**5.1** *cag* **PAI (Pathogenicity Island)**

Although the *H. pylori* infection nearly always triggers chronic active gastritis, most affected patients are free of apparent health signs and have no other complications [52]. This lead to the belief that some strains could be more virulent than others. Early studies of variations of *H. pylori* strains demonstrated the capacity of such virulent strains to cause morphological changes, vacuolizations and successive degeneration from in vitro-cultivated cells. This pathogenicity is linked [53]. This activity was then linked to the presence of a protein with a molecular mass of approximately 140 kDa that was named CagA (for "cytotoxinassociated gene A").

The CagA protein is a highly immunogenic protein encoded by the *cagA* gene [54]. About 50–70 percent of *H. pylori* strains have this gene [55] and is a marker for the presence of a genomic PAI of about 40 kb that, depending on the strain analyzed, encodes between 27 and 31 proteins [54]. Strains carrying the Cag PAI are called CagA+ strains, as their ability to cause major antibody titers against the CagA marker protein is widely recognized in patients. In CagA patients, inflammation is typically higher and the probability of developing a signs (peptic ulcer or gastric cancer) in western populations is considerably higher [56], though not in Asian populations [57]. While CagA+ strains are associated with a higher risk of ulceration, gastritis and gastric cancer, cag PAI strains are also associated with a higher risk of peptic ulcer or gastrointestinal cancer, even though at a smaller frequency.

Eighteen cag PAI-coded proteins are used to form a type IV secretive unit that forms a structure like a syringe that is able to penetrate gastric epithelial cells and to promote the translocation of CagA, peptidoglycan, and probably other bacterial components into host cells [58] (**Figure 3**). Once delivered inside the cell, the CagA protein is phosphorylated at tyrosine residues in EPIYA motifs [59] by Src family kinases [60]. Phosphorylated CagA interacted then with a number of host signaling molecules, including tyrosine phosphate SHP-2, which results in morphological changes in the epithelial cells [61].

Apoptosis of T cells is impaired by the cag PAI since the immune response is also affected [62]. The association of type IV formation with the host cell also results in pro-inflammatory cytokines in epithelial cell induction [63].

It was originally believed that this proinflammatory cytokines are caused by a CagA protein itself, but nowadays CagA only plays, if any, a minor role in triggering them [63]. It is possible that the intimate contact with the IV-type form contributes to peptidoglycan leak into the eukaryotic cell [64] (**Figure 3**), although it cannot be ruled out completely that the activation of the IL-8 signaling cascade results from the translocation of a thus-far-unknown bacterial factor [63].

Tyrosine Phosphorylation is necessary for binding CagA to SHP-2 within the CagA EPIYA motif. [65]. The number of EPIYA tyrosine phosphorylation motives within the CagA proteins of various *H. pylori* isolates varies considerably. CagA is specifically correlated with the amount of repetitions of tyrosine phosphorylation. [66]. Strains with a larger number of CagA repetitions cause more marked morphological changes in cultivated epithelial cells [67] and an increased risk of gastric carcinogenesis being correlated. CagA also interacts through SH2 domains with the

#### **Figure 3.**

*Schematic representation of the different roles of the Cag type IV secretion system in immune modulation, cell proliferation, and morphological changes [42].*

c-terminal Src kinase, which contributes to c-tyrosine Src's kinase inactivation. This inactivation, since it mediates CagA tyrosine phosphorylation, leads to a decrease in CagA phosphorylation, thereby creating a feedback loop to control CagA behavior [65]. This cross talk of host-pathogen results in tested virulence and can thus serve to colonize the host for a lifetime.

#### **5.2 VacA vacuolating cytotoxin**

Around 50% of all strains of *H. pylori* secrete VacA, a highly immunogenic 95 kDa protein that is causing massive vacuolisation of epithel cells in vitro [68]. VacA is a key factor in both peptic ulceration and gastric cancer pathogenesis. Though VacA is not necessary for in vitro growth of *H. pylori* the murine gastric colonization by *H. pylori* has been shown to make a substantial contribution [69].

The activities of VacA include development of the membrane channel, endosomal and lysosomal disorders, incorporate cell receptor signaling effects and cytoskeleton-related interference with cell-dependent functions, apoptosis induction and immune regulation (**Figure 4**). Although vacuolization is readily observed in vitro, it does not seem to occur *in vivo* [42]. The VacA protein is formed with a protoxin of 140 kDa and is broken into the shape of 95 kDa as it is secreted.

While all strains have a functional vacA gene, the vacuolating activities among strains differ considerably [69]. This is due to the sequence heterogeneity within the *vacA* gene at the signal region (s) and the middle region (m). The s region of the gene, which encodes the signal peptide, occurs as either an s1 or s2 type, whereas the m region, which contains the p58 cell binding domain, exists as an m1 or m2 type [70].

In the cell epithelial membrane, VacA forms pores that cause urea and anions to be released from the host cells. It also enhances transcellular penetration, resulting in nutrient and cation releases [71]. Interestingly, a major portion of the secreted toxin does not go to the environment, but is bound to the outer membrane of *H. pylori*. These toxin clusters are passed to the host cell surface following bacterial

#### **Figure 4.**

*The VacA protein influences cellular processes via different routes, thus assisting in chronic colonization of the gastric mucosa by* H. pylori*. (1) Surface-bound VacA may be directly delivered to the cell membrane. Secreted VacA may either (2) bind to a cell membrane receptor and initiate a proinflammatory response, (3) be taken up directly by the cell and be trafficked to the mitochondria and induce apoptosis, (4) be taken up by pinocytosis and induce vacuolization, (5) form a membrane channel, resulting in leakage of nutrients to the extracellular space, or (6) pass through the tight junctions and inhibit T-cell activation and proliferation. (Modified with permission from* Nature Reviews Microbiology *[23].*

interaction with their host cells and have their toxic effects. This touch-dependent mechanism for direct delivery proposes the inclusion in bacterial-cell contact of particular receptors. However, such a receptor has not yet been identified [72].

Secreted VacA can be further processed into a 33-kDa N-terminal fragment and a 55-kDa C-terminal fragment through proteolytic cleavage. In the development of anion channels, the N-terminal protein plays an important role while C-terminal proteins mediate cell binding [73].

In spite of the proteolytic cleavage, these fragments remain noncovalently associated with each other [74]. Spontaneously purified VacA forms oligomeric aggregates and disassembles in the active monomers of pores in the cell membrane after exposed to acidic pH. Spontaneously purified VacA forms oligomeric aggregates and disassembles in the active monomers of pores in the cell membrane after exposed to acidic pH but are likely to be an *in vitro* artifact [75].

While several VacA-mediated effects are induced by membrane binding and pore formation, VacA also reaches the cytosol, then accumulates in the mitochondrial inner membrane and causes apoptosis, activating endogenous mitochondrial channels [76]. The proapoptotic influence of VacA is based on the cell type and may be restricted to gastric epithelial cells like parietal cells. This may result in reduced acid secretion, thereby predisposing for development of gastric cancer [77].
