ORIGINAL ARTICLE
Virulence Factors of Helicobacter pylori Luigina Cellini 1 and Gianfranco Donelli2 From the 1Laboratory of Medical Bacteriology, Department of Biomedical Sciences, UniversitaÁ `G. D’Annunzio’, Chieti and 2Laboratory of Ultrastructures, Istituto Superiore di SanitaÁ, Rome, Italy Correspondence to: Dr. Luigina Cellini, Dept. Biomedical Sciences, UniversitaÁ `G. D’Annunzio’, Via dei Vestini, 66100 Chieti, Italy. Fax » 39 0871 3555282; E-mail:
[email protected]
Microbial Ecology in Health and Disease 2000; Suppl 2: 259– 262 This review focuses on the main virulence factors characterizing Helicobacter pylori strains. Several pathogenic factors are important for the establishment and maintenance of H. pylori infection. Among those present in all isolates are the production of urease and phospholipases, the presence of agella, the ability to attract neutrophils, the expression of iceA gene and a number of adhesins that ensure tissue-specic colonization. In addition, a subset of H. pylori strains is characterized by: i) a potent toxin (VacA) able to cause vacuolar degeneration of target cells by interfering with intracellular membrane fusion; and ii) the pathogenicity island (PAI), named CagPAI that encodes for a putative secretory mechanism in which the cagA gene encodes an immunodominant antigen that is associated with cytotoxin expression.
INTRODUCTION Helicobacter pylori colonizes the human stomach early in life even if only decades later the related pathology may be expressed. Both the spiral-shaped bacterium and its coccoid form (1 –5) persist in a unique biological niche within the gastric mucus layer (6) and are able to cause a strong inammatory state and lesions of the gastric mucosa (7) and to drive a relevant immune response in a restricted percentage of hosts (8, 9). In fact, disease occurs only in about 15% of people infected, with the development of gastritis, gastric glandular athrophy, duodenal and gastric ulcers, gastric adenocarcinoma or MALT lymphoma (10). Genomic and phenotipic features of different strains, presumably together with the transient condition of the gastric microenvironment, allow the expression of virulence factors which enable some strains, rather than others, to cause disease (7, 10). H. pylori expresses its pathogenicity through: (i) adhesion to gastric epithelium; (ii) colonization of the mucous gel layer increasing the permeability to hydrogen ions and pepsin; (iii) penetration in and distruction of intercellular junctions; (iv) invasion of gastric glands and canaliculi of parietal cells; (v) evasion of host immune defences; (vi) secretion of enzymes and production of cytotoxins (11). Several virulence factors (Table I) contribute to the pathogenicity of H. pylori. UREASE Urease is an important virulence factor for H. pylori and is critical for bacterial colonization of the human gastric © Taylor & Francis 2000. ISSN 1403-4174
mucosa. H. pylori urease metabolizes urea producing ammonia to neutralise the microenvironment in which the bacterium resides (12). The presence of cytoplasmatic urease activity suggests a role of this enzyme in assimilation of organic nitrogen (12, 13). The ammonia production can damage the gastric mucosa through the disruption of tight junctions and the alteration of permeability of gastric epithelium. Moreover, urease stimulates activation of mononuclear phagocytes and production of inammatory cytokines (14). The native H. pylori urease consists of a nickel-containing hexameric molecule with a molecular mass of approximately 540 kDa made up of two subunits: UreA [30 kDa] and UreB [62 kDa]. The urease gene cluster contains nine genes, including ureA and ureB structural genes (15). PHOSPHOLIPASES H. pylori phospholipases induce generation of products such as lysolecithin which disrupt the protective phospholipid-rich layer on the apical membrane of mucus cells (16). FLAGELLA The presence of agella is an essential factor of colonization in H. pylori. Aagellate strains are not able to colonize gnotobiotic piglets (17). H. pylori possesses two to six polar agella characterized by two types of agellin proteins coded by aA and aB genes that are required for full motility and persistent infection of the gastric mucosa (18). A recent study demonstrated that agellar biosynthesis and urease activity may be linked (19). Microbial Ecology in Health and Disease
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NEUTROPHIL ACTIVATING PROTEIN H. pylori is able to activate neutrophils and to increase neutrophil adherence to endotelial cells through the expression of a 150 kDa activating protein (Hp-Nap), made up of 10 identical subunits, coded by the napA gene (20). ADHESINS It is widely accepted that H. pylori adheres to receptors in the gastric epithelium by means of adhesins. Several specic receptors are involved in these mechanisms including lipids, gangliosides and sulfated carbohydrates, and different types of adhesins have been characterized (21). An interesting study revealed the presence of a protein (coded by babA and babB genes) able to bind the human blood group antigen Lewis b (Leb) to human gastric epithelial cells (22). Furthermore, the chemical structure of LPS of some strains of H. pylori has been found to mimic Lewis x and Lewis y blood group antigens expressed in the gastric mucosa; this may serve to downregulate the immune response in patients with acute and chronic infections (23). IceA The gene encoding IceA has been identied in isolates from patients with peptic ulcer, independently of the vacA and cagA genotype (24) (see below). The expression of iceA is induced by adherence of H. pylori to gastric ephitelium (25). DNA sequencing has revealed the presence of two families: iceA1 and iceA2. Strains with the iceA1 gene are most frequently associated with peptic ulceration and increase the production of IL-8 (26). VACUOLATING CYTOTOXIN (VacA) One of the primary virulence factors described for H. pylori is VacA (27). VacA is an oligomeric toxin composed of 87 kDa active subunits obtained by treatment at low pH. An antiserum produced against these puried proteins neutralizes the cytotoxic activity (27). All strains of H.
pylori possess the vacA gene and about 50–60% of them express a fully virulent cytotoxin (28, 29) able to induce acidic vacuoles in the cytoplasm of eukaryotic cells (11). The toxin causes vacuolar degeneration of target cells by interfering with intracellular membrane fusion. The vacuolation mechanism involves the stimulation of adenosinetriphosphate dependant proton pump and of a small GTPase called rab7 (30– 32). Furthermore, VacA induces an inactivation of energy metabolism followed by mitochondrial damage, leading to impairment of the cell cycle in gastric epithelial cells (33). VacA is immunolocalised in the periplasm and outer membrane of whole bacteria and also in vesicles and outer membrane blebs (34). Several different families of vacA alleles characterize H. pylori and encode products with different activities. Mosaicism in vacA alleles is expressed by ve vacA subtypes of which three concern signal sequence regions (s1a, s1b and s2 ) and two middle region motifs (m1 and m2 ) (35, 36). vacA genotypes are important in vivo because of the diversity-pathogenicity relationship among H. pylori strains (36). The s1a strains produce higher levels of cytotoxin with more severe gastric inammation and duodenal ulceration than the other two allelic s types (37). The m1 middle region allele is more frequently associated with a higher level of gastric damage as compared with the m2 form and it is toxic for Hela cells (38). Moreover, the distribution of vacA genotypes and the association with expression of pathogenicity is also related to different geographical areas (39, 40). Cag PATHOGENICITY ISLAND H. pylori strains isolated from gastric epithelium can be classied in at least two groups, named type I and type II, on the basis of genotypic and phenotypic differences. Infections by type I strains are associated with the more severe forms of disease with respect to the less virulent type II strains (41, 42). Two type I H. pylori strains, 26695 (43) and J99 (44), have been entirely sequenced and they
Table I Virulence factors of Helicobacter pylori Factor
Gene
Function
Urease
ure operon
Phospholipase Flagella Nap Adhesins IceA VacA cag PAI
gene flaA, aB napA babA1, babA2 iceA1, iceA2 vacA 31 genes coding for type IV secretion system
CagA
cagA (part of Cag PAI)
Mucosal toxicity, gastric acid neutralisation, assimilation of organic nitrogen Disruption of the gastric mucosal barrier Bacterial motility Neutrophil activation Leb binding to gastric epithelium Homologue of NIa III restriction endonuclease Cytotoxicity for gastric epithelium C-X-C chemokine family increasing neutrophilic inltration into gastric epithelium Immunodominant antigen
H. pylori virulence
differ from type II strains by the presence of a 40 Kb locus, containing 31 genes, inserted into the chromosomal glutamate-racemase gene, named Cag pathogenicity island (abbreviated CagPAI or Cag region) (41). This secretory system is involved in the induction of increased gastric mucosal levels of members of the C-X-C chemokine family, which includes the neutrophil chemoattractant IL-8 (45, 46), and promotes neutrophilic inltration into the gastric epithelium. It has recently been demonstrated that multiple genes in the left half of the CagPAI are required for transcription of the IL-8 gene in gastric epithelial cells and that this is related to the activation of protein tyrosine kinase (47). Several CagPAI genes are homologous to genes of other pathogens that encode for subunits of the specialized type IV secretory system that deliver bacterial virulence factors across the bacterial membrane to the surface or into host cells (42). H. pylori containing CagPAI is associated with the development of chronic active gastritis (26), peptic ulceration (48) and atrophic gastritis with an increased risk of gastric cancer (49). Before the characterization of CagPAI, the development of clinical disease related to H. pylori was associated with the expression of cagA gene (50). CagA is described as an immunodominant antigen with a molecular mass of 120 kDa able to express the cytotoxin encoded by vacA. This gene is at one end of the CagPAI and is considered the marker of its presence (41). Recently, the presence of CagA positive H. pylori infection has been related to food allergy (51). In fact, the enhanced mucosal and inammatory lesions could increase the epithelial permeability with a non selective passage of allergens which, in atopic subjects, could stimulate the IgE response. The incidence of CagPAI positive strains is 60– 70% all over the world, except in Korea and Japan where it is nearly 100% (52, 53). Recent studies (54) have demonstrated the presence of both Cag positive and Cag negative strains in the same patient, suggesting a dynamic equilibrium among strains in which the prevalence of one type over the other modulates the expression of the disease. ACKNOWLEDGEMENTS This review has been carried out with nancial support from the Commission of the European Communities, Agriculture, and Fisheries (FAIR), specic RTD programme PL98-4230 ‘Intestinal Flora: Colonization Resistance and Other effects’. It does not reects its views and in no way anticipates the Commission’s future policy in this area. The careful assistance of Emanuela Di Campli and Donatella Lombardi in preparation of the manuscript is gratefully acknowledged.
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