胃幽門桿菌為一螺旋狀的微耗氧革蘭氏陰性菌，主要寄居在胃部黏膜層的上皮細胞，是引起胃癌的危險因子。脂多醣 (lipopolysaccharide) 被認為是與胃幽門桿菌粘附，和在受感染的宿主上具有調節免疫反應的能力。此革蘭氏陰性菌在各個成長階段中，連續地釋放出外膜囊泡(outer membrane vesicles)，且在囊泡構造上含有脂多醣，其潛在作用與細菌的生存和致病機制有極大相關性。
先前，我們實驗室發現發現 HP0859 基因可轉譯成 ADP-L-glycero-D- manno-heptose-6-epimerase，為參與 ADP-L-D-heptose 生合成途徑所需酵素之一。在本篇研究報告中，我們進一步發現 HP0859 基因缺失的突變菌株不僅表現出了被截短的脂多糖結構，而且其外膜囊泡的產量大幅降低。令人驚訝地，在胃幽門桿菌中主要的兩種毒素蛋白蛋白，cytotoxin associated gene A protein (CagA) 和 vacuolating cytotoxin A protein (VacA)，也也同時被發現存在胃幽門桿菌 26695 正常野生菌株所產生的外膜囊泡中。但是在 HP0859 基因缺失的突變菌株所產生的外膜囊泡中，此兩種毒素蛋白顯著性地減少。為了進一步探討脂多醣結構的改變在外膜囊泡的形成及對 CagA/VacA 功能上的影響， vacA 基因缺失的突變菌株、 cagA 基因缺失的突變菌株和 vacA/cagA 雙基因缺失的突變菌株也一同被建構完成。並且從建構的突變菌株群萃取其外膜囊泡後，與胃腺癌細胞 (AGS cells) 共同培養，進一步探討感染後的現象。胃腺癌細胞在與胃幽門桿菌正常野生菌株所產生的外膜囊泡共同培養後，胃腺癌細胞呈典型的蜂鳥狀形態及細胞空泡形成。此外，當胃腺癌細胞被 cagA 基因缺失的突變菌株所產生的外膜囊泡感染後，胃腺癌細胞顯著減少了蜂鳥狀的細胞外型。當胃腺癌細胞與 vacA 基因缺失的突變菌株所產生的外膜囊泡共同培養後，胃腺癌細胞的細胞空泡形成的現象大量減少了。當胃腺癌細胞與 HP0859 基因缺失的突變菌株所產生的外膜囊泡共同培養後，細胞型狀並沒有顯著性的改變。當胃腺癌細胞與基因缺失的突變株群的外膜囊泡共同培養後，我們也進行了總活性氧化物 (total reactive oxygen species)、粒線體活性氧化物 (mitochondrial reactive oxygen species) 及粒線體細胞膜電位 (mitochondrial membrane potential)的分析實驗。我們發現在胃幽門桿菌所產生的外膜囊泡上致病因子可能存在著拮抗作用，可能為協助細菌能夠更長時間的感染宿主，使宿主不會因單一毒素過度傷害而提早死亡。

Helicobacter pylori, a Gram-negative, spiral-shaped, microaerophilic bacterium that can inhabit various areas of human stomach and duodenum, has been considered as a risk for inducing human gastric cancer. Lipopolysaccharide (LPS) is thought to be associated with H. pylori adhesion and has the capability of modulating the immune response of infected hosts. This bacterium continuously sheds outer membrane vesicles (OMVs) during different phases of growth and these vesicles containing LPS have been suggested to have potential roles in bacterial survival and pathogenesis.
Previously, our laboratory has demonstrated that the hp0859 gene encodes an ADP-L-glycero-D-manno-heptose-6-epimerase, which is involved in the synthesis of ADP-L-D-heptose for the assembly of LPS inner core in H. pylori. In this study, we first demonstrated that the HP0859 knockout mutant (WTΔ0859) not only exhibited a truncated LPS structure but also decreased its OMV production level. Surprisingly, two major H. pylori toxins, cytotoxin associated gene A (CagA) and vacuolating cytotoxin A (VacA), were both present in OMVs derived from H. pylori 26695 wild type strain but significantly lesser amounts were present in OMVs derived from WTΔ0859. To further investigate the effects of the LPS structure on OMV formation and CagA/VacA functions, WTΔVacA, WTΔCagA, and WTΔVacA/ΔCagA double knockout mutants were also constructed. The OMVs isolated from these mutants were co-cultured with AGS cells for infection studies. The AGS cells showed a typical hummingbird phenotype and cellular vacuolation after co-culturing with OMVs derived from H. pylori 26695 wild type strain. In contrast, the AGS cells significant lost the hummingbird phenotype when infected with OMVs isolated from the WTΔCagA, and the vacuolation phenomenon significant reduced when co-culturing with OMVs from WTΔVacA. When the AGS cells were treated with OMVs from WTΔ0859, they neither showed the hummingbird phenotype nor cellular vacuolation morphological change. We also conducted total reactive oxygen species (tROS), mitochondrial reactive oxygen species (mROS) and mitochondrial membrane potential (MMP) assays on AGS cells co-culturing with OMVs derived from these mutants, and found that there may exist a functional antagonism between the OMV-associated CagA and the OMV-associated VacA to control the entry of these two toxins into the host cell along with affecting the mitochondrial functions of the infected gastric epithelial cells.

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