Expert Review of Anti-infective Therapy

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Role of vacuolating cytotoxin A in Helicobacter

pylori infection and its impact on gastric
pathogenesis

Shamshul Ansari & Yoshio Yamaoka

To cite this article: Shamshul Ansari & Yoshio Yamaoka (2020): Role of vacuolating cytotoxin A in
Helicobacter�pylori infection and its impact on gastric pathogenesis, Expert Review of Anti-infective
Therapy, DOI: 10.1080/14787210.2020.1782739

To link to this article: https://doi.org/10.1080/14787210.2020.1782739

Accepted author version posted online: 14

Jun 2020.

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Publisher: Taylor & Francis & Informa UK Limited, trading as Taylor & Francis Group

Journal: Expert Review of Anti-infective Therapy

DOI: 10.1080/14787210.2020.1782739
Article type: Review

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Role of vacuolating cytotoxin A in Helicobacter pylori infection and its impact on gastric

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pathogenesis

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Shamshul Ansari1 and Yoshio Yamaoka2, 3, 4, 5

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Department of Microbiology, Chitwan Medical College, Bharatpur 44200, Chitwan, Nepal
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Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine,
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Idaigaoka, Hasama-machi, Yufu, Oita 879-5593, Japan
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Global Oita Medical Advanced Research Center for Health, Idaigaoka, Hasama-machi, Yufu,
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Oita 879-5593, Japan

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Department of Medicine, Gastroenterology and Hepatology Section, Baylor College of
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Medicine, 2002 Holcombe Blvd., Houston, TX 77030, USA

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Borneo Medical and Health Research Centre, Universiti Malaysia Sabah, Kota Kinabaru, Sabah
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88400, Malaysia
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*Corresponding author:
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Yoshio Yamaoka,

Head of Department and Dean of Faculty of Medicine

Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine,

1-1 Idaigaoka, Hasama, Oita 879-5593, Japan

Phone: 81 97 586 5742; Fax: 81 97 586 5749

E-mail: yyamaoka@oita-u.ac.jp

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Abstract

Introduction: Helicobacter pylori causes, via the influence of several virulence factors,

persistent infection of the stomach, which leads to severe complications. Vacuolating cytotoxin

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A (VacA) is observed in almost all clinical strains of H. pylori; however, only some strains

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produce the toxigenic and pathogenic VacA, which is influenced by the gene sequence

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variations. VacA exerts its action by causing cell vacuolation and apoptosis. We performed a

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PubMed search to review the latest literatures published in English language.

Areas covered: Articles regarding H. pylori VacA and its genotypes, architecture,
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internalization, and role in gastric infection and pathogenicity are reviewed. We included the
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search for recently published literature until January 2020.
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Expert opinion: H. pylori VacA plays a crucial role in severe gastric pathogenicity. In addition,

VacA mediated in vivo bacterial survival leads to persistent infection and an enhanced bacterial
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evasion from the action of antibiotics and the innate host defense system, which leads to drug

evasion. VacA as a co-stimulator for the CagA-phosphorylation may exert a synergistic effect
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playing an important role in the CagA mediated pathogenicity.

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Keywords: H. pylori, pathogenicity, VacA, VacA-mediated apoptosis, vacuolation

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Article highlights

• H. pylori infects at least half of the world population; however, the prevalence varies in

different geographic areas, from nearly 90% in some areas to less than 10% in others.

• H. pylori causes severe complications such as gastritis and peptic ulcer diseases and

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increases the risk of developing gastric cancer and mucosa-associated lymphoid tissue

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lymphoma.

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VacA-mediated pathogenicity is genotype-dependent.

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• To exert its effect, VacA is internalized into gastric epithelial cells via receptor-

dependent mechanism.

•
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VacA enhances in vivo bacterial survival through a transient receptor potential mucolipin
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1-mediated mechanism leading to the failure of eradication therapy.

• Internalized VacA induces multiples cellular effects such as cell vacuolation,

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mitochondrial stress and dysfunction, membrane potential depolarization, autophagy,

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inhibition of T cell activity, and apoptosis. Moreover internalized VacA stimulator for the
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CagA-phosphorylation.
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 1. Introduction

Helicobacter pylori is estimated to colonize the gastric epithelium of approximately half of the

world population. A recent study has discovered that the global prevalence is 44.3%. However,

prevalence varies among different geographical locations. For example, it is as high as 89.7% in

Nigeria and as low as 8.9% in Yemen [1]. This variation in prevalence among countries is

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affected by socio-economic status, level of urbanization, and sanitation during childhood [2].

Epidemiological data suggest that H. pylori is the greatest risk factor for the development of

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severe gastric complications [3]. In the gastric lumen, H. pylori encounters a highly acidic

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environment, wherein it prefers to colonize specific sites, such as the antral region. However, to

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successfully colonize the gastric epithelium in these harsh conditions, H. pylori harbors a well-
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developed mechanism to overcome the acidic conditions and successfully commence permanent

infection [4]. Permanent infection leads to the development of several gastro-duodenal

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complications such as chronic gastritis, duodenal ulcer, gastric ulcer, gastric cancer, and gastric

mucosa-associated lymphoid tissue (MALT) lymphoma [5]. Approximately 90% of all duodenal
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ulcers and 70–90% of all gastric ulcers occur because of H. pylori infection [6]. Successful
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eradication therapy of H. pylori infection decreases the recurrence of peptic ulcer diseases
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compared to no therapy [7]. However, virulence factors, together with the gastric environment

and host genetic make-up, influence the development of severe complications such as peptic
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ulcer diseases and gastric cancers, which may take several decades to develop [8]. One of the
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most important virulence factors is vacuolating cytotoxin A (VacA), produced by almost all H.

pylori strains, which is associated with the severity of gastric complications. Using the

keywords; “VacA” alone as well as in the combinations with other words such as “pylori,

structure, receptors, pathogenicity, and gastric colonization, we performed a PubMed search. The
publications with only abstract, case reports, commentaries, and editorials were excluded. Of the

literatures displayed we mainly focused on articles with the full text in English to compile the

latest information published in scientific literatures in order to get a deeper understanding of the

role and mechanism of VacA in the development of severe gastric complications.

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2. VacA and its allelic diversity

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H. pylori VacA is a pore-forming cytotoxin that interacts with gastric epithelial cells and plays a

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crucial role in pathogenicity [9, 10]. After its initial formation as a 140 kDa protoxin, VacA is

secreted through the autotransporter system. The mature VacA that is secreted from H. pylori

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consists of an 88 kDa monomer that undergoes limited proteolysis to yield two fragments; an N-
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terminal p33 domain (amino acids 1 to 311) and a C-terminal p55 domain (amino acids 312 to

821), which are linked by a flexible loop that is sensitive to limited proteolysis in vitro (Figure
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1A) [11, 12]. Moreover, ∼15 Å resolution cryo-electron microscopy (cryo-EM) has been used to
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elucidate the structure of various VacA oligomers and reveals the interactions between the p33
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and p55 domains for VacA assembly [13]. The p55 domain performs host cell receptor binding

activity, whereas the p33 domain is responsible for pore-forming activity [14]. The 2.4 Å crystal
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structure of the p55 domain reveals a predominant right-handed β-helix [15]. Furthermore, the 19

Å cryo-EM structure of the VacA dodecamer reveals the p55 domain as the peripheral arms of
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the flower-like structure and the p33 domain as the central core [16]. Further investigation has
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shown that the binding interface of p33-p55 is approximately 1,300 Å2 and involves two salt

bridges (H276-D360 and E295-K353) at the loops of the β-helix, as well as three side-chain

hydrogen bonds (H276-D360, D281-N363, and E295-K353) [17]. Moreover, the laterally

connected β-strands are stabilized by the main chains of the two domains (residues 275–293 and
residues 352–380), which are close enough to form multiple main-chain hydrogen bonds [18].

Multiple tryptophan residues (W49, W80, W82, W90, and W96) in the p33 domain play an

important role in the membrane-binding process [19–22]. However, the entire p33 domain and

111 N-terminal amino acid residues in the p55 domain are required to achieve efficient

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vacuolating capability [23].

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vacA contains three heterogenic regions with significant sequence variations, designated as the

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“s” region, which represents the sequence variation in the 5’-end signal sequence; “m” region,

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which represents the sequence variation in the middle of the p55 domain; and “i” region, which

was identified recently in a survey conducted in the Iranian population and represents the

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sequence variation in the intermediate region located between the “s” and “m” region in the p33
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domain [24–26]. Studies conducted on clinical H. pylori strains have shown further sequence

variation within the “s” region, designated as the s1a, s1b, s1c, and s2 sub-families; within the
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“m” region, designated as the m1 and m2 sub-families; and within the “i” region, designated as
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the i1, i2, and i3 sub-families [24, 26–28]. Some studies have also discovered two additional
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regions of variation: the “d” region, located between the “i” and “m” regions, either without a

69–81 base pair deletion (d1) or with this deletion (d2); and the “c” region at the 3’-end of vacA,
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either with a 15 base pair deletion (c1) or without this deletion (c2) (Figure 1B) [29, 30]. The

detailed allelic diversity has been reviewed by Tran et al. [31]. It has been shown that the
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vacuolation capability of VacA is enhanced by the combination of divergent sequences in the

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three regions (“s”, “m”, and “i”). One study has reported the vacuolating capabilities of

genotypes that possess combinations of “s” and “m” regions. The combination “s1/m1” exhibits

high vacuolating activity, “s1/m2” exhibits intermediate activity, and s2/m2 exhibits no such

activity, which indicates a lower vacuolating capability of VacA with a genotype that possesses
s2 sequence variation than with a genotype that possesses s1 sequence variation [24]. An

additional 12 amino acids in the N-terminal sequence result in a predominantly hydrophilic

nature in the s2 region, whereas the strongly hydrophobic s1 region does not possess this amino

acid segment. This hydrophilic s2 region may cause impairment in anion-selective channel

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formation and cell vacuolation; thus, a genotype with the s1 sequence possesses higher

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vacuolating capabilities than the s2 sequence [28]. Moreover, the lower efficiency of the s2

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genotype to export VacA from the cytoplasmic membrane to the periplasmic space compared to

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the s1 genotype supports the lower vacuolating activity of s2 compared to s1 [32]. Furthermore,

more VacA is secreted in s1 than in s2 genotypes because of the elevated vacA transcription by

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strains with the s1 sequence [33]. A higher prevalence of vacA with s1a, m1, and i1 H. pylori
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genotypes has been observed in clinical settings than other genotypes [25, 34]. A recent meta-

analysis conducted in a Western population also reported an increased risk for the development
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of gastric cancer in populations infected with H. pylori that possesses vacA with s1 or m1

specific sequences [35]. In Middle Asia and Middle East Asia, there is a higher risk of gastric
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cancer development (OR = 10.9–15.0) if H. pylori strains harbor vacA i1 than if H. pylori
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harbors other vacA genotypes [36]. Studies have also shown that the sequence variations in both
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the p33 (i1 and i2) and p55 (m1 and m2) domains of the VacA protein influence cellular tropism

and toxicity, probably due to the alteration of the cellular receptor binding ability [37–39].
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Winter et al. in an in-vitro study also found an extensive vacuolation of RK13 cells by strains
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with s1i1 mutants whereas the minimum vacuolation was found by the strains with s1i2 or s2i2

mutants [40].

3. Cellular receptors for VacA

The binding of VacA to the plasma membrane of the host cells, achieved via interaction with the

receptors on the plasma membrane, is the first step towards pathogenicity. One experiment

suggests that the monomers of VacA bind to the host plasma membrane, and oligomerization

takes place to form snowflake-shaped hexamers [41]. Both domains, p33 and p55, participate in

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cell receptor binding and oligomerization [42–46].

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The C-terminal domain of VacA, which is activated in the low pH environments of the stomach

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[47], contains the receptor binding sites. Transmembrane proteins, such as receptor protein

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tyrosine phosphatases (RPTPα and RPTPβ) and low-density lipoprotein receptor-related protein-

1 (LRP1), which are expressed on gastric epithelial cells and hypothalamus, serve as VacA

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receptors [48–51]. These receptors interact with VacA and contribute to the internalization of
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VacA into the target cells (Figure 2).
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In addition to its role as a receptor for VacA, RPTPβ controls several cellular processes, such as

cell migration and differentiation, as well as synaptogenesis [52–54]. RPTPβ is primarily

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expressed in brain tissue; however, studies have shown that it is also expressed in gastric tissue,
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particularly in the submucosal and muscle layers [55, 56]. LRP1 interacts with VacA, which

causes an accumulation of reactive oxygen species that leads to the activation of the Akt
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pathway. Akt activation results in the formation of autophagic vacuoles via the MDM2-mediated
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elimination of p53. However, the middle region of VacA with the m2 genotype is not able to
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bind to LRP1, whereas the m1 genotype is able to bind LRP1 [48, 57].

Other molecules, of different cell types, that interact with VacA include CD18 on T-lymphocytes

and multimerin 1 on platelets. Once VacA binds to the cell surface molecules, it interacts with

CD18 and moves to lipid rafts for internalization [48]. H. pylori infection is associated with
immune thrombocytopenic purpura; however, its detailed mechanism remains unknown.

Increased expression of CD62P on platelets has been reported in H. pylori-infected mice and

humans, which is decreased by H. pylori eradication therapy [58, 59]. Satoh et al., using an

immune-precipitation method, reported that CD62P expression on platelets is mediated by

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binding with multimerin 1. This was identified as a candidate VacA receptor for platelet

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activation [60]. The internalization of VacA activates signal transduction pathways and

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contributes to cell death, which leads to gastric ulceration [48]. Other receptors that facilitate

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VacA binding include epidermal growth factor receptor, β2 integrin, fibronectin, and

sphingomyelin [48, 61].

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4. Oligomerization of VacA
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VacA oligomerization is important for insertion into the lipid membrane bilayer to form ion

channels. After its secretion, VacA assembles into water-soluble single-layer or double-layer
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flower-shaped oligomers that contain six (hexamer) or seven (heptamer) copies of protomers [13,
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16]. Structural analysis has shown that the angle of protomer-protomer interactions is the major
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difference between hexamers and heptamers, with a larger binding interface in hexamers than in

heptamers [62]. A recent study suggests that the water-soluble VacA oligomer first dissociates
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into monomers at the low pH conditions provided in the gastric lumen. Here, these monomers
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bind to the host cell membrane, insert into the lipid bilayer, oligomerize, and finally form

membrane channels [63]. For oligomerization, the protomers interact via the residues present in

both the p33 and p55 domains. As the structure of VacA is mostly rolling β-strands connected by
flexible loops, an extended loop in the p55 domain of one protomer contacts the p33 domain of

the adjacent protomer, and a α-helix is sandwiched between the protomers [62].

One study showed that VacA s1/i1/m1 oligomerizes as a chiral hexamer on the host cell

membrane. This VacA is inserted into the lipid bilayer and is structurally distinct from soluble

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oligomers. A prominent structural difference in the central region of the oligomers has been

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observed between soluble and membrane-bound VacA hexamers. The same study also

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demonstrated that the insertion of the membrane-bound p33 domain into the membrane, which is

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independent of toxin oligomerization, is an essential step in hexameric pore formation and causes

the structural differences between the membrane-bound and soluble VacA hexamers [64]. In

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addition to the three N-terminal GXXXG repeats in the p33 domain, a motif that facilitates the
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oligomeric interactions between transmembrane helices, several other regions of VacA are

required for pore formation [65, 66]. A study that utilized a computationally generated structural
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model of the predicted VacA transmembrane domain sequence suggested that packing together
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of these N-terminal GXXXG repeating motifs takes place to form the anion channel [67].
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However, for channel and cell-vacuolating activity, the 32 N-terminal hydrophobic non-polar

amino acid residues of the p33 domain, the proline residue at position 9 (P9), and the GXXXG
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motifs are required [68, 69]. In the VacA m region, localized in a cryo-EM map study, although

sequence variations due to deletions and insertions alter the loops that interact with the surface of
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the cell, these sequence variations are not confined to individual loops or β-strands. It has been
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predicted that, during membrane contact, approximately 18 loops are involved, and the combined

efforts exerted by these protein-lipid membrane interactions along the length of p88 increase the

avidity of VacA to bind to the cell membrane [62].

5. VacA membrane insertion

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Despite the receptor-mediated interactions of VacA, the positively charged membrane-facing

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surface of the VacA hexamer may also facilitate membrane attachment by simple electrostatic

attraction that favors contact with anionic phospholipids [18]. Once VacA contacts the cell

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surface, it inserts into the membrane to form anion-selective membrane channels that require a

stretch of hydrophobic amino acids at the N-terminal end of the p33 domain [41, 68–72].

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H. pylori VacA, as well as many other bacterial protein toxins, utilize lipid rafts for
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internalization as the first step towards the intoxication of host cells [73]. Lipid rafts are host cell

membrane microdomains that are enriched in cholesterol, glycosylphosphatidylinositol-anchored

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proteins, and sphingolipids, which are advantageous targets for VacA as they concentrate the
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receptors for oligomeric toxins [74–77]. A recent study has shown that the p55 domain on its
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own binds to lipid rafts for internalization, whereas the role of lipid rafts for the p33 domain

remains to be determined [78].

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6. Role of VacA in H. pylori infection

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In the stomach, H. pylori encounters harsh conditions of extremely low pH, of approximately

2.0, in which most organisms are killed. To successfully establish a persistent infection, a

bacterium must survive the acidic environment. H. pylori has developed well-defined

mechanisms; urease dependent such as neutralization of acidic condition by the urease and
maintaining the periplasmic pH around 6.1 by acid acclimation, and urease independent

mechanisms such as the role of gastric mucus, helical shape of bacterial, the role of flagella,

chemotactic activity, and the role of several proteins; to counteract this harsh condition [4]. In

addition to extracellular survival mechanisms, H. pylori survives inside gastric cells via the

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inhibition of autophagy.

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VacA induces autophagy; however, prolonged exposure of VacA to cells can disrupt

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autophagosome maturation, which causes the defective accumulation of autophagosomes

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containing-bacteria within infected cells. An In vitro study has suggested the role of VacA in the

synthesis of large bacteria-containing vacuoles in infected cells caused by the fusion of late

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endocytic compartments. Vacuoles and autophagosomes acquire a lysosomal marker, lysosomal
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Associated Membrane Protein 1, and induce an acidic pH to indicate their fusion; however, these

fused compartments lack lysosomal hydrolases such as cathepsin D and are not degradative.
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These dysfunctional vacuoles and non-degradative compartments constitute an intracellular

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niche that provides a suitable environment for H. pylori survival in vivo. This VacA-generated
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intracellular niche protects the bacteria from antibiotic therapy and allows evasion from immune-

mediated killing, which enables re-colonization of H. pylori and a persistent infection [79]. A
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recent study has suggested that the transient receptor potential membrane channel mucolipin 1

(TRPML1), an endolysosomal calcium channel, is disrupted by the inhibition of lysosomal and

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autophagic killing caused by VacA s1m1i1 isoform, which promotes the intracellular niche and
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bacterial survival [80–82]. H. pylori VacA also plays a role in the flow of ions and nutrients from

the mucosa towards the gastric lumen, which causes an alteration in gastric epithelium cell

integrity; thus, VacA plays an important role in the survival of H. pylori and establishment of a

persistent infection [83]. Additionally, the VacA-mediated disruption of epithelial cell tight
junctions enhances the distribution of VacA in the lamina propria, where it encounters T cells

recruited to the infection sites. Inside the T cells, VacA blocks the Ca2+ calmodulin dependent

phosphatase calcineurin suppression of nuclear factor (NF) translocation. Therefore, the

suppression of several proteins results in the inhibition of T cell activation and proliferation,

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which induces cell cycle arrest. This protects the bacteria from immune clearance, thereby

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establishing persistent infection [84, 85]. The harboring of vacA in almost all H. pylori strains

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suggests its crucial role in the colonization and persistence of H. pylori within the gastric mucosa

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[85].

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7. VacA mediates gastric pathogenicity

VacA, due to its variety of effects that are exerted on the host cells, are termed as the muti-
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functional toxin. After its internalization into the gastric epithelial cells, VacA accumulates

inside different cellular compartments and induces multiple cellular effects including cell
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vacuolation [86], depolarization of membrane potential [87], dissipation of mitochondrial

transmembrane potential leading to mitochondrial dysfunction and cell apoptosis [88–90],

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activation of mitogen-activated protein kinases [91], autophagy [90], inhibition of T cell

activities [92, 93], activation of mitogen-activated protein kinase pathways [94], and activation
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of proapoptotic factor Bcl-2 associated X protein (Bax) via Bax transfer to mitochondria which
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results in apoptosis and cell death [95–97]. Most of these effects occur because of the formation

of VacA-mediated anion-selective channels in the cell membrane [94]. The alteration of the host

inflammatory response, mediated by VacA, can suppress T cell activation. The toxin can also
induce an inflammatory response mediated by the activation of NF-kB resulting in the up-

regulation of interleukin-8 [98].

Several studies have observed the development of precancerous lesions, such as intestinal

metaplasia (IM), in individuals infected with the H. pylori strains that possess the vacA i1

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genotype and it was suggested that the amount of VacA produced during infection has a large

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implication for disease severity. Therefore, studies investigating vacA mRNA levels in a human

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gastric biopsy found that the vacA i1 allele is associated with gastric inflammation and

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precancerous change, whereas no correlation was observed between peptic ulcer disease in

subjects infected with strains that harbored the vacA i2 genotype and IM [99, 40].

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Cytotoxin-associated gene A (CagA) is an effector protein that is expressed by the virulent
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strains possessing the cag pathogenicity island (PAI). After its synthesis, the CagA is
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translocated into the gastric epithelial cells via type 4 secretion system (T4SS) and the EPIYA

(glutamic acid-proline-isoleucine-tyrosine-alanine)-motif undergoes tyrosine phosphorylation

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[100]. Tyrosine phosphorylation of intracellular cytotoxin-associated gene A (CagA) is a crucial

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step for gastric carcinogenicity [101]. CagA and VacA interaction has been suggested to affect

the severity of gastroduodenal diseases [102]. Although CagA has been shown to reduce VacA
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entry into the cell, suggesting an antagonistic interaction between VacA and CagA, isogenic
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cagA and vacA mutant strains induce greater vacuolation and a more pronounced hummingbird
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phenotype than wild-type strains [103–107]. Furthermore, a recent in-vitro study showed

enhanced CagA phosphorylation after RPTPα binding by VacA in AZ-521 cells, thus explaining

the increased cellular carcinogenicity. In the study, AZ-521 cells infected with wild-type H.

pylori strains induced CagA phosphorylation, whereas no such phosphorylation was induced if
infected with vacA gene-disrupted mutant strains. Additionally, this CagA phosphorylation

capability was restored when purified VacA was added, which indicates the possible role of

VacA in the induction of CagA phosphorylation in AZ-521 cells via the VacA-RPTPα-Src

signaling pathway [108]. Therefore, VacA-dependent CagA phosphorylation may be important

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in H. pylori-induced gastric pathogenicity. It has also been reported that accumulation of both

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total and phosphorylated CagA occurs in the presence of VacA, which is important for gastric

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carcinogenicity [109]. Another study has reported that a significantly large number of patients

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show a precancerous lesion, such as IM, as well as gastric cancer, when infected with strains that

express both CagA and VacA [110]. In H. pylori-infected individuals, VacA is important for the

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transformation of normal gastric cells to metaplastic cells, which indicates the role of VacA as a
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marker for the risk of H. pylori-induced gastric malignancy.

The alteration of several cellular proteins has been linked with the development and progression
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of cancer. Cortactin (cortical actin-binding protein), a filamentous actin-binding protein, is an

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activator of actin-related protein complexes (ARP)2/3 and important molecular link between
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signal transduction pathways and the cytoskeleton [111–115]. In cancer tissue samples, the

expression of the cortactin gene is abnormally high and is associated with cancer cell metastasis
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[116–119]. In a in-vitro study, AGS gastric epithelial cells that expressed cortactin and were

treated with VacA showed a significant increase in the percentage of apoptotic cells and
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expression of the proapoptotic protein Bax, whereas a decrease in the expression of the
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antiapoptotic protein Bcl-2 was observed, suggesting the role of VacA in association with

cortactin for gastric pathogenicity [120]. Similarly, the role of connexin 43 (Cx43), a member of

the human Cx family, is responsible for VacA-induced cell death and gastric pathogenicity [121,

122]. The Cx-family proteins are membrane proteins that form channels at gap junctions to
regulate development and homeostasis via intercellular communication, cell-cell channel

formation, and exchange of signaling molecules [123–125]. A recent in-vitro study demonstrated

the role of Cx43 in VacA-induced AZ-521 cell death via a Rac1/ERK-dependent pathway in H.

pylori-infected gastric mucosa [126]. Elevated Cx43 level in H. pylori-infected gastric tissue is

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observed, which is associated with erosive gastritis.

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8. African Enigma

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The prevalence of H. pylori shows the geographical variation with a higher prevalence in

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developing countries including majority of African countries when compared with the developed
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countries. Although, the H. pylori infection is endemic in Africa, a lower prevalence of the

gastro-duodenal diseases especially gastric cancer has been reported [127].

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In this continent, gastritis has been reported as the most common gastroduodenal disease
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associated with H. pylori infection whereas its progression to atrophic gastritis and cancer in
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these populations is quite similar to that reported in the Western countries and America [128].

The age-standardized incidence of gastric cancer rates are significantly higher in East-Asian
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regions (45.3 per 100,000 persons per year) when compared with rates in North America (7.6 per

100,000 persons per year) and North Europe (9.3 per 100,000 persons per year) [129]. The
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incidence of gastric cancer reported across many regions of Africa is comparable to that reported
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in America and Europe. Therefore, the “African enigma” referred to a higher prevalence of H.

pylori infection with a rare development of gastric adenocarcinoma [130]. Moreover, the strains

isolated from most of the African regions have been found to be predominantly the virulent types
possessing the key virulent factors that play a major role in the development of gastric cancer

such as CagA and VacA with s1 genotypes [131, 132-134].

Despite of the possibilities of several factors, the differences in intake of particular foods, co-

existence of other infection or gut microbiota composition have been speculated to differ the

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gastric cancer rates between African countries and other parts of the world [135]. A study on co-

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infection of mice with the Heligosomoides polygyrus which is an extracellular mouse parasite

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and Helicobacter felis has found an alteration in the immune response to a greater extent with

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down-regulation of Th1 dependent IgG2a serum antibody when compared with the Th2

dependent IgG1 response leading to a reduced H. pylori mediated gastric atrophy suggesting a

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role of other common infections in Africa that might affect the outcome of the H. pylori infection
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[136].
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9. Conclusion
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This review concludes that H. pylori vacA possesses sequence polymorphisms at multiple sites
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that affect the genotype of vacA. In turn, VacA-dependent pathogenicity is influenced by these

genotypes. Moreover, in addition to its role in gastric pathogenicity, VacA enhances the in vivo
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survival of H. pylori. However, a deeper understanding of the role of VacA in gastric

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pathogenicity requires more detailed experimental studies.

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10. Expert opinion

H. pylori VacA has been discussed regarding its pathogenic activity to enhance the cellular

effects of H. pylori and its role in intracellular bacterial survival, which pose challenges for the

eradication of H. pylori. However, current knowledge of the VacA-mediated mechanism is not

sufficient to clearly elucidate the mechanism of VacA; therefore, other studies are needed to

T
understand the mechanism of VacA pathogenicity. Additional experimental studies are also

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required to rule out the possibilities of other bacterial, as well as environmental factors that play

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a role in reducing the effects of the VacA.

SC
H. pylori infection occurs worldwide and leads to serious conditions such as ulcer diseases,

gastric cancers, and MALT-lymphoma [5]. Eradication therapy is the only effective treatment

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that reduces the occurrence of such severe diseases [7]. Institutions that have strictly followed
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the diagnostic and therapeutic guidelines have recently witnessed a reduction in the prevalence

of ulcer and gastric cancer diseases. However, it is difficult to achieve the desired and predicted
M

efficacy of eradication therapy. Additionally, environmental and bacterial factors influence

D

eradication therapy. H. pylori VacA interferes with intracellular killing of bacteria. Usually,
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bacteria entering a cell are killed by phagolysosome fusion. However, H. pylori VacA enhances

the formation of dysfunctional vacuoles and non-degenerative phagolysosomal compartments,

EP

which lead to a favorable niche for the survival of bacteria [80]. Recently, TRPML1-mediated

inhibition of autophagic killing was determined to lead to the in vivo survival of H. pylori [83].
C

The intracellular hiding of bacteria during stresses and their protection from the action of
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antibiotics and natural host defenses may be an additional hurdle to the treatment of H. pylori-

mediated pathogenicity. This is because protection will reduce the efficacy of eradication

therapy. From this perspective, there will be a reduced efficacy than expected because of the
VacA medicated bacterial hiding. Moreover, most clinical strains possess vacA; thus, this

reduced efficacy prediction applies worldwide.

Recently, a few studies have aimed to uncover deeper insights into the role of VacA in gastric

pathogenicity. Although some studies have elaborated on the association of VacA with disease

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pathogenicity, structural and functional architecture, and gastric infection recurrence, we

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encourage researchers to conduct more studies to solve the unknown aspects of the VacA

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mechanism.

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Funding

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This work was supported by the Ministry of Education, Culture, Sports, Science and Technology
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under grant numbers 15H02657, 16H06279, 16H05191, and 18KK0266; National Institutes of

Health under grant number DK62813.

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Declaration of interest
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The authors have no relevant affiliations or financial involvement with any organization or entity
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with a financial interest in or financial conflict with the subject matter or materials discussed in

the manuscript. This includes employment, consultancies, honoraria, stock ownership or options,
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expert testimony, grants or patents received or pending, or royalties.

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Reviewer disclosures
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Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contributions
Conceptualization, Y.Y.; methodology, Y.Y. and S.A.; literature review, S.A.; original draft

writing, S.A.; draft supervision, review, and editing, Y.Y.; funding acquisition, Y.Y.

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IP
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SC
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Figure legends

Figure 1. (A) The VacA vacuolating cytotoxin A protoxin is produced by H. pylori and the
signaling domain allows transportation to the bacterial inner membrane, whereas the
autotransporter domain allows translocation to the bacterial outer membrane. The mature 88 kDa
VacA is cleaved by an unknown protease into an N-terminal p33 domain and a C-terminal p55
domain. (B) Allelic diversity showing s1 and s2 polymorphic region in signal (s) sequences; i1,
i2, and i3 polymorphic sequences in the intermediate (i) region; d1 and d2 sequences in d region;

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m1 and m2 sequences in the middle (m) region; and c1 and c2 sequences in the (c) region.

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Figure 2. The VacA produced by the H. pylori binds with the transmembrane proteins RPTPα,
RPTPβ, and LRP1 leading to the synthesis of autophagic vacuoles. In addition, the RPTPβ after

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binding with the VacA can affect the cellular processes such as the cell migration, differentiation
and synaptogenesis whereas the LRP1 after binding with the VacA causes accumulation of
reactive oxygen species (ROS) leading to the synthesis of autophagic vacuoles through the

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activation of AKT pathway and MDM2. In T-lymphocytes, the VacA is internalized through
binding with the surface protein CD18 and its recognition with the lipid rafts. In platelets, the
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binding of VacA to the multimerin1 causes expression of CD62P.
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