**5. VacA proteins**

the pathogenesis of *H. pylori*-related diseases, in specific peptic ulceration and distal gastric adenocarcinoma. The accurate function of VacA in these diseases is still under research, but VacA may contribute to the capacity of *H. pylori* to colonize and persist in the human gastric mucosa and may also contribute immediately to gastric epithelial damage. Therefore, VacA is a purpose for therapeutic intervention and a candidate for inclusion in a vaccine against

Vacuolating cytotoxin (vacA) is the most commonly identified virulence factor among *H. pylori* strains. VacA belongs to the group of genes with mutable genotypes related to damage to gastric epithelial cells. This gene exists in almost all strains of *H. pylori*. This gene is polymorphic and contains variable signal regions (type s1 or type s2) and midregions (type m1 or type m2) [27] and intermediate regions (i1 and i2 alleles, and the rare i3 allele) [28] (**Figure 1**). There are various levels of its cytotoxicity that is caused by the variety of signal (s) and mid (m) regions of vacA gene [29]. S region variations are more related to the vacuolating activity of vacA, and m region variations have effect on binding of the toxin to the host cells, as reasons are

A copy of the toxin gene, vacA, exists in all *H. pylori* strains. The vacA transcript is monocistronic. Transcriptional start point in this gene is located about 119 nucleotides upstream from the ATG start codon [25, 30]. The capacity of *H. pylori* to induce vacuolation in epithelial cells abrogates by insertional mutagenesis of vacA. In addition, it interrupts a number of other vacA-induced toxic effects [24, 25, 31]. Alleles of vacA from about 25 different *H. pylori* strains

have been sequenced and range from 3864 to 3933 nucleotides in length [8, 24, 32–34].

**4. The association of vacA types with cytotoxin production**

Significant genetic diversity exists between vacA alleles from different strains. These alleles could be classified into various families. The most significant studied form of VacA is encoded

*H. pylori*.

**3. The** *vacA* **gene**

**Figure 1.** VacA gene.

contributed to define cell specificity [29].

62 Helicobacter Pylori - New Approaches of an Old Human Microorganism

VacA encodes a protein with a mass of about 140 kDa; however, under denaturing conditions, the mature secreted VacA toxin drifts as a band of almost 90 kDa [7, 8, 24–26]. A comparison of the amino-terminal sequence of the mature secreted toxin with that predicted for the protoxin shows that a 33-amino-acid amino-terminal signal sequence is cleaved during the procedure of VacA secretion. Investigations by antisera raised against various regions of recombinant VacA show that a polypeptide of about 33 kDa isolated from the carboxy-terminal portion of the protoxin stays localized to the bacteria and is not secreted [26]. This carboxy-terminal portion of VacA seems to contain amphipathic β-sheets capable of forming a β-barrel structure and has a terminal phenylalanine-containing motif that is available in several outer membrane proteins [25]. These qualities, with a pair of cysteine residues nearly the carboxy-terminus of the mature secreted protein, are specification of a family of secreted bacterial proteins called autotransporters [35]. Autotransporters for export across the bacterial outer membrane do not need any auxiliary proteins. By studying the Neisseria gonorrhoeae IgA1 protease, we achieved this information of autotransporter export. Translocation of IgA1 protease through the bacterial cytoplasmic membrane is achieved via a Sec-mediated process and is accompanied by cleavage of an amino-terminal signal peptide. After inserting the carboxy-terminal β-barrel domain into the outer membrane, it functions as a pore through that the residue of the molecule passes. The mature secreted IgA1 protease is produced by autoproteolytic cleavage. The carboxy-terminal domain stays related to the outer membrane [36].

Primary studies showed, despite mature VacA monomers are about 90 kDa in mass, that the toxin exists as a much larger complex or aggregate under nondenaturing conditions [7]. Lupetti et al. investigated the ultrastructure of purified VacA using deep-etch electron microscopy. They illustrated that the toxin forms into large flower-shaped complexes that appear to consist of a central ring surrounded by six or seven "petals" [37]. An accurate view of the surface of VacA oligomers is presented by three-dimensional reconstructions of these deepetch metal copies (**Figure 2**) [38]. Moreover, the classical flower-like complexes, VacA, could be assembled into other type of complex, that is named a "flat form," which includes of six or seven petals without a notable central ring [37, 39].

carboxy terminus of the protein. Proteolytic cleavage occurs at a site containing multiple charged amino acids [26]. In fact, the 37 and 58 kDa fragments of VacA are considered as

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

Burroni et al. manufactured an *H. pylori* mutant in order to specify whether cleavage of VacA into 37 and 58 kDa fragments is needed for toxin activity. In this *H. pylori* mutant, the region of vacA encoding the 46 amino acids flanking the VacA cleavage site was removed [48]. Because of this fact that this mutant VacA was entirely active, it is informing that cleavage of the exposed loop is not required for activity. While the wild-type VacA produced by the parent strain prefer to form seven-sided complexes, in contrast, the mutant prefers to form six-sided complexes. This revealed that deleting the exposed loop presented structural

In experiments wherever mutant forms of vacA under the control of a eukaryotic promoter have been expressed from plasmids in the cytosol of epithelial cells, it has been explained that the minimal region of VacA is required for vacuolating activity [11]. These experiments revealed that the epithelial cell lines, which transfected with plasmid constructs encoding either the full-length ~90 kDa secreted toxin or amino- or carboxy-terminally truncated fragments. In addition, these experiments represented that a VacA protein lacking most of the carboxy-terminal 58 kDa domain preserved complete vacuolating activity [49, 50]. By eliminating 10 amino acids from the amino-terminus, activity was entirely abolished, and by eliminating 6 amino acids from the amino-terminus, activity was only in part abolished [49, 50]. The minimal VacA domain that presented complete vacuolating activity when expressed intracellularly was a peptide containing amino acids 1–422, which is the 37-kDa domain plus a fragment of the 58-kDa domain [50]. The 37 kDa fragment was inactive in alone, but coexpression of this fragment with a fragment including the amino-terminal 165 amino acids of the 58 kDa fragment resulted in complete vacuolating activity [50]. A conceivable explanation for the importance of the VacA amino terminus was determined by hydrophobicity plots. In fact, the only hydrophobic region in VacA is amino acids 1–32 of

A *H. pylori* vacA partial deletion mutant was produced, which lacked codons for amino acids 6–27, for more research [51]. The structure of mutant VacA did not have variations compared

In addition, alanine scanning mutagenesis showed that point mutations at proline 9 or glycine 14 entirely abrogated VacA activity [52]. Another factor that abrogated toxin activity is the addition of an amino-terminal hydrophilic extension to VacA [53]. As a result, it is obvious

It is demonstrated that amino acid sequences located in the carboxy-terminal portion of the

that the amino-terminal hydrophobic region has an important role in toxin activity.

subunits or domains of the holotoxin [47].

this region, and it is long enough to span a membrane.

**7. Receptor binding region**

mature protein mediated binding of VacA to cells.

to wild-type VacA; however, mutant VacA lacked cytotoxic activity.

restriction.

**Figure 2.** VacA oligomer.

The petals that contain the flat form generally radiate from the center of the complex with a specific clockwise chirality. Several models have suggested clarifying the assembly of VacA into flower-like complexes and chiral flat forms. In one of these models, the flower-like forms are proposed to contain six or seven monomers of about 90 kDa [38, 39]. In another model, the flower-like forms are considered to be dodecamers or tetradecamers of VacA monomers of about 90 kDa, and flat forms are proposed to be hexamers or heptamers [37]. In acidic or alkaline pH, VacA oligomers separate into monomeric parts of approximately 90 kDa, each measuring of about 6 by 14 nm [37, 40, 41]. This pH-mediated disassembly is related by a marked enhance in VacA cytotoxic activity [39, 41–43].

This opinion exists that VacA monomers have more cytotoxic activity than water-soluble VacA oligomers. Subsequent researches about VacA structure have been undertaken using atomic force microscopic imaging of purified toxin bound to supported lipid bilayers [44].

Two-dimensional crystalline arrays of VacA on lipid bilayers include an arranged array of hexagonal central rings connected by thin connectors to peripheral domains.

In-frame deletions in the portion of VacA encoding the amino-terminal region of the toxin produced mutant strains of *H. pylori*. Mutant strains of *H. pylori* express truncated VacA proteins. These proteins are secreted, though fail to oligomerize and lack recognizable cytotoxic activity [45, 46]. VacA Δ91–330 is a mutant VacA protein that has water-soluble dimeric form, which has an ultrastructural appearance similar to that of the peripheral petals of VacA oligomers [45]. The peripheral petals of VacA oligomers can be consistent with the carboxyterminal portion of the mature secreted VacA polypeptide.

#### **6. Functional domains in VacA**

The purified ~90 kDa VacA toxin through extended storage or incubation with trypsin break down into ~37 and ~58 kDa components, which are isolated from the amino terminus and carboxy terminus of the protein. Proteolytic cleavage occurs at a site containing multiple charged amino acids [26]. In fact, the 37 and 58 kDa fragments of VacA are considered as subunits or domains of the holotoxin [47].

Burroni et al. manufactured an *H. pylori* mutant in order to specify whether cleavage of VacA into 37 and 58 kDa fragments is needed for toxin activity. In this *H. pylori* mutant, the region of vacA encoding the 46 amino acids flanking the VacA cleavage site was removed [48]. Because of this fact that this mutant VacA was entirely active, it is informing that cleavage of the exposed loop is not required for activity. While the wild-type VacA produced by the parent strain prefer to form seven-sided complexes, in contrast, the mutant prefers to form six-sided complexes. This revealed that deleting the exposed loop presented structural restriction.

In experiments wherever mutant forms of vacA under the control of a eukaryotic promoter have been expressed from plasmids in the cytosol of epithelial cells, it has been explained that the minimal region of VacA is required for vacuolating activity [11]. These experiments revealed that the epithelial cell lines, which transfected with plasmid constructs encoding either the full-length ~90 kDa secreted toxin or amino- or carboxy-terminally truncated fragments. In addition, these experiments represented that a VacA protein lacking most of the carboxy-terminal 58 kDa domain preserved complete vacuolating activity [49, 50]. By eliminating 10 amino acids from the amino-terminus, activity was entirely abolished, and by eliminating 6 amino acids from the amino-terminus, activity was only in part abolished [49, 50]. The minimal VacA domain that presented complete vacuolating activity when expressed intracellularly was a peptide containing amino acids 1–422, which is the 37-kDa domain plus a fragment of the 58-kDa domain [50]. The 37 kDa fragment was inactive in alone, but coexpression of this fragment with a fragment including the amino-terminal 165 amino acids of the 58 kDa fragment resulted in complete vacuolating activity [50]. A conceivable explanation for the importance of the VacA amino terminus was determined by hydrophobicity plots. In fact, the only hydrophobic region in VacA is amino acids 1–32 of this region, and it is long enough to span a membrane.

A *H. pylori* vacA partial deletion mutant was produced, which lacked codons for amino acids 6–27, for more research [51]. The structure of mutant VacA did not have variations compared to wild-type VacA; however, mutant VacA lacked cytotoxic activity.

In addition, alanine scanning mutagenesis showed that point mutations at proline 9 or glycine 14 entirely abrogated VacA activity [52]. Another factor that abrogated toxin activity is the addition of an amino-terminal hydrophilic extension to VacA [53]. As a result, it is obvious that the amino-terminal hydrophobic region has an important role in toxin activity.

#### **7. Receptor binding region**

The petals that contain the flat form generally radiate from the center of the complex with a specific clockwise chirality. Several models have suggested clarifying the assembly of VacA into flower-like complexes and chiral flat forms. In one of these models, the flower-like forms are proposed to contain six or seven monomers of about 90 kDa [38, 39]. In another model, the flower-like forms are considered to be dodecamers or tetradecamers of VacA monomers of about 90 kDa, and flat forms are proposed to be hexamers or heptamers [37]. In acidic or alkaline pH, VacA oligomers separate into monomeric parts of approximately 90 kDa, each measuring of about 6 by 14 nm [37, 40, 41]. This pH-mediated disassembly is related by a

This opinion exists that VacA monomers have more cytotoxic activity than water-soluble VacA oligomers. Subsequent researches about VacA structure have been undertaken using atomic force microscopic imaging of purified toxin bound to supported lipid bilayers [44].

Two-dimensional crystalline arrays of VacA on lipid bilayers include an arranged array of

In-frame deletions in the portion of VacA encoding the amino-terminal region of the toxin produced mutant strains of *H. pylori*. Mutant strains of *H. pylori* express truncated VacA proteins. These proteins are secreted, though fail to oligomerize and lack recognizable cytotoxic activity [45, 46]. VacA Δ91–330 is a mutant VacA protein that has water-soluble dimeric form, which has an ultrastructural appearance similar to that of the peripheral petals of VacA oligomers [45]. The peripheral petals of VacA oligomers can be consistent with the carboxy-

The purified ~90 kDa VacA toxin through extended storage or incubation with trypsin break down into ~37 and ~58 kDa components, which are isolated from the amino terminus and

hexagonal central rings connected by thin connectors to peripheral domains.

marked enhance in VacA cytotoxic activity [39, 41–43].

64 Helicobacter Pylori - New Approaches of an Old Human Microorganism

**Figure 2.** VacA oligomer.

terminal portion of the mature secreted VacA polypeptide.

**6. Functional domains in VacA**

It is demonstrated that amino acid sequences located in the carboxy-terminal portion of the mature protein mediated binding of VacA to cells.

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

recognized by both inactive and activated VacA exist; in addition, specific VacA receptors

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

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

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

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 antigen-

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 dysfunc-

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

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

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].

are affected. By utilizing the isogenic mutant strains, this is confirmed

exist that are variably expressed in different cell lines.

accumulate and persist for some days [63, 64].

processing compartment of B lymphocytes [72, 73].

tion [74].

1000–1200 Ω/cm2

separate and independent effects.

are derived from these sections [66, 67].

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