ReviewVacuolating cytotoxin A (VacA) – A multi-talented pore-forming toxin from Helicobacter pylori
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)
- et al.
Multiple Oligomeric States of the Helicobacter pylori vacuolating toxin demonstrated by cryo-electron microscopy
J. Mol. Biol.
(2002) - et al.
Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration
J. Biol. Chem.
(1995) - et al.
Contaminated water as a source of Helicobacter pylori infection: a review
J. Adv. Res.
(2015) - et al.
Intoxication strategy of Helicobacter pylori VacA toxin
Trends Microbiol.
(2012) - et al.
Structural analysis of the oligomeric states of Helicobacter pylori VacA toxin
J. Mol. Biol.
(2013) - et al.
Induction of apoptosis and expression of apoptosis related genes in human epithelial carcinoma cells by Helicobacter pylori VacA toxin
Toxicon
(2003) - et al.
Purification and characterization of the vacuolating toxin from Helicobacter pylori
J. Biol. Chem.
(1992) - et al.
Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains
J. Biol. Chem.
(1994) - et al.
Helicobacter pylori in health and disease
Gastroenterol
(2009) - et al.
Mimicry of a host anion channel by a Helicobacter pylori pore-forming toxin
Biophys. J.
(2005)
High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy
J. Struct. Biol.
Clinical relevance of Helicobacter pylori vacA and cagA genotypes in gastric carcinoma
Best. Pract. Res. Clin. Gastroenterol.
Both the p33 and p55 subunits of the Helicobacter pylori VacA toxin are targeted to mammalian mitochondria
J. Mol. Biol.
VacA from Helicobacter pylori: a hexameric chloride channel
FEBS Lett.
Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori vacuolating toxin
J. Biol. Chem.
The acid activation of Helicobacter pylori toxin VacA: structural and membrane binding studies
Biochem. Biophys. Res. Commun.
Helicobacter pylori genotypes may determine gastric histopathology
Am. J. Pathol.
Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity
J. Biol. Chem.
In search of the Helicobacter pylori VacA mechanism of action
Toxicon
Helicobacter pylori VacA: a new perspective on an invasive chloride channel
Microbes Infect.
3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin
J. Mol. Biol.
A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer
Gastroenterol
Interaction of Helicobacter pylori with host cells: function of secreted and translocated molecules
Curr. Opin. Microbiol.
Sticky socks: Helicobacter pylori VacA takes shape
Trends Microbiol.
Integrin subunit CD18 is the T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin
Cell Host Microbe
Involvement of Syntaxin 7 in human gastric epithelial cell vacuolation induced by the Helicobacter pylori-produced cytotoxin VacA
J. Biol. Chem.
Inhibition of the vacuolating and anion channel activities of the VacA toxin of Helicobacter pylori
FEBS Lett.
Interactions between p-33 and p-55 domains of the Helicobacter pylori vacuolating cytotoxin VacA
J. Biol. Chem.
Functional Properties of the p33 and p55 Domains of the Helicobacter pylori vacuolating cytotoxin
J. Biol. Chem.
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.
Membrane topology of VacA cytotoxin from H. pylori
FEBS Lett.
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.
Helicobacter pylori virulence factors in gastric carcinogenesis
Cancer Lett.
Protein-tyrosine phosphatase, RPTP, is a Helicobacter pylori VacA receptor
J. Biol. Chem.
Essential domain of receptor tyrosine phosphatase (RPTP ) for interaction with Helicobacter pylori vacuolating cytotoxin
J. Biol. Chem.
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.
Identification of the minimal intracellular vacuolating domain of the Helicobacter pylori vacuolating toxin
J. Biol. Chem.
Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies
Mikrobiyol. Bul.
Epidemiology of Helicobacter pylori infection in dyspeptic Ghanaian patients
Pan. Afr. Med. J.
Functional association between the Helicobacter pylori virulence factors VacA and CagA
J. Med. Microbiol.
Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori comprise two geographically widespread types, m1 and m2, and have evolved through limited recombination
Curr. Microbiol.
The pathogenesis of Helicobacter pylori-induced gastro-duodenal diseases
Annu. Rev. Pathol.
The versatility of the Helicobacter pylori vacuolating cytotoxin VacA in signal transduction and molecular crosstalk
Toxins
Appropriate use of special stains for identifying Helicobacter pylori: Recommendations from the Rodger C. Haggitt Gastrointestinal Pathology Society
Am. J. Surg. Pathol.
Animal model reveals potential waterborne transmission of Helicobacter pylori infection
Helicobacter
Helicobacter pylori and gastric cancer
Dig. Dis.
Polymorphism in the Helicobacter pylori CagA and VacA toxins and disease
Gut Microbes
Helicobacter pylori: epidemiology and routes of transmission
Epidemiol. Rev.
Endosome-mitochondria juxtaposition during apoptosis induced by H. pylori VacA
Cell Death Differ.
Pathogenesis of Helicobacter pylori infection
Helicobacter
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2020, Protein Expression and PurificationCitation Excerpt :However, no VacA homolog has been reported in Helicobacter species or other bacteria so far, although there was one previous study reporting that the deduced amino acid sequence of the cloned vacA gene contains a ~10-residue C-terminal segment that is commonly identified in several other bacterial proteins, including proteases and/or surface proteins from other Gram-negative bacteria [14]. Most common polymorphisms are signal (s) region which resides at the N-terminus of VacA, and also intermediate (i) and mid (m) regions which are located within the N-terminal and C-terminal domains, respectively [13]. Among them, the ‘mid’ region variants, particularly m1 and m2, are frequently associated with severe pathogenicity and detected at similar incidences in Asia, Europe or elsewhere [15–20].
Oral pH sensitive GNS@ab nanoprobes for targeted therapy of Helicobacter pylori without disturbance gut microbiome
2019, Nanomedicine: Nanotechnology, Biology, and MedicineImportance of the Cys<sup>124</sup>−Cys<sup>128</sup> intermolecular disulfide bonding for oligomeric assembly and hemolytic activity of the Helicobacter pylori TlyA hemolysin
2019, Biochemical and Biophysical Research CommunicationsCitation Excerpt :H. pylori infection significantly increases gastric cancer risk and about 3% of infected patients developed gastric cancer, making H. pylori the only bacterial pathogen classified as a class I carcinogen [1]. H. pylori contains a wide range of virulence factors which are essential for continual infection and disease development such as urease, catalase, lipopolysaccharides, adhesins, the cytotoxin-associated gene A and vacuolating cytotoxin A [2]. TlyA is a hemolysin/cytolysin encoded by HP1086 gene (Gene ID: 899622) within H. pylori strain 26695 (NC_000915.1) [3].
Preventive effect of anti-VacA egg yolk immunoglobulin (IgY) on Helicobacter pylori-infected mice
2018, VaccineCitation Excerpt :Most studies focused on the production of IgY against H. pylori whole-cells, H. pylori-lysate, or purified urease because of its unique relevance to H. pylori infection [29]. VacA toxin is involved in disease progress and colonization by H. pylori of the stomach and is a potential and promising candidate for antigen in a vaccine against H. pylori [30]. Until now, no studies have described the use of anti-VacA IgYs for preventing H. pylori infection, although some reports showed that VacA can be used as an antigen for oral vaccination [31,32].
Helicobacter pylori point-of-care diagnosis: Nano-scale biosensors and microfluidic systems
2017, TrAC - Trends in Analytical ChemistryCitation Excerpt :The VacA protein also localizes to the mitochondrial membrane and induces release of cytochrome c from mitochondria leading to the activation of an apoptotic cascade. This cellular destruction sends signals to the immune system by the production of cytokines, leading to tissue infiltration of immune cells [19,22]. Lastly, release of free radicals from the granulocytes causes the damage.