Elsevier

Toxicon

Volume 118, August 2016, Pages 27-35
Toxicon

Review
Vacuolating cytotoxin A (VacA) – A multi-talented pore-forming toxin from Helicobacter pylori

https://doi.org/10.1016/j.toxicon.2016.04.037Get rights and content

Highlights

  • Vacuolating cytotoxin VacA is an important virulence factor of H. pylori.

  • Main activity of VacA is the formation of ion channels in membranes.

  • VacA induces apoptosis by a yet incompletely characterized mechanism.

  • A compilation of findings focused on structure-function relations is presented.

Abstract

Helicobacter pylori is associated with severe and chronic diseases of the stomach and duodenum such as peptic ulcer, non-cardial adenocarcinoma and gastric lymphoma, making Helicobacter pylori the only bacterial pathogen which is known to cause cancer. The worldwide rate of incidence for these diseases is extremely high and it is estimated that about half of the world's population is infected with H. pylori. Among the bacterial virulence factors is the vacuolating cytotoxin A (VacA), which represents an important determinant of pathogenicity. Intensive characterization of VacA over the past years has provided insight into an ample variety of mechanisms contributing to host-pathogen interactions. The toxin is considered as an important target for ongoing research for several reasons: i) VacA displays unique features and structural properties and its mechanism of action is unrelated to any other known bacterial toxin; ii) the toxin is involved in disease progress and colonization by H. pylori of the stomach; iii) VacA is a potential and promising candidate for the inclusion as antigen in a vaccine directed against H. pylori and iv) the vacA gene is characterized by a high allelic diversity, and allelic variants contribute differently to the pathogenicity of H. pylori. Despite the accumulation of substantial data related to VacA over the past years, several aspects of VacA-related activity have been characterized only to a limited extent. The biologically most significant effect of VacA activity on host cells is the formation of membrane pores and the induction of vacuole formation.

This review discusses recent findings and advances on structure-function relations of the H. pylori VacA toxin, in particular with a view to membrane channel formation, oligomerization, receptor binding and apoptosis.

Introduction

Given the enormous impact on human health, Helicobacter pylori is now among the best characterized bacterial human pathogens and intensive research efforts are devoted to the analysis of the molecular pathobiology of this organism. Helicobacter pylori, a Gram-negative, spiral-shaped, microaerophilic organism is able to persistently colonize the human stomach for decades and causes serious chronic diseases such as dyspepsia, gastric atrophy, peptic ulcer disease, gastric adenocarcinoma and gastric cell lymphoma (Cover and Blaser, 2009, Montecucco and Rappuoli, 2001, Suerbaum and Michetti, 2002). Striking differences are associated with gastric cancer prevalence in different geographical locations. Gastric cancer is the third leading type of cancer worldwide and it is the fifth most common cancer in Europe (Ferlay et al., 2010). The risk of developing gastric cancer is strongly correlated to the prevalence of H. pylori–associated atrophic gastritis (Wen and Moss, 2009). H. pylori is now considered as the most common bacterial infectious agent of relevance to human health (Atherton, 2006, Ferlay et al., 2010) and it is obvious that a pathogen with this impact on human health became a priority target for the extensive analysis of its molecular physiology. The clinical presentation of infections with H. pylori is determined by a complex interaction of multiple factors such as strain diversity, host genetic predisposition, environmental factors and nutrition (Wen and Moss, 2009). H. pylori is contagious and most likely transmitted via oral-oral, fecal-oral, and gastro-oral (mediated by a reflux of gastric juice) routes (Boehnke et al., 2015, Brown, 2000, El-Sharouny et al., 2015, Khalifa et al., 2010, Krueger et al., 2015, Rakhmanin and German, 2014, Santiago et al., 2015, Tirodimos et al., 2014, Yokota et al., 2015). Epidemiological data related to disease prevalence have to be interpreted with caution as a large proportion of infected individuals (approx. 80%) can remain symptomless over long periods of time. Moreover, diagnostic procedures appropriate for H. pylori infections may be limited or not available at all in some developing countries (Akguc et al., 2014, Batts et al., 2013, Shrestha et al., 2014, Taylor and Blaser, 1991, Vilaichone et al., 2014). Nevertheless, current information on the incidence of H. pylori-infections suggests a high burden to public health care, especially in developing countries where acquisition of the disease appears to occur at higher rates than in developed countries (Aziz et al., 2015, Weaver, 1995, Archampong et al., 2015). In addition, infections during the childhood seem to be more frequent in developing countries where up to 50% of children (5 years of age) and 90% of adults are infected (Khalifa et al., 2010). Marked geographic differences in H. pylori prevalence were previously attributed to differential acquisition rates during early childhood and developing countries display higher incidences of H. pylori infections during childhood (<10 years of age) than developed countries (Pounder and Ng, 1995).

The vacuolating cytotoxin VacA represents an important determinant of pathogenicity with highly complex interactions between H. pylori and the epithelial cells of the gastric mucosa which have now become a paradigm for host–pathogen association. Virulence factors are generally defined as molecules produced by pathogens that contribute to colonization, attachment to host cells, evasion of the host immune response and consumption of nutrients. Among the major H. pylori virulence factors are flagellin, urease, catalase, mucinase, lipase, neutrophil activating protein (NAP), outer membrane proteins (OMP), lipopolysaccharides, cytotoxin cytotoxin-associated gene pathogenicity island (cagPAI) and the vacuolating cytotoxin VacA (Essawi et al., 2013, Rieder et al., 2005). Some actions of the VacA toxin are apparently antagonized by opposing effects mediated through CagA (Argent et al., 2008). Highly pathogenic (“type I”) strains of H. pylori display a constant association of VacA and CagA and it was speculated that CagA-VacA interaction would be required to promote long-term colonization of the stomach by ameliorating the detrimental effects of the bacterial virulence factor (Oldani et al., 2009). The cagPAI covers approximately 40 kb and comprises 30 genes including a type IV bacterial secretion system utilized by the CagA protein, which contributes to a pro-inflammatory response. However, recent studies have shown that CagA plays only a minor role, if any, during the release of pro-inflammatory cytokines such as interleukin-8 (Fischer et al., 2001, Kusters et al., 2006).

Regulatory effects exerted by CagA depend on tyrosine phosphorylation by host kinases. Accumulation of unphosphorylated CagA triggers an anti-apoptotic mechanism at the mitochondria without affecting the intracellular trafficking of the toxin, whereas phosphorylated CagA apparently prevents VacA to reach its intracellular target compartments (Oldani et al., 2009). Although it seems plausible that the proinflammatory and anti-apoptotic effects of CagA are contributors to the development of peptic ulcer and gastric cancer, the progress of gastroduodenal diseases is probably attributable to a multitude of bacterial virulence factors as well as various host factors (de Bernard and Josenhans, 2014). It is interesting to note that the cagPAI pathogenicity island is not present in all strains of H. pylori and humans infected with cagPAI-negative strains apparently remain symptomless (Ferreira et al., 2014).

While there is a number of excellent reviews published recently on host-pathogen interactions of H. pylori (Backert and Tegtmeyer, 2010, Bornschein and Malfertheiner, 2014, Cid et al., 2013, Yamaoka and Graham, 2014), this review will focus mainly on structure-mechanism relations of the VacA toxin from H. pylori. For a comprehensive overview, the reader is directed to previous reviews summarizing the history of key findings obtained for VacA (Boquet and Ricci, 2012, Isomoto et al., 2010, Palframan et al., 2012). We apologize to authors whose work we have failed to cite owing to space constraints.

Section snippets

VacA – structure

The VacA cytotoxin represents a multifunctional protein of about 860 amino acid residues which displays structural, mechanistic and functional features unrelated to other known bacterial toxins (Cover and Blanke, 2005). The vacA gene is present as sole chromosomal copy in all strains investigated to date and can vary in length from 3.86 to 3.94 kbp. The gene sequences encoding VacA show considerable variations and some strains were identified which ostensibly express a functionally inactive

Oligomerization and receptor binding

The crystal structure of the VacA p55 domain has been determined at a resolution of 2.4 Å and consists of a right-handed β-helix (residues 355–735) and a small C-terminal globular domain (residues 736–811) (Fig. 2) (Gangwer et al., 2007). The p55 structure has two main parts: residues 355–735 within p55 represent a right-handed parallel β-helix and a small globular domain at the C –terminus (residues 736–811) exhibits mixed α/β secondary structure elements. This structure is consistent with the

Membrane insertion, vacuolation and apoptosis

VacA is able to insert into lipid membranes and form low-conductance pores which display moderate selectivity for anions over cations (Iwamoto et al., 1999). The mechanism of VacA insertion into biological membranes and the structural requirements for pore formation are not well understood at present and no 3-dimensional structural model based on crystallographic data for the p33 pore forming domain is available. However, it is well established that the ultimate result of ion channel formation

Allelic diversity

Strains of H. pylori can demonstrate considerable variations in their production of VacA cytotoxin activity due to extensive polymorphisms in vacA gene structure. Clinical isolates of H. pylori were reported that fail to express a functionally VacA protein due to internal duplications, deletions, 1 bp insertions or nonsense mutations (Ito et al., 1998). A section of the vacA gene which exhibits maximum sequence diversity corresponds to approximately 800 bp in the middle of the gene within the

Conclusions

Currently the VacA cytotoxin of H. pylori is intensively investigated for a number of reasons. The structural and mechanistic properties of VacA are unrelated to any other known bacterial toxin. The VacA toxin is an important virulence factor for the bacterial colonization of the stomach and the development of gastroduodenal diseases. The gene encoding VacA is characterized by a high degree of allelic diversity and variants of the toxin have been linked to different risks of diseases caused by

Acknowledgments

The authors would like to thank Prof. Dr. Wanpen Chaicumpa, Department of Parasitology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, and Prof. Dr. Chanan Angsuthanasombat, Institute of Molecular Biosciences, Mahidol University, for critical discussions and helpful advice. Anchalee Nirachanon is acknowledged for excellent secretarial assistance. This work was supported by grant RSA55800047 (to GK) and Institute Research Grant IRG5780009 from the Thailand Research Fund

References (102)

  • C. El-Bez et al.

    High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy

    J. Struct. Biol.

    (2005)
  • R.M. Ferreira et al.

    Clinical relevance of Helicobacter pylori vacA and cagA genotypes in gastric carcinoma

    Best. Pract. Res. Clin. Gastroenterol.

    (2014)
  • J.H. Foo et al.

    Both the p33 and p55 subunits of the Helicobacter pylori VacA toxin are targeted to mammalian mitochondria

    J. Mol. Biol.

    (2010)
  • H. Iwamoto et al.

    VacA from Helicobacter pylori: a hexameric chloride channel

    FEBS Lett.

    (1999)
  • M.S. McClain et al.

    Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori vacuolating toxin

    J. Biol. Chem.

    (2003)
  • M. Molinari et al.

    The acid activation of Helicobacter pylori toxin VacA: structural and membrane binding studies

    Biochem. Biophys. Res. Commun.

    (1998)
  • C. Nogueira et al.

    Helicobacter pylori genotypes may determine gastric histopathology

    Am. J. Pathol.

    (2001)
  • P.I. Padilla et al.

    Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity

    J. Biol. Chem.

    (2000)
  • E. Papini et al.

    In search of the Helicobacter pylori VacA mechanism of action

    Toxicon

    (2001)
  • J. Rassow et al.

    Helicobacter pylori VacA: a new perspective on an invasive chloride channel

    Microbes Infect.

    (2012)
  • J.M. Reyrat et al.

    3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin

    J. Mol. Biol.

    (1999)
  • J.L. Rhead et al.

    A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer

    Gastroenterol

    (2007)
  • G. Rieder et al.

    Interaction of Helicobacter pylori with host cells: function of secreted and translocated molecules

    Curr. Opin. Microbiol.

    (2005)
  • X. Sewald et al.

    Sticky socks: Helicobacter pylori VacA takes shape

    Trends Microbiol.

    (2008)
  • X. Sewald et al.

    Integrin subunit CD18 is the T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin

    Cell Host Microbe

    (2008)
  • J. Suzuki et al.

    Involvement of Syntaxin 7 in human gastric epithelial cell vacuolation induced by the Helicobacter pylori-produced cytotoxin VacA

    J. Biol. Chem.

    (2003)
  • F. Tombola et al.

    Inhibition of the vacuolating and anion channel activities of the VacA toxin of Helicobacter pylori

    FEBS Lett.

    (1999)
  • V.J. Torres et al.

    Interactions between p-33 and p-55 domains of the Helicobacter pylori vacuolating cytotoxin VacA

    J. Biol. Chem.

    (2004)
  • V.J. Torres et al.

    Functional Properties of the p33 and p55 Domains of the Helicobacter pylori vacuolating cytotoxin

    J. Biol. Chem.

    (2005)
  • H.-J. Wang et al.

    Expression and binding analysis of GST-VacA fusions reveals that the C-Terminal ∼100-Residue segment of exotoxin is crucial for binding in HeLa cells

    Biochem. Biophys. Res. Commun.

    (2000)
  • X. Wang et al.

    Membrane topology of VacA cytotoxin from H. pylori

    FEBS Lett.

    (2000)
  • L.T. Weaver

    Royal society of tropical medicine and hygiene meeting at manson house, London, 16 February 1995. Aspects of Helicobacter pylori infection in the developing and developed world. Helicobacter pylori infection, nutrition and growth of west African infants

    Trans. R. Soc. Trop. Med. Hyg.

    (1995)
  • S. Wen et al.

    Helicobacter pylori virulence factors in gastric carcinogenesis

    Cancer Lett.

    (2009)
  • K. Yahiro et al.

    Protein-tyrosine phosphatase, RPTP, is a Helicobacter pylori VacA receptor

    J. Biol. Chem.

    (2003)
  • K. Yahiro et al.

    Essential domain of receptor tyrosine phosphatase (RPTP ) for interaction with Helicobacter pylori vacuolating cytotoxin

    J. Biol. Chem.

    (2004)
  • E. Yamasaki et al.

    Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome c release and cell death, independent of vacuolation

    J. Biol. Chem.

    (2006)
  • D. Ye et al.

    Identification of the minimal intracellular vacuolating domain of the Helicobacter pylori vacuolating toxin

    J. Biol. Chem.

    (1999)
  • M. Akguc et al.

    Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies

    Mikrobiyol. Bul.

    (2014)
  • T.N. Archampong et al.

    Epidemiology of Helicobacter pylori infection in dyspeptic Ghanaian patients

    Pan. Afr. Med. J.

    (2015)
  • R.H. Argent et al.

    Functional association between the Helicobacter pylori virulence factors VacA and CagA

    J. Med. Microbiol.

    (2008)
  • J.C. Atherton et al.

    Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori comprise two geographically widespread types, m1 and m2, and have evolved through limited recombination

    Curr. Microbiol.

    (1999)
  • J.C. Atherton

    The pathogenesis of Helicobacter pylori-induced gastro-duodenal diseases

    Annu. Rev. Pathol.

    (2006)
  • S. Backert et al.

    The versatility of the Helicobacter pylori vacuolating cytotoxin VacA in signal transduction and molecular crosstalk

    Toxins

    (2010)
  • K.P. Batts et al.

    Appropriate use of special stains for identifying Helicobacter pylori: Recommendations from the Rodger C. Haggitt Gastrointestinal Pathology Society

    Am. J. Surg. Pathol.

    (2013)
  • K.F. Boehnke et al.

    Animal model reveals potential waterborne transmission of Helicobacter pylori infection

    Helicobacter

    (2015)
  • J. Bornschein et al.

    Helicobacter pylori and gastric cancer

    Dig. Dis.

    (2014)
  • D.R. Bridge et al.

    Polymorphism in the Helicobacter pylori CagA and VacA toxins and disease

    Gut Microbes

    (2013)
  • L.M. Brown

    Helicobacter pylori: epidemiology and routes of transmission

    Epidemiol. Rev.

    (2000)
  • F. Calore et al.

    Endosome-mitochondria juxtaposition during apoptosis induced by H. pylori VacA

    Cell Death Differ.

    (2010)
  • T.P. Cid et al.

    Pathogenesis of Helicobacter pylori infection

    Helicobacter

    (2013)
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