The Helicobacter pylori cag Pathogenicity Island Protein CagN Is a Bacterial Membrane-Associated Protein That Is Processed at Its C Terminus

Helicobacter pylori infects the human stomach and is associated with an increased risk for diseases ranging from gastritis to peptic ulcer and gastric cancer. While some aspects of infection outcome are related to diet and host genetic factors, the strongest indicator of a severe disease risk is infection with a bacterial strain harboring the cytotoxin-associated gene pathogenicity island (cag PAI) (5). This 40-kb cassette of approximately 28 genes encodes a type IV secretion system (T4SS) and its substrate, CagA. During infection, CagA is delivered into host cells in a T4SS-dependent manner and induces multiple changes in host cell signaling which are thought to contribute directly to disease (reviewed in reference 6). The correlation between cag PAI+ strains and disease has generated great interest in the function of the H. pylori T4SS. Several bacterial pathogens and symbionts alike employ T4SS for delivery of protein or DNA effectors into host cells (10). Of the 28 genes carried by the cag PAI, 6 have homology to the T4SS of Agrobacterium tumefaciens, the best-studied model of type IV secretion. The remaining genes are unique to the H. pylori cag PAI, and a function has been proposed for only a few of these (8, 26, 37). Infection of cultured gastric epithelial cells with cag PAI+ H. pylori strains results in interleukin-8 (IL-8) induction and a dramatic cellular elongation. The discovery of mutations that blocked CagA delivery but not IL-8 induction led to the hypothesis that a second secreted effector molecule existed that was responsible for IL-8 induction. However, systematic deletion of cag PAI genes identified only four classes of mutants: those that had no effect on CagA delivery or IL-8 induction, those that blocked both CagA delivery and IL-8 induction, those that blocked CagA delivery but not IL-8 induction, and those that had an intermediate and variable effect on both CagA delivery and IL-8 induction (16, 31). No cag PAI mutants that allowed CagA delivery but blocked IL-8 induction were identified, ruling out any cag-encoded proteins as candidates for an IL-8-specific inducing effector. Recent experiments have implicated peptidoglycan, acting through the cytoplasmic receptor Nod1, as the secreted factor that induces IL-8 (39), while others have shown that in some strain backgrounds CagA itself is responsible for some IL-8 induction (7). These deletion studies were an important first step in understanding the functions of individual members of the cag PAI, yet many questions about the functions of individual cag PAI genes remain unanswered. The cag PAI genes whose deletion does not alter CagA delivery and IL-8 secretion in tissue culture cells may encode delivered effector proteins with unknown activities in infection. Other pathogens that employ a T4SS in their infection process, such as A. tumefaciens, Legionella pneumophila, and Bartonella henselae, deliver multiple effector proteins into host cells (22, 29, 38). Alternatively, these genes may encode proteins with redundant functions in type IV secretion. Mutations that resulted in intermediate and variable levels of CagA delivery and IL-8 induction are promising candidates for proteins important in T4SS assembly and function. These genes may encode accessory proteins required for substrate translocation (such as chaperones) or may even encode additional substrates themselves. Here, we report a detailed characterization of one such gene, HP0538 or cagN. A cagN mutant was reported by Fischer et al. to have an intermediate and variable CagA delivery and IL-8 induction phenotype (16). We found that CagN is localized to the bacterial membrane and is not a substrate for the T4SS. We then analyzed the CagN protein itself and found that it is cleaved at its C terminus. This processing event is independent of other cag PAI proteins and is only partially blocked by mutations of the predicted cleavage site. Finally, we show that a cagN mutant is not deficient for CagA-induced AGS cell elongation. We conclude that CagN either serves a redundant function in T4SS or is required for some other, uncharacterized aspect of pathogenesis.