Mechanism of H. pylori intracellular entry: an in vitro study.
Mechanism of H. pylori intracellular entry: an in vitro study.
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DOI：
10.3389/fcimb.2012.00013
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发表时间：
2012
影响因子：
5.7
通讯作者：
Dubois A
中科院分区：
文献类型：
--
作者：
Liu H;Semino-Mora C;Dubois A
大多数幽门螺杆菌存在于胃上皮细胞表面和粘液中，但有一小部分幽门螺杆菌进入宿主上皮细胞和免疫细胞。为探讨裸露侵袭素在宿主细胞进入中的作用，构建了J99菌株的Δ裸露缺失衍生子，并用聚合酶链式反应和荧光原位杂交方法对转化子进行了验证。用野生型(WT)菌株J99或其Δ裸露突变体接种ags细胞，用庆大霉素保护试验测定与细胞结合并存在于细胞内的细菌的比例。我们观察到，无论是幽门螺杆菌结合在AGS细胞膜上的密度还是细胞内的幽门螺杆菌密度都没有显著差异。为了进一步探索这一发现，将每种培养物的不同小室固定在戊二醛中，进行透射电子显微镜和免疫金电镜观察。这种对“经典”庆大霉素试验的补充表明，与Δ裸露等位基因感染的细胞相比，WT感染的ags细胞中存在更多的细胞内Hp，而膜结合的Hp更少。因此，两组中细胞内和膜结合的幽门螺杆菌的总数是相似的。由于没有其他类似的透射电子显微镜研究，目前尚不清楚我们的观察结果是否能被其他人结合起来复制，然而，我们的观察表明，“经典的”庆大霉素保护试验不够敏感，不足以分析幽门螺杆菌的细胞进入，而加入透射电子显微镜的测试表明，nudA在幽门螺杆菌进入AGS细胞的体外过程中起作用。此外，invasin基因的缺失似乎限制了幽门螺杆菌对AGS细胞表面的作用，在那里它可能对庆大霉素具有部分保护作用。相反，这种特殊的环境可能会使幽门螺杆菌更容易受到宿主防御和治疗干预的影响，并且不太容易触发正常的免疫、致癌和其他发育反应途径。
The majority of Helicobacter pylori reside on gastric epithelial cell surfaces and in the overlying mucus, but a small fraction of H. pylori enter host epithelial and immune cells. To explore the role of the nudA invasin in host cell entry, a ΔnudA deletion derivative of strain J99 was constructed and transformants were verified by PCR and by fluorescence in situ hybridization. AGS cells were inoculated with either wild type (WT) strain J99 or its ΔnudA mutant to determine the fraction of bacteria that were bound to the cells and were present inside these cells using the gentamicin protection assay. We observed no significant difference between either the density of H. pylori bound to AGS cell membranes or the density of intracellular H. pylori. To further explore this finding, separate chambers of each culture were fixed in glutaraldehyde for transmission electron microscopy (TEM) and immunogold TEM. This addition to the “classical” gentamicin assay demonstrated that there were significantly more intracellular, and fewer membrane-bound, H. pylori in WT-infected AGS cells than in ΔnudA allele infected cells. Thus, the sum of intracellular and membrane-bound H. pylori was similar in the two groups. Since no other similar TEM study has been performed, it is at present unknown whether our observations can be reproduced by others Taken together however, our observations suggest that the “classical” gentamicin protection assay is not sufficiently sensitive to analyze H. pylori cell entry and that the addition of TEM to the test demonstrates that nudA plays a role in H. pylori entry into AGS cells in vitro. In addition, deletion of the invasin gene appears to limit H. pylori to the AGS cell surface, where it may be partly protected against gentamicin. In contrast, this specific environment may render H. pylori more vulnerable to host defense and therapeutic intervention, and less prone to trigger normal immune, carcinogenic, and other developmental response pathways.
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DOI：
10.1186/1478-811x-6-2
发表时间：
2008-08-06
期刊：
Cell communication and signaling : CCS
影响因子：
--
作者：
Schneider S;Weydig C;Wessler S
通讯作者：
Wessler S
影响因子：
4.8
作者：
Luo, Yihui;Liu, Yan;Yan, Jie
通讯作者：
Yan, Jie
影响因子：
3.1
作者：
Kwok, T;Backert, S;Meyer, TF
通讯作者：
Meyer, TF
影响因子：
64.8
作者：
Alm, RA;Ling, LSL;Trost, TJ
通讯作者：
Trost, TJ
通过实时 RT-PCR 和原位杂交特异性、灵敏地检测生物样本中的幽门螺杆菌。
影响因子：
3.7
作者：
Liu H;Rahman A;Semino-Mora C;Doi SQ;Dubois A
通讯作者：
Dubois A