。最後，將可表現FLAG-IpaB和FLAG-IpaB803融合蛋白的表現質體轉染到HCT116後，感染 SHB2308或大腸桿菌，發現轉染細胞的細胞核與細胞質中皆可偵測到SHB2308或大腸桿菌。 Shigella flexneriinfect epithelial cells of human gut, causing acute inflammation response with abdomen pain, diarrhea and bloody stool. The bacteria carry a virulence plasmid containing many virulence genes including ipaB, which encodes invasion plasmid antigen B (IpaB). When S. flexneri enter human gut, M cells and the underlying macrophages engulf the bacteria to form phagosomes. The bacteria secrete IpaB inside phagosome, helping the bacteria to escape from the phagosome and initiating the death of macrophage. The purpose of this study was to investigate the effects of IpaB on humancolorectal carcinoma cell line HCT116.
An S. flexneri Taiwan strain SH2308 and its ipaB knock-out mutant strainSHB2308 were incubated with HCT116 cells for 2 and 4 hours, and in situ fluorescence immunological staining was carried out. Upon observation under a confocal microscope, it was found that SH2308 entered cytoplasm and nucleus of the cells, but SHB2308 did not. The C terminus of IpaB contains several hydrophobic domains. The expression plasmid that could express the fusion protein of EGFP (enhanced green fluorescence protein) and either IpaB or its truncated version IpaB803, which does not carry the C-terminal hydrophobic domains of IpaB, was transfected into HCT116 cells. The cells were incubated for 24 hours and in situ fluorescence immunological staining was carried out. Both the IpaB-EGFP and IpaB803-EGFP fusion proteins expressed well either in cytoplasm or in both cytoplasm and nucleus of the HCT116 cells. Percentages of the fluorescent cells that were either suspended in medium or adherent to the bottom of the cultured dishes were checked. About 10% and 53% of the suspended cells expressed IpaB-EGFP and IpaB803-EGFP, respectively, and less than 1% and 38% of theadherent cells expressed IpaB-EGFP and IpaB803-EGFP, respectively. This result indicates that IpaB-EGFP and IpaB803-EGFP cause cells to lose the adherence character. The expression plasmid that could express either the FLAG-IpaB or FLAG-IpaB803 fusion protein was transfected into HCT116 cells, followed by co-cultured with SHB2308 or E. col. In situ fluorescence immunological staining showed that both bacteria could successfully enter the cytoplasm and nucleus of the HCT116 cells