探討幽門螺旋桿菌GroES之羧基端對人類PBMC及KATO-Ⅲ細胞IL-8表現之影響

Abstract

幽門螺旋桿菌被證實會造成胃部之慢性發炎並且與許多胃部之疾病(例如胃炎、胃潰瘍、十二指腸潰瘍與胃癌)有很大之關聯，至目前為止雖然有很多致病因子被研究出來，但是幽門螺旋桿菌的致病機制至今還尚未完全釐清。 在實驗室先前的研究中，發現了幽門螺旋桿菌新的致病因子GroES，而且GroES對胃癌病人血清有很高的抗原性，同時也會使得周邊血液單核球細胞釋放前發炎細胞激素，並對胃上皮細胞產生免疫反應及細胞增生之現象，而此造成之現象可能與胃癌的形成有關。GroES是一個熱休克蛋白，並且在細胞中與GroEL形成一個co-chaperone。經由文獻搜尋後得知，Escherichia coli或是其他微生物之 GroES並不會造成細胞之免疫反應，而幽門螺旋桿菌之GroES所造成細胞之免疫反應是否與其蛋白質序列有關，因此，我們分析了不同物種之GroES的蛋白質序列，發現幽門螺旋桿菌之GroES在羧基端有28個胺基酸片段延伸，而這片段是在其他物種中從未發現。 因此，我們想進一步了解此胺基酸片端是否會造成細胞產生免疫反應，所以我們將全長GroES中的後28個胺基酸片段刪除 (deletion)，並藉由分析周邊血液單核球細胞以及胃上皮細胞所釋放之IL-8，發現這樣的刪除會使得原本GroES的喪失誘導細胞釋放IL-8之能力，並且使得GroES無法與胃上皮細胞表面進行結合。再進一步的分析後，我們發現這一段28個胺基酸之片段，會引發周邊血液單核球細胞之IL-8的釋放，似乎這一段胺基酸片段是GroES活性的重要來源，並進一步造成宿主細胞之免疫反應。

Helicobacter pylori (H. pylori) which can cause chronic inflammation is now emerging as a risk factor in the development of chronic gastritis, duodenal ulcer, gastric ulcer, and gastric cancer (1-2%). Although many virulence factors of H. pylori have been reported, but the pathogenic mechanism of H. pylori still remains unclear. In the previous study, we have found a novel GC-associated virulence factor which is GroES of H. pylori and this protein may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation. GroES, a heat shock protein family is well known as a co-chaperone with GroEL.

In the previous study of Escherichia coli GroES, it showed significant activity only on the expression and release of IL-6 in keratinocytes and E-selectin in monocytes and endothelial cells. So It sound like that there is no immunogencity in Escherichia coli GroES-treat cells. H. pylori GroES contains 118 amino acids. After comparing the amino acid sequences, the difference among GroES of different species was that there are extra 28-amino-acid in the carboxyl terminus of Helicobacter pylori GroES. This region was not present in any of the other bacterial GroES homologues characterized. We were interested in the contribution of the 28-amino-acid carboxyl terminus of H. pylori GroES in the effect of inflammation. We deleted the 28-amino-acid carboxyl terminus of H. pylori GroES and purified the protein that contains 1-90 amino acids of H. pylori GroES〈△rGroES(1-90)〉. We investigated the IL-8 expression in △rGroES(1-90)-treated PBMC (peripheral blood mononuclear cell) and gastric epithelial cell (KATO-Ⅲ). By comparing with wild type H. pylori and E. coli GroES, we found that deletion of C-terminal amino acid (91-118) of H. pylori GroES reduce IL-8 expression in both cells. It can be observed that △rGroES(1-90) doesn’t bind to surface of KATO-Ⅲ cell but H. Pylori rGroES(1-118) does.

The synthesized peptide of H. pylori C-terminal amino acid (91-118) can induce IL-8 expression in PBMC. This peptide may contribute to the activity of H. pylori GroES whereas △rGroES(1-90) has no effect on IL-8 expression. Therefore, GroES seems to be involved in the inflammatory response of H. Pylori infection through its carboxyl terminus of 28-amino-acid.