Helicobacter pylori infection has been recognized as a worldwide problem. H. pylori infection is the most prevalent cause of chronic gastritis and has been related to peptic ulcer disease and gastric cancer. It is considered that H. pylori infects half of the world’s population. Several virulence factors are produced by H. pylori in which each of them is related to an increase in the risk of disease development. The vacuolating cytotoxin (VacA) is one of these virulence factors. The first defined action of VacA was induction of intracellular vacuolation. VacA uses a variation in other effects on target cells, such as disruption of mitochondrial functions, stimulation of apoptosis, and blockade of T-cell proliferation, for the induction of vacuolation. In addition, VacA has an important role for colonization of H. pylori in vivo.
- Helicobacter pylori
- vacuolating cytotoxin (VacA)
Study on this microaerophilic spiral-shaped bacterium is interesting.
For the first time in 1982, two Australian scientists Barry Marshall and Robin Warren identified
Multiple virulence factors are produced by
A type IV secretion system is encoded in the cag pathogenicity island (cagPAI), and it replaces CagA into gastric epithelial cells. It causes morphological changes and proinflammatory cytokine secretion .
The gastric mucosa of almost 50% of the world’s population has colonized by
The two independent
The 26695 genome was 24 kb larger than J99. However, both the J99 and 26695 genomes possessed a total (G + C)% of 39%. There is some similarity in J99 and 26695, such as average lengths of coding sequences, coding density, and the bias of initiation codons. The genome of J99, consistent with the genome of strain 26695, had no clearly recognizable origin of replication. Near the origin of replication in prokaryotes, specific genes, including dnaA, dnaN, and gyrA, are often detected. However, these genes are not in close nearness either to each other or to the repeated heptamer that was determined as nucleotide number one in both published
Additional evidence is that this position may be regarded as the replication origin achieved from using an algorithm that analyzes the bias of short oligomers whose direction is preferentially skewed around the replication origin of prokaryotes .
Leunk et al. found massive vacuolar degeneration of various cultured epithelial cell lines in supernatants from broth cultures of
Subsequent to the primary characterization of the toxin and its gene, research has focused on VacA structure, the mechanisms underlying VacA’s toxic activity, naturally happening differences between VacA proteins produced by various strains of
Studies on VacA has expanded, not only because of its potential as a novel tool for exploring features of eukaryotic cell biology but also mainly because of its supposed function in the pathogenesis of
Vacuolating cytotoxin (vacA) is the most commonly identified virulence factor among
A copy of the toxin gene, vacA, exists in all
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 by type s1/m1 vacA alleles. s1/m1 vacA alleles typically encode VacA proteins, related to a high level of vacuolating cytotoxin activity , while s1/m2 strains have moderate toxin production . In fact, s1/m2 strains that have an i1 allele are able to induce vacuolation. However, s1/m2 strains that have an i2 allele are not able to induce vacuolation . s2/m2 strains have rare or even absent toxin production .
By comparing vacA s1 and m1 strains with vacA s2 and m2 strains, which are less virulent, it was revealed that
5. VacA proteins
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, 25, 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 . 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 . 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 . 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 .
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 . 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” . An accurate view of the surface of VacA oligomers is presented by three-dimensional reconstructions of these deep-etch metal copies (Figure 2) . 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].
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 . 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, 42, 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 .
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
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 . In fact, the 37 and 58 kDa fragments of VacA are considered as subunits or domains of the holotoxin .
Burroni et al. manufactured an
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 . 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 . 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 . 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.
In addition, alanine scanning mutagenesis showed that point mutations at proline 9 or glycine 14 entirely abrogated VacA activity . Another factor that abrogated toxin activity is the addition of an amino-terminal hydrophilic extension to VacA . As a result, it is obvious that the amino-terminal hydrophobic region has an important role in toxin activity.
7. Receptor binding region
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
Investigations by naturally occurring and engineered m1/m2 chimeric proteins  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.
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 . 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 .
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 . However, saturable binding has not been indicated with classical ligand binding assays with 12I-labeled VacA . 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 . 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 . 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.
Both 58 and 37 kDa regions are needed for VacA internalization . VacA should be preactivated by disposal of acid or alkali, in order to be internalized . 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 . 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 . VacA caused decrement of the degradative power of HeLa cell lysosomes and also decrement of the antigen-processing 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 .
Subsequently, disposal to VacA, transepithelial electrical resistance (TER) reduces, occurred with an increase in transepithelial flux of low-molecular-weight molecules . 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 . 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 . 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 anion-selective channels [76, 77].
Patch clamp analysis of HeLa cells indicates that VacA forms plasma membrane channels with features similar to those perceived in model membranes . 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 . 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 . 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 . VacA induces an increased apical anion secretion in Caco-2 cells, and this also is blocked by NPPB , implying that it is also because of VacA anion channel formation.
A toxin that has damaging outcomes on epithelial cells is produced by
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, 89, 90, 91].
VacA has a significant role in the pathogenesis of