Evolution and function of genetic polymorphisms in the Helicobacter pylori type IV secretion pilus protein CagL in gastric carcinogenesis

Background The cytotoxin-associated genes ( cag ) type IV secretion system (T4SS) of the gastric pathogen Helicobacter pylori constitutes a key determinant in gastric cancer (GC) development. The CagL protein represents a VirB5 ortholog that acts as a signalling factor at the surface of the T4SS pilus, which facilitates binding to host target cells to inject T4SS substrates such as the CagA oncoprotein and ADP-heptose. Previous studies reported the presence of a hypervariable region in CagL at amino acid positions 58 and 59 with a proposed role in GC progression, however, studies using a large number of H. pylori strains as well as detailed functional experiments are yet missing. Methods Here, we investigated the CagL status in 2,267 worldwide H. pylori genomes from an evolutionary point of view, concerning gastric disease status, and directly compared the Y58/E59 versus N58/K59 polymorphism in CagL in the GC-associated H. pylori strain 7.13 with regard to T4SS-pilus formation and cancer signalling during infection of gastric epithelial cells in vitro and infection of Mongolian gerbils in vivo. Results Structural analysis showed that the genetic polymorphism, located in an amino acid loop flanked by two α-helices, affects CagL conformation and functional activity. Remarkably, both strains revealed similar robust T4SS-pilus formation, but functional differences. We demonstrate that wild-type H. pylori 7.13 encoding N58/K59 CagL triggered GC development in Mongolian gerbils and characteristic GC signalling events in gastric epithelial cells, including CagA phosphorylation, activation of MAP kinases (p38, ERK1/2 and JNK), β-catenin and cell motility as well as pronounced induction of pro-inflammatory transcription factor NF-κB-dependent interleukin-8 (IL-8) production and DNA double-strand breaks (DSBs), whereas H. pylori encoding Y58/E59 CagL elicited markedly attenuated responses. Conclusions These data suggest that the Y58/E59 mutation impairs translocation of CagA into host cells, but retains T4SS-pilus formation and ADP-heptose-dependent NF-κB activation. This implies that CagL mutation may act as molecular regulator of the T4SS for “finetuning” CagA delivery, host inflammation and other oncogenic responses. Interestingly, this scenario is similar to previously reported CagY, another T4SS-pilus protein, which can cause the gain or loss of IL-8 production by genetic polymorphisms that occur through recombination events.