Research Center of Translational Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan 250013, Shandong Province, China.
National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
World J Gastroenterol. 2024 Nov 7;30(41):4449-4460. doi: 10.3748/wjg.v30.i41.4449.
() persistently colonizes the human gastric mucosa in more than 50% of the global population, leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma. Cytotoxin-associated gene A (CagA) protein, an important oncoprotein, has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus, which play crucial roles in pathogenesis. Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer.
To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function.
We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894. The gene was amplified and mutated into -NT and -NE (sequence characteristics of strains from nongastric cancer patients), cloned and inserted into pAdtrack-CMV, and then transfected into AGS cells. The expression of and its mutants was examined using real-time polymerase chain reaction and Western blotting, cell elongation cell counting, F-actin cytoskeleton visualization using fluorescence staining, and interleukin-8 (IL-8) secretion enzyme-linked immunosorbent assay.
The results revealed that pAdtrack/ induced a more pronounced hummingbird phenotype than pAdtrack/-NT and pAdtrack/-NE (40.88 ± 3.10 32.50 ± 3.17, < 0.001 and 40.88 ± 3.10 32.17 ± 3.00, < 0.001) at 12 hours after transfection. At 24 hours, pAdtrack/NE induced significantly fewer hummingbird phenotypes than pAdtrack/ and pAdtrack/NT (46.02 ± 2.12 53.90 ± 2.10, < 0.001 and 46.02 ± 2.12 51.15 ± 3.74, < 0.001). The total amount of F-actin caused by pAdtrack/ was significantly lower than that caused by pAdtrack/NT and pAdtrack/NE (27.54 ± 17.37 41.51 ± 11.90, < 0.001 and 27.54 ± 17.37 41.39 ± 14.22, < 0.001) at 12 hours after transfection. Additionally, pAdtrack/ induced higher IL-8 secretion than pAdtrack/NT and pAdtrack/NE at different times after transfection.
Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity, which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic .
（）在全球超过 50%的人口中持续定植于胃黏膜，导致各种胃十二指肠疾病，从慢性胃炎到胃癌不等。细胞毒素相关基因 A（CagA）蛋白是一种重要的癌蛋白，其羧基末端具有高度多态性的 Glu-Pro-Ile-Tyr-Ala 片段，在发病机制中发挥关键作用。我们之前的研究表明，位置 893 和 894 处的氨基酸缺失与胃癌显著相关。
研究位置 893 和 894 处的氨基酸缺失对 CagA 功能的影响。
我们从一位患有位置 893 和 894 氨基酸缺失的胃癌患者中选择了一个具有代表性的 HZT 菌株。扩增 基因并突变为-NT 和-NE（非胃癌患者菌株的序列特征），克隆并插入 pAdtrack-CMV，然后转染 AGS 细胞。使用实时聚合酶链反应和 Western blot 检测 及其突变体的表达，通过荧光染色可视化 F-肌动蛋白细胞骨架，使用酶联免疫吸附试验检测白细胞介素-8（IL-8）分泌。
结果显示，与 pAdtrack/-NT 和 pAdtrack/-NE 相比，pAdtrack/在转染后 12 小时诱导更明显的蜂鸟表型（40.88 ± 3.10 对 32.50 ± 3.17， < 0.001 和 40.88 ± 3.10 对 32.17 ± 3.00， < 0.001）。在 24 小时时，pAdtrack/NE 诱导的蜂鸟表型明显少于 pAdtrack/和 pAdtrack/NT（46.02 ± 2.12 对 53.90 ± 2.10， < 0.001 和 46.02 ± 2.12 对 51.15 ± 3.74， < 0.001）。pAdtrack/引起的 F-肌动蛋白总量明显低于 pAdtrack/NT 和 pAdtrack/NE（27.54 ± 17.37 对 41.51 ± 11.90， < 0.001 和 27.54 ± 17.37 对 41.39 ± 14.22， < 0.001）在转染后 12 小时。此外，与 pAdtrack/NT 和 pAdtrack/NE 相比，pAdtrack/在不同时间转染后诱导更高的 IL-8 分泌。
位置 893 和 894 处的氨基酸缺失增强了 CagA 的致病性，这对于揭示 CagA 的致病机制和鉴定高致病性 的生物标志物至关重要。
