**8. Activity of VacA**

Epithelial cell vacuolation in vitro occurs by VacA; however, this does not cause cell death quickly. Cell death in human gastric epithelial cells that are exposed to high doses of toxin is reported after 2 days [56]. On the other hand, cell death does not normally happen in immortalized cell lines exposed to the toxin. As an example, incubation of AZ-521 gastric epithelial cells with VacA for several hours causes decreased mitochondrial ATP production and decreased oxygen utilization but does not result in cell death [57].

The exact mechanisms of binding and uptake of VacA by cells are not clearly understood yet. The prototypic s1/m1 form of VacA binds to HeLa cells in a saturable manner recognized by flow-cytometry analysis [58]. However, saturable binding has not been indicated with classical ligand binding assays with 12I-labeled VacA [43]. Activation of VacA by acid treatment significantly increases its vacuolating activity but does not remarkably increase its binding to HeLa or Baby Hamster Kidney (BHK) cells [40, 58]. However, binding of the toxin to the gastric cell line AZ-521 is increased by acid activation [41]. A number of specific VacA receptors were proposed. Activated VacA binds to a 250 kDa receptor protein-tyrosine phosphatase β (RPTPβ) in the AZ-521 system that regulates intracellular tyrosine phosphorylation [41, 59].

Autotransporters are a family of secreted bacterial proteins, which are determined by mentioned features, together with a pair of cysteine residues near the carboxy-terminus of the mature secreted protein. It is suggested that RPTPβ has an important role in binding VacA to cells and following intoxication. Treatment of the HL-60 cell line with phorbol 12-myristate 13-acetate (PMA) causes stimulation of RPTPβ expression that is occurred with stimulation of VacA sensitivity [60]. BHK-21 cells are insensitive to VacA, but transfection with expression vectors including the RPTPβ gene can make them sensitive. Antisense oligonucleotides in PMA-treated HL-60 cells lead to ablation of RPTPβ synthesis. As a result, a considerable reduction occurs in VacA-induced vacuolation. An unidentified 140 kDa protein in AZ-521 and AGS cells and the epidermal growth factor receptor in HeLa cells [61, 62] are two other particular VacA receptors. These evidence suggested that multiple surface-binding sites recognized by both inactive and activated VacA exist; in addition, specific VacA receptors exist that are variably expressed in different cell lines.

Investigations on the purified 58 kDa fragment from a mutant *H. pylori* strain represent that this protein binds to HeLa cells with kinetics similar to those of the intact toxin [45]. The binding of VacA to cells is inhibited by polyclonal antiserum reactive with the 58 kDa domain [54]. Several natural forms of VacA have significant divergent amino acid sequences in the 58 kDa domain, which are called m2 forms. These forms cause vacuolation in a more confined range of cultured epithelial cell lines. Differences in cell binding would be a reason for this [34]. VacA with a type m2 58 kDa domain, that did not cause HeLa cell vacuolation when applied externally, affected vacuolation when expressed from a plasmid in the HeLa cell cytoplasm. This indicates that m2 VacA is entirely active but cannot get to its site of action. This would be

Investigations by naturally occurring and engineered m1/m2 chimeric proteins [55] propose that an ~40 amino acid region near the amino-terminal end of the 58 kDa domain is required

Epithelial cell vacuolation in vitro occurs by VacA; however, this does not cause cell death quickly. Cell death in human gastric epithelial cells that are exposed to high doses of toxin is reported after 2 days [56]. On the other hand, cell death does not normally happen in immortalized cell lines exposed to the toxin. As an example, incubation of AZ-521 gastric epithelial cells with VacA for several hours causes decreased mitochondrial ATP production and

The exact mechanisms of binding and uptake of VacA by cells are not clearly understood yet. The prototypic s1/m1 form of VacA binds to HeLa cells in a saturable manner recognized by flow-cytometry analysis [58]. However, saturable binding has not been indicated with classical ligand binding assays with 12I-labeled VacA [43]. Activation of VacA by acid treatment significantly increases its vacuolating activity but does not remarkably increase its binding to HeLa or Baby Hamster Kidney (BHK) cells [40, 58]. However, binding of the toxin to the gastric cell line AZ-521 is increased by acid activation [41]. A number of specific VacA receptors were proposed. Activated VacA binds to a 250 kDa receptor protein-tyrosine phosphatase β (RPTPβ) in the AZ-521 system that regulates intracellular tyrosine phosphorylation [41, 59]. Autotransporters are a family of secreted bacterial proteins, which are determined by mentioned features, together with a pair of cysteine residues near the carboxy-terminus of the mature secreted protein. It is suggested that RPTPβ has an important role in binding VacA to cells and following intoxication. Treatment of the HL-60 cell line with phorbol 12-myristate 13-acetate (PMA) causes stimulation of RPTPβ expression that is occurred with stimulation of VacA sensitivity [60]. BHK-21 cells are insensitive to VacA, but transfection with expression vectors including the RPTPβ gene can make them sensitive. Antisense oligonucleotides in PMA-treated HL-60 cells lead to ablation of RPTPβ synthesis. As a result, a considerable reduction occurs in VacA-induced vacuolation. An unidentified 140 kDa protein in AZ-521 and AGS cells and the epidermal growth factor receptor in HeLa cells [61, 62] are two other particular VacA receptors. These evidence suggested that multiple surface-binding sites

because of inability to bind to the cell [11].

66 Helicobacter Pylori - New Approaches of an Old Human Microorganism

**8. Activity of VacA**

for HeLa cell vacuolation and can have a role in HeLa cell binding.

decreased oxygen utilization but does not result in cell death [57].

Both 58 and 37 kDa regions are needed for VacA internalization [45]. VacA should be preactivated by disposal of acid or alkali, in order to be internalized [43]. Internalization happens through an energy-dependent process; the exact nature of which is not clear. However, it may be a receptor-mediated endocytosis. VacA molecules localize in membrane vesicles, after internalization [54]. Then localized VacA molecules are transported along the endocytic pathway to vacuolar-type (V-) ATPase-positive late endosomes and lysosomes. In this state, they accumulate and persist for some days [63, 64].

The first defined action of VacA was induction of intracellular vacuoles [64, 65]. The vacuolar membranes include both late endosomal and lysosomal markers, indicating that the vacuoles are derived from these sections [66, 67].

The complete activity of V-ATPase and the existence of weak bases are needed for the formation of VacA-induced vacuoles, which indicated that vacuoles are derived from the accumulation of weak bases within acidic sections, and with water influx and swelling followed [63, 64, 68, 69]. Moreover, the membrane traffic regulator rab7 and the actin-cytoskeleton-associated Rac1 are two small GTP-binding proteins that involved in vacuole biogenesis [70, 71]. Rac1 and rab7 are related with the membrane of VacA-induced vacuoles. The expression of rab7 or Rac1 dominant negative mutants inhibits vacuolization, and the expression of rab7 or Rac1 dominant positive mutants potentiates vacuolization. It has been proposed that membrane fusion events and the cytoskeleton supporting late endosomal sections regulated vacuole development. VacA destructs the transport of acidic hydrolases to lysosomes and causes the release of these enzymes into the extracellular medium in HeLa cells [72]. VacA caused decrement of the degradative power of HeLa cell lysosomes and also decrement of the antigenprocessing compartment of B lymphocytes [72, 73].

VacA is unable to vacuolate epithelial monolayers of MDCK I, T84, or epH4 cells on porous filters. In addition, MDCK I, T84, or epH4 cells do not show signs of endolysosomal dysfunction [74].

Subsequently, disposal to VacA, transepithelial electrical resistance (TER) reduces, occurred with an increase in transepithelial flux of low-molecular-weight molecules [74]. There are some reasons, which propose that VacA modulates the resistance of these model epithelia through a paracellular effect. These reasons include the size selectivity of this increased epithelial permeation, lack of accompanying vacuolation, and lack of redistribution of junctional proteins. Just epithelial cell monolayers capable of expanding a TER higher than 1000–1200 Ω/cm2 are affected. By utilizing the isogenic mutant strains, this is confirmed that the effect is dependent on VacA [31]. In MDCK cells, m2 type of VacA decreases TER. However, it does not lead to vacuolation in this cell line even when cells are nonconfluent [31]. It is corroborated that vacuolation and increased permeability of monolayers are separate and independent effects.

VacA constructs ion channels in model lipid bilayers and cell plasma membranes. This occurrence may underlie all the other consequences of VacA. Acidic conditions cause disassembly of the inactive VacA oligomer, which permits insertion of the toxin into lipid bilayers [66, 73, 75].

Investigations with planar model membranes represent that membrane insertion is followed by the formation of voltage-dependent, low-conductance (10–30 pS in 2 M KCl), and anionselective channels [76, 77].

proved by most of these studies from outside Asia that s1 strains are more often associated

*H. pylori* vacA s1 and m1 strains are related to higher levels of inflammation in the gastric mucosa and increased risk for gastric atrophy and carcinoma. In addition, it was revealed that the determinant of cytotoxicity i1 allele is related to gastric carcinoma. So, evaluation of characterization of this region as a determinant of the clinical outcome of *H. pylori* infection

*Helicobacter pylori* has been investigated since its first culture in 1982 from a gastric biopsy. Cytotoxin-associated gene A (CagA) and the vacuolating cytotoxin (VacA) are the virulence factors which are produced by *H. pylori* and are related to an increase in the risk of disease extension [9]. VacA has a significant role in the pathogenesis of *H. pylori*-associated diseases, especially in peptic ulceration and distal gastric adenocarcinoma. For this reason, VacA has been studied widely. VacA is still under examination in order to find out its accurate role in these diseases. However, VacA makes *H. pylori* able to colonize in the human gastric mucosa. Moreover, VacA could have a role in gastric epithelial damage. For this reason, VacA is a target for therapeutic intervention and also is considered for usage in a vaccine against *H. pylori*.

\*, Abbas Doosti<sup>2</sup>

and Reza Ranjbar<sup>3</sup>

VacA Genotype in *Helicobacter pylori* http://dx.doi.org/10.5772/intechopen.81203

with peptic ulceration or gastric carcinoma than s2 strains [32, 81, 88–91].

could be used [28].

**10. Conclusion**

**Author details**

, Amirhossein Sheikhshahrokh1

Baqiyatallah University of Medical Sciences, Tehran, Iran

Gastroenterology. 2008;**134**(1):306-323

\*Address all correspondence to: amirhossein.sheikhshahrokh@yahoo.com

2 Biotechnology Research Center, Shahrekord Branch, Islamic Azad University,

3 Molecular Biology Research Center, Systems Biology and Poisonings Institute,

[1] Control, C.F.D. and Prevention. CDC Health Information for International Travel 2014:

[2] Amieva MR, El–Omar EM. Host-bacterial interactions in *Helicobacter pylori* infection.

The Yellow Book. Oxford, United Kingdom: Oxford University Press; 2013

1 Young Researcher Club, Islamic Azad University, Shahrekord, Iran

Elnaz Saeidi1

Shahrekord, Iran

**References**

Patch clamp analysis of HeLa cells indicates that VacA forms plasma membrane channels with features similar to those perceived in model membranes [78]. Different anion channel blockers inhibit VacA channels in vitro with various powers and are able to prevent and partially inverse vacuolation of HeLa cells [78, 79], informing an essential role of the anion channel in vacuolation [41]. With permitting anions to permeate into late endosomes, the endocytosed VacA channel increases the turnover of the electrogenic V-ATPase that causes accumulation of weak bases and leads to vacuole formation by water influx [80, 81]. Because of that, internalization of surface-bound VacA is required for the further development of vacuolation; this hypothesis is acceptable [43]. Vacuolation in this model can be considered as a side effect of the massive accumulation of endocytosed VacA channels in endolysosomes. With 5-nitro-2-(3-phenylpropylamine) benzoic acid (NPPB), VacA epithelial permeabilization of MDCK I cells can be partly prevented and reversed, the most efficient blocker of VacA channels, implying that epithelial permeabilization, similar to vacuolation, is less important for the formation of apical anion channels [78]. VacA induces an increased apical anion secretion in Caco-2 cells, and this also is blocked by NPPB [82], implying that it is also because of VacA anion channel formation.

#### **9. Discussion**

*H. pylori* is considered as a significant cause of chronic active gastritis, peptic ulcer, and atrophic gastritis. It is related to an enhanced risk of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT). VacA is a virulence factor related to peptic ulcer. Moreover, oral administration of VacA leads to gastric mucosal damage in mice. It could be concluded that VacA might contribute to epithelial cell damage or peptic ulceration in H. pylori-infected humans [83].

A toxin that has damaging outcomes on epithelial cells is produced by *H. pylori*. In addition, colonization of *H. pylori* has contributed in the development of peptic ulceration. By this information, it could be concluded that VacA directly harms the gastric and duodenal epithelium in vivo; therefore, it leads to ulcers. *H. pylori* strains which have vacuolating cytotoxin activity in vitro are more often associated with disease than *H. pylori* strains which are noncytotoxic strains. There is a significant association between vacuolating activity and peptic ulcer disease, which is revealed by some studies all over the world [32, 84–86]; however, this association is not true in all situations. Often noncytotoxic *H. pylori* isolates from patients with peptic ulceration, and cytotoxic *H. pylori* isolates from patients without peptic ulceration. However, explanation of these studies depends on some factors.

There have been many studies on the relationship between specific vacA genotypes and diseases, which are developed by multiple vacA genotypes and explained by polymerase chain reaction (PCR)-based methodology for discrimination between them [32, 81, 87]. It was proved by most of these studies from outside Asia that s1 strains are more often associated with peptic ulceration or gastric carcinoma than s2 strains [32, 81, 88–91].

*H. pylori* vacA s1 and m1 strains are related to higher levels of inflammation in the gastric mucosa and increased risk for gastric atrophy and carcinoma. In addition, it was revealed that the determinant of cytotoxicity i1 allele is related to gastric carcinoma. So, evaluation of characterization of this region as a determinant of the clinical outcome of *H. pylori* infection could be used [28].

#### **10. Conclusion**

Investigations with planar model membranes represent that membrane insertion is followed by the formation of voltage-dependent, low-conductance (10–30 pS in 2 M KCl), and anion-

Patch clamp analysis of HeLa cells indicates that VacA forms plasma membrane channels with features similar to those perceived in model membranes [78]. Different anion channel blockers inhibit VacA channels in vitro with various powers and are able to prevent and partially inverse vacuolation of HeLa cells [78, 79], informing an essential role of the anion channel in vacuolation [41]. With permitting anions to permeate into late endosomes, the endocytosed VacA channel increases the turnover of the electrogenic V-ATPase that causes accumulation of weak bases and leads to vacuole formation by water influx [80, 81]. Because of that, internalization of surface-bound VacA is required for the further development of vacuolation; this hypothesis is acceptable [43]. Vacuolation in this model can be considered as a side effect of the massive accumulation of endocytosed VacA channels in endolysosomes. With 5-nitro-2-(3-phenylpropylamine) benzoic acid (NPPB), VacA epithelial permeabilization of MDCK I cells can be partly prevented and reversed, the most efficient blocker of VacA channels, implying that epithelial permeabilization, similar to vacuolation, is less important for the formation of apical anion channels [78]. VacA induces an increased apical anion secretion in Caco-2 cells, and this also is blocked by NPPB [82], implying that it is also because of

*H. pylori* is considered as a significant cause of chronic active gastritis, peptic ulcer, and atrophic gastritis. It is related to an enhanced risk of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT). VacA is a virulence factor related to peptic ulcer. Moreover, oral administration of VacA leads to gastric mucosal damage in mice. It could be concluded that VacA might contribute to epithelial cell damage or peptic ulceration in H. pylori-infected

A toxin that has damaging outcomes on epithelial cells is produced by *H. pylori*. In addition, colonization of *H. pylori* has contributed in the development of peptic ulceration. By this information, it could be concluded that VacA directly harms the gastric and duodenal epithelium in vivo; therefore, it leads to ulcers. *H. pylori* strains which have vacuolating cytotoxin activity in vitro are more often associated with disease than *H. pylori* strains which are noncytotoxic strains. There is a significant association between vacuolating activity and peptic ulcer disease, which is revealed by some studies all over the world [32, 84–86]; however, this association is not true in all situations. Often noncytotoxic *H. pylori* isolates from patients with peptic ulceration, and cytotoxic *H. pylori* isolates from patients without peptic ulceration. However,

There have been many studies on the relationship between specific vacA genotypes and diseases, which are developed by multiple vacA genotypes and explained by polymerase chain reaction (PCR)-based methodology for discrimination between them [32, 81, 87]. It was

selective channels [76, 77].

68 Helicobacter Pylori - New Approaches of an Old Human Microorganism

VacA anion channel formation.

explanation of these studies depends on some factors.

**9. Discussion**

humans [83].

*Helicobacter pylori* has been investigated since its first culture in 1982 from a gastric biopsy. Cytotoxin-associated gene A (CagA) and the vacuolating cytotoxin (VacA) are the virulence factors which are produced by *H. pylori* and are related to an increase in the risk of disease extension [9].

VacA has a significant role in the pathogenesis of *H. pylori*-associated diseases, especially in peptic ulceration and distal gastric adenocarcinoma. For this reason, VacA has been studied widely. VacA is still under examination in order to find out its accurate role in these diseases. However, VacA makes *H. pylori* able to colonize in the human gastric mucosa. Moreover, VacA could have a role in gastric epithelial damage. For this reason, VacA is a target for therapeutic intervention and also is considered for usage in a vaccine against *H. pylori*.
