Chapter 31The cag Pathogenicity Island

The cag Pathogenicity Island of H. pylori

H. pylori is a gram-negative spiral-shaped human pathogen that colonizes the antrum and the corpus of the stomach. Many virulence factors have been described in the past decade. These factors enable the bacterium to survive in the extreme acidic environment of the gastric tract, to reach the more neutral environment of the mucous layer, and to resist the human immune response, resulting in persistence. Most of the infections occur during childhood, with only a minority of all infections progressing to pathological states.

In this chapter we will focus on the functions that are encoded by a 40-kb chromosomal region with the features of a PAI (1, 9, 54). The H. pylori PAI was originally named cag (cytotoxin-associated gene) since it was thought to be associated with expression of the vacuolating toxin (VacA). However, it was later shown that both factors, VacA and the PAI, are independent of each other, even though cag-negative strains often do not express VacA (53). Interestingly, biopsies from patients with severe gastric diseases including chronic active gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer contain the cagA gene in more than 90% of all the cases, establishing a direct correlation of the presence of the cagA gene with disease (7, 13, 15). On the genetic level, the cag PAI is flanked by a 31-bp direct repeat, which contains the recombination site and corresponds to the last nucleotides of the glr gene (9) (Fig. 1). The module also forms the core of the left and right ends of an insertion sequence common in H. pylori, the IS605 element. Depending on the strain, different numbers of insertion sequences are associated with the PAI. Often, however, the PAI is split into subregions, cagI and cagII, which are interrupted by two IS605 elements. Between the two insertion sequences, intervening DNA is present. As a consequence, strains with many insertions often resemble type II strains (less virulent) more than type I (more virulent, PAI-containing) strains with respect to virulence attributes. Additional genetic instability of cag results from deletions and inversions (27). In some cases, the cag PAI can even be lost completely due to DNA transfer of an empty site from a cag-deficient strain into a type I strain and subsequent homologous recombination (30).

Figure 1

Genetic organization of the cag pathogenicity island. The open reading frames within the 40-kb cag PAI are depicted as arrows with the cag gene designations (single letters) below and names of homologs above. The whole genome gene designations are given (more...)

There are 31 open reading frames predicted within the cag region (Fig. 1). One of these open reading frames encodes the immunodominant antigen CagA, which is localized to the 3′ end of the island (12, 52). CagA was identified as the first protein of the PAI and appeared to be a major virulence factor (43). The molecular size of cagA is variable and depends on the number of repeats of a 102- to 108-bp motif that is repeated with specific strains. At the protein level, the absence of repeats corresponds to a molecular weight of 128 kDa, while every repeat up to a maximum of four repeats shifts the molecular weight of CagA by approximately 4 kDa (59). Comparison of strains with differences in the number of repeats isolated from patients with gastric cancer suggested that strains with more repeats were associated with higher levels of CagA antibody, more severe degrees of atrophy, and reduced survival in a low pH (pH 3). The repeats have a modular structure, which is a result of duplications and rearrangements of DNA from the central region of the cagA gene. However, the number, sequence, and position of the modules can vary, depending on the geographic region from which the strain was isolated. These local differences appear to be a result of frequent genetic recombination events and accumulation of mutations within the cagA gene that were maintained by geographic segregation. Besides CagA, the cag PAI encodes a helicobacter-specific type IV secretion system.

Type IV Secretion Systems and Their Functions

One of the most exciting research areas in microbial pathogenesis over the past decade has been the exploration of type III secretion systems. Many gram-negative pathogens including Salmonella, Yersinia, and Shigella species, pathogenic E. coli, and many plant pathogens use a type III system to export bacterial virulence proteins from the bacterial cytoplasm directly into the host cell (26, 31, 35). The exported molecules, called effectors, interfere with a variety of basic signaling pathways of the host cell (20). The effectors are species specific. We also know that at least one other secretion system, the type IV secretion system, is able to translocate virulence proteins into the host cells. It is important to note that the evolutionary origin of the two systems is different. Type III probably evolved from the flagellar system (37), while type IV systems originated from conjugative apparatuses (10, 11, 58). Whereas flagella are organelles of motility, the conjugation systems allow the exchange of DNA between bacteria. However, both systems also have important features in common. It is thought that both can transport proteins through a complex channel structure directly through the inner and outer bacterial membranes into the cytoplasm of the host cell. Furthermore, they use chaperones or chaperone-like molecules to stabilize the effectors and energize the protein export by ATP-hydrolysis; their polymerization is transient since it is induced upon cell contact. The variety of substrates and the secretion mechanism itself, however, seem to be less uniform for the type IV system. While type III systems exclusively export monomeric proteins, type IV machinery may export multisubunit and nucleoprotein complexes. In the case of the pertussis toxin, for example, sec-dependent transport through the inner membrane is coupled with type IV-driven transport through the outer membrane and substrate secretion occurs only into the supernatant.

Up to now, the group of pathogens encoding a type IV secretion system with demonstrated virulent functions is composed of five species: Agrobacterium tumefaciens (virB and virD4), Bordetella pertussis (ptl), Legionella pneumophila (icm/dot), Brucella suis (virB), and H. pylori (cag). Other type IV secretion systems with probable importance for pathogenesis were identified in the genome of Rickettsia prowazekii (3), Bartonella henselae, and Actinobacillus actinomycetemcomitans. The homologies among the Vir homologs in the different pathogens are mainly found in the structural core of the secretion apparatus. However, in contrast to type III secretion systems, the components of type IV systems are not interchangeable between the different bacterial species as shown by complementation experiments. Apart from the homologous components of the core, every system contains additional factors that are specific for each individual pathogen.

Our knowledge of the localization of the Vir homologs within the secretion system is mainly obtained from the extensive work done on A. tumefaciens. On the basis of present knowledge, we divide individual components in several classes. VirD4, VirB4, and VirB11 homologs are localized in the inner bacterial membrane and encode proteins with ATPase activity. VirD4 is also thought to couple protein–protein interactions. Further classes involve (i) transglycosidases (VirB1) that may create holes in the murein sacculus to allow putative pilus components to assemble on the surface of the bacteria, (ii) periplasmic scaffolds consisting of the lipoprotein VirB7 associated with VirB9, (iii) cytoplasmic chaperones that bind and stabilize the substrate (VirD1 as chaperone for VirD2), (iv) the VirB2 pilus subunit (32), and (v) the substrates for translocation. In Agrobacterium sp. the type IV secretion system is formed on the bacterial surface after induction with phenolic compounds and at low temperature (6, 22). Three substrates are known to be translocated into the plant cell. VirE2 binds the 5′-end of the T-DNA and is injected into the plant cell as a nucleoprotein particle. Inside the host cell, an additional translocated protein, the single-strand DNA-binding protein VirE2, binds to the T-DNA and protects it from degradation. VirE2 and the F-box protein VirF are translocated independently of DNA and have a function within the plant cell that contributes to the tumor formation. This allows speculation about the origin of DNA transfer systems that might eventually have evolved from protein transfer systems. It was shown that the secretion signal for VirF and VirD2 is located in the C-terminal half of the proteins, where a common Arg-Pro-Arg motif was recognized (55). VirD2 and VirE2 carry nuclear targeting signals and mediate the transport of the T-DNA complex into the nucleus where the T-DNA integrates into the plant cell genome. The genetic information of the T-DNA is then translated into proteins, which interfere with the plant cell physiology and ultimately lead to plant cell tumors (crown gall).

As mentioned above, B. pertussis secretes the five-subunit pertussis toxin across the bacterial outer membrane into the supernatant using a type IV secretion apparatus (12, 14, 56). The toxin then binds to the receptor on the epithelial surface and is subsequently internalized. Inside the host cell, the toxin ADP-ribosylates heterotrimeric G proteins (Gi), thus interfering with receptor kinase signaling pathways.

The icm/dot genes of L. pneumophila, the causative agent of Legionnaire's disease and Pontiac fever, were identified during mutational studies to identify genes essential for intracellular growth and macrophage killing (49, 50). The substrates that are mediating these events are not yet described. The type IV secretion system of Legionella sp. contains only two genes, icmE and dotB, which share homology with the VirB homologs of Agrobacterium sp. Fourteen additional genes have homologs in bacterial conjugation systems, and not surprisingly, the Icm/Dot system has been shown to also mediate movement of the plasmid RSF1010. Thus, the type IV system of Legionella sp. has maintained both functions, one for DNA and one for protein export.

Homologs of most of the VirB proteins, including a VirB2 homolog, were recently discovered in B. suis (41). Mutants in virB5, virB9, or virB10 were highly attenuated in an in vitro infection model with human macrophages. This led to the conclusion that the virB region is essential for the intracellular survival and multiplication of B. suis (41). In H. pylori several homologs of VirB and VirD4 are present. These include the ATPases VirB4 (CagE) and VirB11 (HP525), which energize the transport of CagA and possible further substrates through the putative transenvelope channel. VirD4 (HP524) is localized in the inner membrane as well and might have a function in the substrate recognition. VirB7 (CagT) and VirB9 (HP528) are possibly located in the outer membrane. CagM might have a chaperone-like function, stabilizing VirB7 (33). VirB8 and VirB10 are probably part of the channel structure.

Pathogenic Functions of the cag Pathogenicity Island

When the genetic analysis of the cag PAI suggested the presence of a type IV secretion system, it was anticipated that this structure should mediate contact to the host cell and possibly induce signal transduction pathways that contribute to the virulence of H. pylori. At the present stage, we differentiate between two basic cag-mediated host cell signaling pathways that are induced upon attachment of H. pylori. One is induced by the immunodominant antigen of H. pylori, the CagA protein, while the second pathway is independent of CagA. Deconvolution microscopy, mutational analysis, and biochemical fractionation experiments have provided evidence that adherent H. pylori translocates CagA into host cells and that this process depends on a functional type IV secretion system (4, 5, 16, 42, 46, 51) (Fig. 2). Inactivation of all cag genes tested, except cagN, abolished CagA translocation.

Figure 2

Activation of CagA and intracellular actin condensation. Translocation is mediated by the cag TFSS (a); activation is dependent by a membrane-associated host kinase (b, c) that phosphorylate CagA at multiple sites; small GTP-binding proteins may be involved (more...)

Inside the host cell, CagA becomes phosphorylated on a tyrosine residue by a yet unidentified host cell kinase (Fig. 2). Although c-src and crude host cell lysates were able to tyrosine-phosphorylate CagA in vitro, the significance of this finding under in vivo conditions remains unclear (4). Cell fractionations showed that the majority of intracellular CagA is found in the insoluble fraction, while a significant amount was associated with host cell membranes. This finding suggests that, following phosphorylation of tyrosine residues, CagA becomes part of an insoluble and probably membrane-associated complex inside the host cell. The CagA tyrosine-phosphorylation site was mapped to an EPIYA motif in the C-terminal half of the protein (Stein et al., unpublished observations). EPIYA is located immediately upstream and within the 102-bp repeat region in the C-terminal part of the protein.

Although the function of CagA on the molecular level is still unknown, it was suggested that CagA is involved in the rearrangements of the actin cytoskeleton, which results in the formation of cup-like structures underneath the adherent bacteria (47, 51). On the molecular level, those changes are mediated by activation of Arp2/3 complexes via small GTPases and N-WASP or N-WASP-like proteins (57). Listeria sp., Shigella sp. (19), and vaccinia virus use variations of this mechanism to move on the tip of actin tails within the host cells (36). Attaching and effacing pathogens, including enteropathogenic and enterohemorrhagic E. coli, remain extracellular and cause actin rearrangements in a similar manner through the host cell membrane (28, 29). The resulting protrusions on the cell surface (pedestals) somehow resemble the cup-like structures induced by H. pylori, although they are clearly different structures.

A second kind of change in the host cell morphology also depends on CagA translocation. A type I wild-type strain, but not the cagA isogenic mutant, induced a scatter factor-like phenotype, which was called the "hummingbird'' phenotype and probably results from activation of small GTPases of the Rho family. It was also shown that cag-positive strains affect gastric epithelial cell proliferation, diminish cell viability, and attenuate apoptosis in vivo (Fig. 3). The fact that cag interferes with the cell cycle might offer a possible explanation for the increased risk of gastric cancer (44). Whether these events also depend on CagA or on additional injected components is still not yet understood.

Figure 3

Cellular responses to CagA activation. Isolated cells forming the structure of an epithelium are represented during contacts with type I strains of Helicobacter pylori (defined as positive for expression, translocation, and activation of CagA). Two predominant (more...)

Among the CagA-independent pathways, the induction of interleukin-8 (IL-8) secretion by gastric epithelial cells was the first signaling event that was connected to infection with type I strains (18). Mutational analysis demonstrated that most of the cag genes were essential for increased IL-8 secretion. Only four cag mutants, cagA, cagF, cagN, and the virD4 homolog did not show any difference in their IL-8 levels when compared to the wild-type strain (9, 17, 18, 39, 48).

IL-8 is the major chemokine that causes the infiltration of neutrophils to the infection site. The result of these processes is a strong inflammatory response in the gastric mucosa. Since H. pylori is one of the few pathogens that chronically infect the host, a continuous inflammation may thus be an important factor for the development of gastric disease. Two cag-dependent pathways were described that lead to the induction of IL-8 secretion in H. pylori. The first one depends on the activation of transcription factor nuclear factor-kappa B (NF-κB) via p21-activated kinase 1 (PAK1). PAK1 associates with and phosphorylates NF-κB interacting kinase (NIK), which then leads to the degradation of the inhibitory subunit IκBa from the NF-κB complex. The released NF-κB dimerizes and enters the nucleus, where it binds and positively regulates the IL-8 promoter. cag mutants that do not activate NF-κB also fail to induce IL-8 secretion (21, 23). A second transcription factor that positively regulates the IL-8 promoter is activator protein 1 (AP-1) (40). The molecular mechanism of AP-1 activation is initiated through small GTPases of the Rho family that activate ERK/MAP kinase cascades. Pathways further downstream involve MKK4 and JNK, and their activation results in phosphorylation of c-Jun and Elk-1 (38, 40). Elk-1 then upregulates c-fos transcription. Homo- and heterodimers of the proto-oncogenes c-Fos and c-Jun finally form a complex with AP-1 that can bind the IL-8 promoter and induce transcription of IL-8. The bacterial molecules that are mediating these inflammatory events are not yet identified. We can imagine that either the contact of the type IV system with the host cell is sufficient to trigger the response or the type IV secretion system exports a factor that mediates the IL-8 secretion.

Recent observations characterized the involvement of the cag PAI in phagocytosis of H. pylori. Mononuclear phagocytes were shown to engulf H. pylori within approximately 4 minutes, but the internalized bacteria were not killed if type I strains were used for the infection. Instead, H. pylori induced homotypic phagosome fusions leading to the formation of large vacuoles. These so-called megasomes contained multiple organisms that were viable for at least 24 h. The process was not induced by cag-negative mutants and was dependent on intact microtubules and bacterial protein synthesis (2). In a different study, a functional type IV secretion system and de novo protein biosynthesis, but not CagA, were required to block phagocytosis through polymorphonuclear leukocytes and monocytes. This effect resembled antiphagocytosis by Yersinia enterocolitica and prevented the engulfment not only of H. pylori but also of latex beads and cells of Neisseria gonorrhoeae (45). This finding suggests that CagA is not the only molecule that is injected into the host cell, but that further cag-encoded factors are also translocated and may be responsible for induction of the antiphagocytotic response.

Conclusions

The function of the cag PAI in H. pylori-associated disease has been the subject of controversy ever since its discovery in 1993. Clinical studies with different outcomes have been performed to test whether the presence of the cag PAI is connected with peptic ulcer disease or gastric cancer. Often these studies were based on PCR amplifications of the cagA gene, which was used as a marker for the cag PAI. In some of these studies, clear proof of the involvement of cag in severe forms of gastric diseases often was not provided. The problem in some of these studies was that cag was examined as a static DNA region (cagA present or not) rather than as a DNA sequence encoding a sophisticated secretion apparatus with the function of injecting virulence factors into the host cell (Fig. 4). The work of various laboratories during the past year therefore has provided better insight into the biological complexity of H. pylori-host interactions and the molecular function of the cag PAI in pathogenesis. Future approaches have to consider that the presence of the cagA gene alone is not equal to virulence. A single point mutation anywhere in the 40-kbp region may abolish the function of the type IV secretion system and CagA transloction into the host cell (Fig. 4).

Figure 4

Mutations affecting CagA activation. (a) Mutations in CagA expression and structure, (b) mutations in the component of the type IV secretion system, and (c) mutations for absence of phosphorylated CagA or for absence or unresponsiveness of intracellular (more...)

We may assume that the majority of type I strains are de facto less virulent due to an abrogated function of the secretion system or of the cagA gene. Additionally, we have to consider that bacteria are just one term of the equation. No matter how many virulence factors they possess, the host has receptors and targets for these factors and allelic polymorphism may dictate resistance. If we restrict our attention to the group of patients with peptic ulcer disease and who are infected with H. pylori, we will find that more than 90% of the individuals carry type I strains. On the basis of the nature of the cag-encoded factors, it is most likely that the translocation of virulence proteins including CagA through the type IV secretion system is one important molecular mechanism by which H. pylori can, in the long term, influence the clinical outcome of the infection.

Acknowledgments

We thank C. Montecucco and W. Pansegrau for helpful suggestions. We also acknowledge S. Guidotti for the unpublished observations on cag deletion. We gratefully acknowledge G. Corsi for the illustrations and C. Mallia for editorial assistance.

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