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1 VacA protein gene structure and genotype 1.1 VacA gene structure TLCover cloned the VacA gene in 1993 and found that it consists of 3864 base pairs, encoding 1287 amino acids, the encoded protein has a molecular weight of 139kDa, which is VacA The precursor molecule includes three regions: (1) a leader signal sequence consisting of 33 amino acid residues at the N-terminus; (2) a mature vacuolating toxin in the middle region; and (3) a 50 kDa polypeptide at the C-terminus, Its amino acid sequence is homologous to the outer membrane proteins of many G+ bacteria. After the N-terminal signal peptide was removed, the C-terminal peptide chain similar to the IgA protease precursor was modified to become a mature 87 kDa VacA. The active toxin can be degraded into two subunits of 37 kDa and 58 kDa.
VacA may be a family of bacterial toxins with AB structure. The B subunit binds to the membrane receptor on the surface of the target cell and mediates the entry of the A subunit into the cytosol. The A subunit specifically binds to the intracellular target molecule and causes the cell. Destruction of important functions, thereby exerting toxin activity [1].
The VacA purified from the liquid culture supernatant is an oligomeric complex with a molecular weight greater than 900 kDa. The complete VacA molecule is petal-like or snow-like, composed of 6 to 7 95 kDa VacA monomers symmetric along the radius [2]. Exposure of VacA to acidic pH can depolymerize oligomeric complexes into monomers and is associated with increased cytotoxicity of VacA.
1.2 VacA genotype can be divided into two different types according to VacA5, the end sequence: s1 (including s1a, s1b2 subtypes), s2; according to the difference between the intermediate sequences can be divided into two types: m1 and M2. The recombinants produced theoretically by these alleles, except for s2/m1 (presumably s2/m1 may be lethal), have been confirmed to exist. Therefore, Hp can be classified into s1/m1, s1/m2, and s2/m2 depending on the VacA sequence. The Hp strain in the United States is dominated by the s1/m1 subtype, while the Chinese is dominated by the s1/m2 subtype [3].
2 VacA biological activity 2.1 VacA vacuolation VacA induces cell vacuolation, and these vacuolar membranes contain markers of late endosomes and late lysosomes [4]. Some intracellular enzymes required for the formation and maintenance of vacuoles have been demonstrated, including vacuolating ATPase [5] and the small GTP-binding protein Rab7 [6] Rac1 [7]. Among them, vacuolar ATPase can regulate the flow of hydrogen ions through the intracellular membrane, so the need for vacuolar ATPase suggests that the pH of the vacuolar precursor cavity must be acidic so that VacA induces cell vacuolation. Rab7 is a GTPase that regulates ion channels on membranes and promotes lysosomal fusion, is located on late endosomes, and is a marker of late endosomes. Supports membrane deposition and homotypic fusion between late endosomes. Rac1 controls the cytoskeletal components that affect membrane trafficking. Even in the absence of vacuolization, VacA can alter the function of the late phagocytic pathway and lysosomes. Studies have shown that the vacuolation of VacA is affected by Hp urease, because urease can decompose urea to produce ammonia, and ammonia can promote the vacuolation of VacA.
2.2 VacA interferes with tissue damage repair mechanism VacA can specifically inhibit cell proliferation and delay ulcer healing. Recent studies have shown that VacA is involved in the signal transduction pathway of epidermal growth factor (EGF) activation. Fujiwara et al [8] found that the dose of VacA toxin that does not affect cell viability can be significant in the study of the interaction of Hp toxin and EGF on gastric cancer KatoIII cells. It inhibits the binding of EGF to the epidermal growth factor receptor (EGFr) and reduces the cell proliferation caused by EGF stimulation by 22%. Pai et al [9] can enhance the expression of EGFr and autophosphorylation after EGF acts on KATOIII cells of gastric cancer, and VacA can inhibit the above process. From the above, VacA interferes with the EGF-mediated signal transduction pathway. It is possible that the binding of VacA to RPTPβ promotes dephosphorylation of the EGF receptor protein tyrosine, resulting in attenuated EGF action, thereby interfering with epithelial repair and delaying ulcer healing.
2.3 Effects of VacA on Immunity Molinari et al [10] studied the effect of VacA on the antigen presentation process of antigen-treated cell prolysin in vivo. The results showed that VacA can specifically inhibit the antigen presentation process mediated by MHC class II antigen. It indicates that VacA can interfere with the body's protective immunity and is conducive to the continuous infection of Hp in the stomach, causing the corresponding clinical symptoms.
2.4 Other effects of VacA on cell function
2.4.1 Causes apoptosis VacA can cause mitochondrial depolarization and partially deplete ATP in cells, but this is not lethal to cells in vitro [11]. Human gastric cells are very sensitive to VacA, and VacA enters mitochondria with apoptosis, which is related to the release of cytochrome c and caspase3 by mitochondria, and cell death occurs 2 days after cell poisoning. In vivo cytotoxicity experiments have shown that there are other stimulating factors, and the apoptosis rate of gastric epithelial cells stimulated by cag+ and VacA-expressing Hp strains in vivo and in vitro is higher [12].
2.4.2 Causes cytoskeletal rearrangement VacA can affect or interact with various components of the cytoskeleton to cause actin rearrangement, Rac-1 changes, and also disrupt the microtubule network. These phenomena are associated with VacA-induced cell proliferation.
2.4.3 Other effects VacA can also increase the second messenger, stimulate the secretion of pepsinogen by AGS cells, and enhance the invasiveness of Hp. However, these findings have yet to be further studied.
Mechanism of action of 3 VacA 3.1 VacA binding to cellular receptors VacA binding to the surface of eukaryotic cells is the first step in cell toxicity. Three cell surface proteins are considered to be receptors for VacA: (1) 140 kDa protein on the surface of AGS cells; (2) EGF receptors in HeLa cells; and (3) RPTPβ. Among these three putative receptors, the interaction of VacA with RPTPβ has been confirmed.
The s1/m1 type VacA can cause vacuolization of many different cells and is therefore considered to be effective in binding to these cell lines. In contrast, s1/m2 VacA can also cause vacuoles in cells of some cell lines and little effect on others. This indicates that the s1/m1 type VacA and the s1/m2 type VacA bind different cell types because the main difference between them is the C-terminus of p58, suggesting that the amino acid sequence required for toxin-binding cells is located in p58 [13]. The antibody of p58 inhibits VacA binding to HeLa cells, and the GST-p58 fusion protein binds to HeLa cells.
3.2 VacA-membrane interaction Studies have shown that [14] activated VacA can be inserted into the liposome membrane but not by the unactivated VacA, which causes the release of ions and fluorescent molecules. Activation of VacA at low pH causes an increase in hydrophobic end exposure because low pH activation is associated with dissociation of soluble VacA oligomers into monomers, and exposure of previously hidden hydrophobic ends may involve membrane insertion. The three regions belonging to VacAp37 become resistant to protease digestion after membrane insertion, suggesting that these three regions are either inserted into the lipid bilayer or translocated into the liposome membrane cavity, with most of the p58 remaining outside the membrane.
3.3 Internalization of VacA VacA is slowly internalized after binding to the cell surface. The p58-mediated binding of surface receptors is not sufficient for the next step of poisoning. In fact, a VacA containing only the deletion mutation of p58 binds to cells but does not endocytosis [15]. Therefore, there may be other interactions during toxin internalization, either mediated by p37 N-terminus or dependent on oligomerization of VacA [16]. Internalization is a necessary step in cell vacuolation.
3.4 VacA activity in cells VacA internalization suggests that it can play a toxic role at the internalization site. To test this hypothesis, HeLa cells were transfected with a plasmid encoding VacA, and when expressed in the cytoplasm, VacA-induced vacuolization was identical to the addition of VacA-induced vacuoles [17]. Immunofluorescence analysis showed that intracellularly expressed VacA was present in a soluble form in the cytoplasm and in the vacuolated chamber. Galmiche et al [18] reported that the full-length VacA and p34 expressed in the cytoplasm of HEp-2 can migrate to the mitochondrial matrix, while p58 remains in the cytoplasm. These evidences indicate that VacA can be translocated through the mitochondrial membrane after entering the cytoplasm.
3.5 Formation of ion channels Experiments have shown that acid-activated VacA inserted into a lipid bilayer can cause channel formation, which is ion-selective. Most scholars believe that the formation of channels plays a direct role in the formation of typical vacuoles induced by VacA. The possible reason is that internalized toxins form anion-selective channels in the late intracellular space, and the increase in anion permeability will The effect of vacuolar ATPase is increased, resulting in the accumulation of CL and permeable amine salts in the intracellular body cavity, which leads to swelling and fusion of the endosomes, and finally formation of vacuoles. Inhibition experiments show that complexes that block the formation of VacA channels also inhibit the formation of vacuoles, and are equally effective, which fully demonstrates that the pore-forming activity of VacA is required for its cytotoxicity. Szabo et al [19] further confirmed that VacA can enhance the ion-selective channel on HeLa cell membrane and depolarize it. The amino terminal hydrophilic region of VacA promotes the insertion of toxins, so VacA lacking the amino terminus neither induces vacuolation nor channels, and protein analysis also shows that p34 plays an important role in channel formation.
4 Clinical significance of VacA
4.1 Clinical relevance of VacA In 1989, Figura [20] compared the production of cytotoxin from Hp strains isolated from 77 patients with gastric ulcer and gastritis. They found that 66.6% of the strains isolated from patients with gastric ulcer produced cytotoxicity, compared with only 30.1% of patients with gastritis. This significant difference suggests that cytotoxin may be closely related to the development of gastric ulcer. In addition, Atherton et al [21] found an important correlation between vacAs1 genotype and gastric ulcer. The vacAs1 genotype also has the same correlation with gastric cancer.
4.2 VacA as a prospect for future vaccines Hp can colonize the stomach of mice. Infection with type I strain can cause gastric lesions similar to human diseases, using purified VacA and E.coil heat-labile enterotoxin as adjuvants. This mouse model protects against infection by type I strains. This indicates that VacA is immunogenic and can be used as a vaccine against Hp infection.
The highly purified VacA from the Hp culture supernatant is biologically active, and rabbits can be induced to produce neutralizing antibodies in rabbits. Its serum studies have shown that it contains most of the antibodies that recognize conformational epitopes. It is indicated that the vacuolating toxin has a conformational change in vivo, so the establishment of an immune response against VacA depends on the natural structure of VacA, and the antiserum against recombinant VacA cannot neutralize the vacuolar activity, and also cannot recognize the natural VacA. Vaccine toxins are only effective if they use intact molecules. However, due to the problem of aggregation, the active VacA has not been synthesized, but the natural VacA can be detoxified by formaldehyde. The formylated VacA can still bind to the target cells, but has no vacuolar activity, although it induces the production of neutralizing antibodies and natural Untreated VacA induced some changes compared to it but was successfully used in conservation experiments.
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Progress in research on Helicobacter pylori vacuolating toxin VacA
 ATCC strain  Helicobacter pylori (Hp) is an important pathogen that was first isolated by Marshall and Warren in 1983. It is a Gram-negative, spiral, micro-aerobic, mainly colonized human gastric mucosa that causes the development of human digestive diseases. Helicobacter pylori infection is globally distributed, and its infection rate is related to local public health conditions. It is reported that about 30% to 50% of adults in western countries are infected with Hp; in China, the infection rate is higher, up to 50%~ 80%, and it has been reported that the proportion of Hp in the population increases with age, and once Hp settles in the human stomach, if it does not take antibacterial treatment, it can last for several decades, seriously endangering the health of the population. Although most Hp-infected individuals are asymptomatic, approximately 10% of those infected have gastroduodenal ulcers, gastric cancer or mucosa-associated tissue (MALT) lymphoma. In 1994, Hp was designated as a Class I carcinogen by WHO, and its secreted protein toxin VacA as a single virulence factor can cause vacuolation, apoptosis, and cytoskeletal rearrangement of target cells, eventually leading to cell death, and thus Hp Important virulence factor. Hp, which was only discovered in 1983, is now one of the most studied pathogens. The more recent studies on Hp virulence factors VacA are summarized below.