Role of the cag-pathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis

Abstract

Helicobacter pylori is a very successful human-specific bacterium worldwide. Infections of the stomach with this pathogen can induce pathologies, including chronic gastritis, peptic ulcers and even gastric cancer. Highly virulent H. pylori strains encode the cytotoxin-associated gene (cag)-pathogenicity island, which expresses a type IV secretion system (T4SS). This T4SS forms a syringe-like pilus structure for the injection of virulence factors such as the CagA effector protein into host target cells. This is achieved by a number of T4SS proteins, including CagI, CagL, CagY and CagA, which by itself binds the host cell integrin member β(1) followed by delivery of CagA across the host cell membrane. A role of CagA interaction with phosphatidylserine has also been shown to be important for the injection process. After delivery, CagA becomes phosphorylated by oncogenic tyrosine kinases and mimics a host cell factor for the activation or inactivation of some specific intracellular signalling pathways. We review recent progress aiming to characterize the CagA-dependent and CagA-independent signalling capabilities of the T4SS, which include the induction of membrane dynamics, disruption of cell-cell junctions and actin-cytoskeletal rearrangements, as well as pro-inflammatory, cell cycle-related and anti-apoptotic transcriptional responses. The contribution of these signalling pathways to pathogenesis during H. pylori infections is discussed.

Figure 1

Model of Helicobacter pylori-induced epithelial-barrier disruption and pathogenesis. The interplay between polarized gastric epithelial cells and a variety of bacterial pathogenicity factors modulates multiple host responses during the course of infection, as indicated. H. pylori expresses several adhesins such as BabA, BabB, SabA, AlpA and AlpB as well as OipA which mediate apical binding of the bacteria (1). Attached H. pylori or those in the mucus secrete virulence factors into the medium (HtrA protease, VacA cytotoxin and others) (2), which could trigger mild opening of tight junctions (TJs) and adherens junctions (AJs) at early time points of infection (3). While internalized VacA causes cellular vacuolization, a hallmark of the ulceration process, HtrA cleaves the junctional protein E-cadherin [8]. Another intriguing possibility of junction disruption could be the transcytosis of basal integrins to the apical surface by early, but unknown, cagPAI-independent signaling (4). Apical exposure of some integrin molecules such as integrin α₅β₁ could stimulate the T4SS pilus to inject CagA and peptidoglycan into cells (5). Injected CagA can then be targeted to TJs and AJs followed by further disruption of these junctions (6). Injected CagA and peptidoglycan (5) in addition to OipA (1) can trigger nuclear factor-kB (NF-κB) activation (7) and the release of proinflammatory cytokines such as IL-8 (8). These cytokines can alter the secretion of mucus and induce changes in gastric-acid secretion and homeostasis. They also attract immune cells to infiltrate from the blood stream into the gastric mucosa (9), where they cause substantial tissue damage at the site of infection (10). H. pylori also express numerous factors to suppress immune cell functions as indicated. The result of the above described processes is local epithelial disruption enabling some H. pylori of entering the intercellular space between adjacent cells and reaching the basal membranes (11). In this manner, the bacteria could access integrins and inject CagA (12). Injected CagA can then induce the massive disruption of cell junctions (13) and loss of cell polarity (14). The induction of metalloproteases (MMPs) might enhance this effect in addition to HtrA. Finally, CagA can induce multiple pathways to trigger host-cell motility and elongation (15) and the onset of mitogenic genes and cell proliferation (16), and it can inhibit apoptosis (17). The interplay of each of these pathways could result in substantial deregulation and oncogenic transformation of gastric epithelial cells in vivo and, thus, are important for H. pylori pathogenesis. Specific steps labeled with question marks are untested or speculative aspects of the model. Other specific abbreviations used: α5β1 (chains of the integrin receptor), cag (cytotoxin-associated genes), ECM (extracellular matrix), IL-8 (interleukin 8), HP0421 (cholesterol-alpha-glucosyltransferase), MΦ (macrophage), NapA (neutrophil-activating protein A), PAI (pathogenicity island), PG (peptidoglycan), RocF (arginase enzyme). For more details see text and glossary in the Supplementary Material (Doc. S1). This model was updated from Wessler and Backert [15] with kind permission from Blackwell Publishing.

Figure 2

Models for the assembly and assembled structure of T4SSs in Agrobacterium tumefaciens and Helicobacter pylori. (A) Proposed assembly of the prototypical Agrobacterium VirB/VirD4 T4SS machinery is shown. The T4SS is a multi-component protein complex spanning the inner and outer membranes of Agrobacterium and other Gram-negative bacteria. Current knowledge of T4SS assembly, cellular localization of its components is shown in a simplified manner. The coupling protein VirD4 and structural components (VirB1-VirB11) are typically required for secretion and are depicted according to their proposed functions. A model for T-pilus assembly in Agrobacterium showing the proposed VirB4-VirB8-VirB5-VirB2 interaction sequence leading to the formation of VirB2-VirB5 complexes followed by T-pilus assembly. The assembled T4SS then triggers the secretion of substrates from the bacterial cytoplasm directly into the cytoplasm of infected host cells or into the extracellular milieu. (B) Model-1 for the assembled T4SS machinery in H. pylori assuming that all VirB1-11 proteins are encoded by the cagPAI and assemble in a similar fashion as proposed for A. tumefaciens [6]. The reported substrates for this T4SS are CagA and peptidoglycan. (C) Model-2 proposes that the T4SS requires basically the same VirB proteins as in panel B with one major difference. The pilus surface is proposed to be covered with CagY molecules. In contrast to VirB10 in many T4SSs, H. pylori VirB10 (CagY) is a very large protein (ca. 250 kDa, domain structure and amino acid positions shown for CagY of strain 26695, accession number NP_207323.1) carrying two transmembrane domains (TM1 and TM2) to form a hairpin-loop structure in the membranes as depicted [10]. Immunogold labelling against the loop region in CagY indicated that this part of the protein is exposed to the extracellular space and is transported to the pilus surface by a yet unknown mechanism [10]. However, recent data demonstrated that the very carboxy-terminus of CagY can bind to the host receptor integrin β₁ [13]. How the latter domain can be exposed to the extracellular space in order to make contact with integrin β₁ is not yet clear and must be clarified in future studies.

Figure 3

Model for the role of H. pylori T4SS in host cell signaling processes which may effect pathogenesis. (A) The H. pylori T4SS pili are induced upon contact with host cells and can inject effector molecules such as the CagA protein and peptidoglycan in a manner dependent on integrin β₁. Injected CagA can then induce cascades as depicted in the panels below. Panel A highlights a multitude of known T4SS-dependent but CagA-independent pathways involved in the activation of receptor and non-receptor tyrosine kinases, pro-inflammatory signalling, Rho GTPase activation, scattering and motility of gastric epithelial cells as well as suppression of histone phosphorylation and H. pylori phagocytosis by immune cells. Two particular T4SS factors have been reported to be involved in some but not all of these responses. The known signaling functions for injected peptidoglycan as well as pilus-exposed or recombinant CagL are shown. For numerous other pathways the actual T4SS factor is yet unknown as also indicated. (B) CagA phosphorylation-dependent and (C) phosphorylation-independent signal transduction events. CagA is injected into the host cell membrane of infected gastric epithelial cells which also requires phosphatidylserine. The tyrosine kinases Src and Abl phosphorylate injected CagA. CagA can then modulate various signaling cascades associated with cell polarity, cell proliferation, actin-cytoskeletal rearrangements, cell elongation, disruption of tight and adherens junctions, pro-inflammatory responses and suppression of apoptosis, as depicted. Black arrows indicate activated sigalling pathways and red arrows correspond to inactivated cascades. Panels B and C were updated from Backert et al. [26] with kind permission from WILEY. For specific abbreviations and terms used in this figure, please see text and glossary in the Supplementary Material (Doc. S1).