Bats Simon H, Metz Felix, Beilmann Johanna, Josenhans Christine
Max von Pettenkofer Institute, Chair for Medical Microbiology and Hygiene, LMU Munich, Pettenkoferstrasse 9a, München 80336, Germany.
Max von Pettenkofer Institute, Chair for Medical Microbiology and Hygiene, LMU Munich, Pettenkoferstrasse 9a, München 80336, Germany; DZIF partner site Munich, Germany.
Int J Med Microbiol. 2025 Jun 23;320:151661. doi: 10.1016/j.ijmm.2025.151661.
The Cag type 4 secretion system (CagT4SS) of Helicobacter pylori is encoded on the cag pathogenicity island (cagPAI) that is present in about 60 % of all strains. It translocates the effector protein CagA, DNA and small bacterial metabolites into human cells. The transport mechanisms of these substrates are not clear and may involve Cag proteins still in search of a function. CagN is a partially surface-exposed CagT4SS protein with a poorly described function. The cagN gene is present in all cagPAI-positive strains and thus likely to be of importance and indispensable for the functionality of the T4SS. CagM is an essential structural component located within the outer membrane core complex of the CagT4SS in the bacterial outer membrane. CagM has a close genomic association and interacts directly with CagN. In this study, we addressed two questions on the basis of prior findings of T4SS-dependent DNA transport and TLR9 activation by H. pylori in host cells. First, we analyzed the role of CagN and CagM in the binding of the presumed CagT4SS substrate DNA. Second, we attempted to elucidate a presumed functional role of CagN in heptose-independent T4SS substrate translocation which may lead to pro-inflammatory activation in human cells. Using electrophoretic mobility shift assays (EMSA) and thermal shift assays (TSA), we found that both CagM and CagN interact with dsDNA. They can also act as nucleases and cleave DNA. Since the transport of substrates through the CagT4SS is likely ATP-driven, we also determined whether CagM and CagN can process ATP, which tested positive for both proteins. Co-incubating H. pylori with human TIFA-k/o cells, which no longer respond to the bacterial translocated effector heptose, but can still be activated by DNA, we established a phenotype of loss of heptose-independent pro-inflammatory activity with H. pylori cagN mutants that could be reversed by complementation. Our results propose an important role for CagN and CagM to bind DNA which might impact or be involved in the transport of substrates such as DNA, through the CagT4SS.
幽门螺杆菌的Cag4型分泌系统（CagT4SS）由cag致病岛（cagPAI）编码，约60%的菌株中存在该致病岛。它将效应蛋白CagA、DNA和小细菌代谢物转运到人类细胞中。这些底物的转运机制尚不清楚，可能涉及仍在寻找功能的Cag蛋白。CagN是一种部分暴露于表面的CagT4SS蛋白，其功能描述甚少。cagN基因存在于所有cagPAI阳性菌株中，因此可能对T4SS的功能至关重要且不可或缺。CagM是位于细菌外膜中CagT4SS外膜核心复合物内的一种必需结构成分。CagM与cagN在基因组上紧密关联且直接相互作用。在本研究中，基于之前关于幽门螺杆菌在宿主细胞中依赖T4SS的DNA转运和TLR9激活的研究结果，我们探讨了两个问题。首先，我们分析了CagN和CagM在假定的CagT4SS底物DNA结合中的作用。其次，我们试图阐明CagN在不依赖庚糖的T4SS底物转运中的假定功能作用，这可能导致人类细胞中的促炎激活。使用电泳迁移率变动分析（EMSA）和热迁移分析（TSA），我们发现CagM和CagN都与双链DNA相互作用。它们还可以作为核酸酶切割DNA。由于底物通过CagT4SS的转运可能由ATP驱动，我们还确定了CagM和CagN是否能处理ATP，结果两种蛋白均呈阳性。将幽门螺杆菌与人TIFA - k/o细胞共同孵育，这些细胞不再对细菌转运的效应物庚糖产生反应，但仍可被DNA激活，我们确定了幽门螺杆菌cagN突变体失去不依赖庚糖的促炎活性的表型，该表型可通过互补逆转。我们的结果表明CagN和CagM在结合DNA方面具有重要作用，这可能影响或参与诸如DNA等底物通过CagT4SS的转运。
