Cag3 Is a Novel Essential Component of the Helicobacter pylori Cag Type IV Secretion System Outer Membrane Subcomplex

Helicobacter pylori strains harboring the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI encodes a type IV secretion (T4S) system required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8). cag PAI genes sharing sequence similarity with T4S components from other bacteria are essential for Cag T4S function. Other cag PAI-encoded genes are also essential for Cag T4S, but lack of sequence-based or structural similarity with genes in existing databases has precluded a functional assignment for the encoded proteins. We have studied the role of one such protein, Cag3 (HP0522), in Cag T4S and determined Cag3 subcellular localization and protein interactions. Cag3 is membrane associated and copurifies with predicted inner and outer membrane Cag T4S components that are essential for Cag T4S as well as putative accessory factors. Coimmunoprecipitation and cross-linking experiments revealed specific interactions with HpVirB7 and CagM, suggesting Cag3 is a new component of the Cag T4S outer membrane subcomplex. Finally, lack of Cag3 lowers HpVirB7 steady-state levels, further indicating Cag3 makes a subcomplex with this protein.Helicobacter pylori infects 50% of the world population. Stomach infection with this bacterium is associated with the development of several gastric diseases, including chronic active gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. Factors influencing disease outcomes are not completely understood, but bacterial, host, and environmental factors have been identified that affect the dynamics of this bacterium-host interaction (30). A hallmark of H. pylori infection is the induction of mucosal inflammation, which is a risk factor for developing more severe pathology (27).Epidemiological studies have established that infection with strains harboring the cag pathogenicity island (PAI) leads to a higher risk for development of severe disease (27). The cag PAI size varies between 35 and 40 kb and encodes 27 putative proteins (1, 13). Several of the encoded proteins share sequence similarities with components of the prototypical type IV secretion (T4S) system VirB/D4 of Agrobacterium tumefaciens (15, 16). Based on research done in A. tumefaciens, the components of the molecular machinery have been divided into channel or core complex components (VirB6, VirB7, VirB8, VirB9, and VirB10), energetic components (VirB11, VirB4, and VirD4), and extracellular appendage components (VirB2 and VirB5). VirB6, VirB8, and VirB10 are components anchored at the inner membrane with domains spanning the periplasm, while VirB7 and VirB9 are located at the outer membrane. Energetic components are located at the inner membrane, and pilus components include the main subunit VirB2 and accessory components, such as VirB5, which functions as an adhesin (15, 16). The VirB/D4 T4S is thought to be energized by the inner membrane ATPases, and this energy is transduced to VirB10 and the outer membrane complex for protein translocation (11). The lipoprotein VirB7 is critical for the stability of HpVirB9 at the outer membrane (19).While the extent of homology of the H. pylori cag T4S components is often limited, sequence analysis has allowed the identification of the VirB11 (HP0525 and HpVirB11), VirB10 (HP0527 and HpVirB10), VirB9 (HP0528 and HpVirB9), and VirD4 (HP0524 and HpVirD4) homologues as summarized in Table S1 of the supplemental material (1, 13, 28). HpVirB9 and HpVirB10 homologies are not distributed along the entire length of the protein. For example, HpVirB10 is a very large protein with only a short domain similar to VirB10. HpVirB10 is also reported to localize on the external surface of the pilus (31), while VirB10 is tethered in the inner membrane. HP0529 (HpVirB6) and HP0530 (HpVirB8) have been assigned as homologs of VirB6 and VirB8, respectively (28). HP0523 (HpVirB1) has lytic transglycosylase activity, supporting its designation as a VirB1 homolog (38). HP0532 (HpVirB7) has a lipoprotein attachment site, suggesting a role as a VirB7 homolog (1, 28), and has been suggested to stabilize a Cag T4S outer membrane subcomplex containing CagM, HpVirB9, and HpVirB10 (28).The activity of the cag PAI-encoded T4S system is responsible for the translocation of the effector protein CagA and induction of proinflammatory chemokine and cytokine secretion, including the chemokine interleukin-8 (IL-8) (7). CagA T4S-mediated translocation into host cells is followed by tyrosine phosphorylation on specific tyrosine phosphorylation motifs (EPIYA motifs) at the C-terminal region of the protein and both phosphorylation-dependent and -independent interference with host cellular pathways. The induction of proinflammatory chemokine production is mediated by a still-uncharacterized Cag T4S-mediated delivery of peptidoglycan into host cells and subsequent activation of Nod receptors (37), and it has also been reported that CagA itself has proinflammatory properties (9). The molecular mechanisms responsible for Cag T4S system assembly and activity remain unclear.Null alleles of the genes with homology to T4S components (HpVirB11, HpVirB4, HpVirB6, HpVirB7, HpVirB8, HpVirB9, and HpVirB10) abolish both CagA translocation and IL-8 induction, with the exception of HpVirD4, which affects CagA translocation but not IL-8 induction (20). Other genes of the island also essential for Cag T4S function do not share sequence or structural homology with known T4S components. More detailed analysis of these Cag T4S essential genes allowed the recent assignment of several proteins as functional homologs of additional VirB components. HP0546 was suggested as a VirB2 homolog, the main subunit of other T4S system pili (3). Ultrastructural work suggested that HpVirB10 is also a major subunit of the Cag T4S system pilus (31, 35), but clear evidence that either HpVirB2 or HpVirB10 is the main pilus subunit is still lacking. CagL (HP0539) has been identified (29) as an adhesin (functionally similar to VirB5) whose binding to host cell receptors is required for activation of the secretion process, and CagF (HP0543) has been characterized as a CagA chaperone (17). CagD (HP0545) has been recently reported as a multifunctional Cag T4S component essential for CagA translocation and full IL-8 secretion induction (12).We have characterized the biochemical role of an additional essential H. pylori-specific gene, HP0522/cag3, in Cag T4S. A previous yeast two-hybrid screen that investigated interactions among cag PAI proteins suggested Cag3 could interact with HpVirB8, HpVirB7, CagM (HP0537), and CagG (HP0542) (10). To begin to understand the molecular basis of Cag3 function in T4S we investigated the subcellular localization of the Cag3 protein and the protein-protein interactions this protein establishes in H. pylori cells. We found evidence suggesting that Cag3 is an integral part of the Cag T4S outer membrane subcomplex required to maintain HpVirB7 levels.