Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island ( cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. Δ cagI and Δ cagL mutant strains fail to form pili, whereas a Δ cagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.
Helicobacter pylori persistently colonizes the stomach in approximately half of the human population. People who are infected with H. pylori strains harboring the cag pathogenicity island (PAI) have an increased risk of developing gastric cancer. The cag PAI encodes a type IV secretion system (T4SS) that is utilized by the bacteria to inject the bacterial oncoprotein CagA into gastric epithelial cells. Related T4SSs found in several other bacteria have been studied in detail, but thus far there has been very little study of the H. pylori cag T4SS. Here, we utilized a mass spectrometry-based approach to reveal co-purification of three constituents of the H. pylori T4SS (CagH, CagI, and CagL) that lack homology to components of T4SSs in other bacterial species. These proteins are essential for CagA translocation into host cells, and scanning electron microscope studies reveal that the proteins are involved in the formation of pili at the bacterial-host cell interface. A conserved C-terminal motif present in CagH, CagI, and CagL is essential for functionality of the T4SS. This study highlights the important role played by unique constituents of the H. pylori cag T4SS, and illustrates the marked variation that exists among bacterial T4SSs.