Helicobacter pylori CagT4SS proteins CagN and CagM bind DNA and CagN is involved in heptose-independent pro-inflammatory substrate translocation by the T4SS.

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.