High expression of <i>Helicobacter pylori</i> VapD in both the intracellular environment and biopsies from gastric patients with severity

Helicobacter pylori is a Gram-negative bacterium that causes chronic atrophic gastritis and peptic ulcers and it has been associated with the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT). One of the more remarkable characteristics of H. pylori is its ability to survive in the hostile environment of the stomach. H. pylori regulates the expression of specific sets of genes allowing it to survive high acidity levels and nutrient scarcity. In the present study, we determined the expression of virulence associated protein D (VapD) of H. pylori inside adenocarcinoma gastric (AGS) cells and in gastric biopsies. Using qRT-PCR, VapD expression was quantified in intracellular H. pylori-AGS cell cultures at different time points and in gastric mucosa biopsies from patients suffering from chronic atrophic gastritis, follicular gastritis, peptic ulcers, gastritis precancerous intestinal metaplasia and adenocarcinoma. Our results show that vapD of H. pylori presented high transcription levels inside AGS cells, which increased up to two-fold above basal values across all assays over time. Inside AGS cells, H. pylori acquired a coccoid form that is metabolically active in expressing VapD as a protection mechanism, thereby maintaining its permanence in a viable non-cultivable state. VapD of H. pylori was expressed in all gastric biopsies, however, higher expression levels (p = 0.029) were observed in gastric antrum biopsies from patients with follicular gastritis. The highest VapD expression levels were found in both antrum and corpus gastric biopsies from older patients (>57 years old). We observed that VapD in H. pylori is a protein that is only produced in response to interactions with eukaryotic cells. Our results suggest that VapD contributes to the persistence of H. pylori inside the gastric epithelial cells, protecting the microorganism from the intracellular environment, reducing its growth rate, enabling long-term infection and treatment resistance.幽门螺杆菌（Helicobacter pylori）是一种革兰氏阴性杆菌，可引发慢性萎缩性胃炎与消化性溃疡，且与胃腺癌及黏膜相关淋巴组织（MALT）的发生发展密切相关。幽门螺杆菌最为显著的特征之一，便是能够在胃部的恶劣环境中存活。它可通过调控特定基因的表达，适应高酸性环境与营养匮乏状态。在本研究中，我们检测了幽门螺杆菌毒力相关蛋白D（VapD）在胃腺癌（AGS）细胞内以及患者胃活检组织中的表达情况。我们采用实时荧光定量逆转录PCR（qRT-PCR）技术，对不同时间点的细胞内幽门螺杆菌-AGS共培养体系，以及患有慢性萎缩性胃炎、滤泡性胃炎、消化性溃疡、伴癌前肠上皮化生的胃炎和胃腺癌的患者的胃黏膜活检组织中的VapD表达水平进行了定量分析。我们的研究结果显示，幽门螺杆菌的vapD基因在AGS细胞内呈现高转录水平，在所有实验中，其表达量随时间推移较基础水平最高可上调至两倍。在AGS细胞内，幽门螺杆菌会形成球形体结构，该结构代谢活跃，可通过表达VapD作为保护机制，从而维持自身处于可存活但不可培养的状态。幽门螺杆菌的VapD在所有胃活检组织中均有表达，但滤泡性胃炎患者的胃窦活检组织中，其表达水平更高（p=0.029）。在年龄大于57岁的患者的胃窦与胃体活检组织中，VapD的表达水平最高。我们发现，幽门螺杆菌的VapD仅在与真核细胞发生相互作用时才会合成。本研究结果表明，VapD可促进幽门螺杆菌在胃上皮细胞内的定植，帮助该微生物抵御胞内环境压力，降低其生长速率，进而实现长期感染与治疗耐受。