Helicobacter Pylori: Phenotypes, Genotypes and Virulence Genes

José Luiz Proença-Modena; Gustavo Olszanski Acrani; Marcelo Brocchi

Disclosures

Future Microbiol. 2009;4(2):223-240. 

In This Article

Genes, Genotypes & Phenotypes of H. pylori Associated with Cell Signaling & Tissue Damage

In the previous section the virulence factors of H. pylori related to infection and colonization of the host have been discussed. In this section, the focus will be on the factors that mediate cell signaling and tissue damage. However, it is important to note that it is generally difficult to establish specific roles for the majority of the virulence genes of H. pylori since the same gene or group of genes may affect pathogenicity in several ways: contributing to the infection, host cell function, persistence and tissue damage. In this section we will discuss the role of the cag pathogenicity island (cagPAI), the vacA and iceA genes, and various genes located in a variable region of the H. pylori genome denoted the 'plasticity region' (PR).

The cag Pathogenicity Island & the Type IV Secretion System

The cagPAI is a genomic region of approximately 40 kb inserted into the glutamate racemase (glr) gene of H. pylori (Table 1).[63] The cagPAI consists of approximately 30 genes that encode proteins that comprise a type IV secretion system (T4SS) used for the translocation of bacterial products directly into the cytoplasm of the host cell.[6,13,63,64,65,66] In some H. pylori strains, the cagPAI region is interrupted by the insertion of IS605, a mobile sequence.[63] We will focus our discussion on the genes in which a function in pathogenicity and clinical outcome is well established, such as the cytotoxin-associated gene (cag)A, cagE, cagT and cagM.

The cagA gene, which is generally used as a marker for the presence of cagPAI, codes for CagA, one of the first proteins to be associated with virulence in H. pylori.[67,68] CagA has no homology with other known proteins, and the presence of CagA has been associated with very severe gastritis, and an increased risk of peptic ulcer disease (including both gastric and duodenal ulcers), atrophic gastritis and gastric cancer, particularly in western populations.[11,41,63,69,70,71,72,73,74,75,76] CagA is recognized by T cells in the gastric mucosa of H. pylori infected patients.[77,78] Indeed, the Th1 polarization of the immune response to CagA and other H. pylori antigens has been associated with peptic ulcers (reviewed in [79]).

The protein CagA is translocated through the type IV secretion apparatus into the cytoplasm of epithelial-gastric cells, where tyrosine residues located in the C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) repeated motifs, present in variable numbers in distinct CagA proteins, are phosphorylated by host Src-family kinases and Abl kinases.[80,81,82,83,84,85,86] The interaction of phosphorylated CagA with host signaling molecules, such as the tyrosine phosphatase Shp-2,[87] C-terminal Src kinase (Csk)[88] and Crk adaptor protein,[89] results in several changes in the host epithelial cell (Figure 1B). The most obvious in vitro effect is the stimulation of a change in cell shape, resulting in the development of cell elongation processes. This elongation has been entitled the hummingbird phenotype,[80] and it may be important in the adherence of H. pylori to host cells as well as their interaction.[80]

The formation of a complex with Shp-2 depends on CagA oligomerization, which is mediated by a domain of 16 amino acids - the CagA multimerization sequence - located in the C-terminal half of the protein.[90] The CagA-Shp-2 complex dephosphorylates and inactivates FAK but activates the Erk signaling pathway, events that are involved in the hummingbird phenotype.[91,92] CagA also inhibits Src by activation of Csk, resulting in the dephosphorylation of actin-binding proteins, leading to actin polymerization and cell elongation.[88,89,93] Cytoskeletal rearrangement is mediated by CagA through activation of Crk and Rac1.[94]

CagA can also interact with cell-signaling molecules in its nonphosphorylated state.[66] This protein interacts with the epithelial tight-junction scaffolding protein ZO-1 and partitioning-defective-1 (PAR1) kinase. The latter is a central regulator of cell polarity, causing disruption of the tight junction and the loss of epithelial barrier function and apical-basolateral polarity.[95,96,97] CagA is also able to interact with E-calderin, activating the Wnt/β-catenin pathway, which may be involved in intestinal transdifferentiation in gastric epithelial cells.[98] In addition, CagA takes part in the activation of the proinflammatory transcription factor NF-κB, an event that likely involves the binding of this protein to Grb-2.[66,99,100] In addition to the properties cited earlier, there are studies demonstrating that CagA affects cell proliferation, through activation of c-Fos and c-Jun,[101] and the development of an immune response, since it induces apoptosis of T cells.[102]

While there is a general association between the presence of CagA and gastric diseases, the number of EPIYA tyrosine phosphorylation motifs varies from one H. pylori isolate to another[83,85,103,104] as stated earlier, and CagA proteins with a greater number of these motifs induce more morphological changes in cultured epithelial cells, such as cell elongation,[105] suggesting a link between the number of these motifs and clinical outcomes.

Other genes with a role in virulence that occur in the cagPAI have also been correlated with gastric diseases, such as cagE, cagT and cagM. The first of these is a 2665 bp gene, initially referred to as picB,[106] which is essential for the induction of IL-8 production by host cells.[106] This gene encodes a 101 kDa polypeptide, identified as a putative ATPase, homologous to the Agrobacterium tumefaciens VirB4 protein.[13,64,65,66] CagE is now recognized as a key structural component of the H. pylori T4SS.[104,107] The cagE gene may be a more accurate marker of intact cagPAI than cagA,[69,108] and its presence has been associated with peptic ulcer disease,[69,108,109] indicating that the detection of cagE can also be used for medical prognosis, since strains with the cagPAI are generally associated with the more severe clinical outcomes such as ulcers and gastric cancer, as discussed previously.

The cagT gene is homologous to the virB7 of A. tumefaciens: it encodes an extracellular lipoprotein located at the base of the T4SS filament. CagT likely helps to stabilize the other Vir proteins through the formation of a disulfide bridge with a VirB9 homolog, thereby playing a crucial role in the H. pylori T4SS.[64,65,66] Some studies have shown a significant association between cagT and peptic ulcer disease.[109,110] This can be explained by the fact that H. pylori strains, lacking the cagT gene, have impaired T4SS machinery, which is unable to induce IL-8 production and translocate CagA into the human gastric adenocarcinoma cell line in vitro.[107]

Mattar et al. observed a correlation between cagM, another gene of the cagPAI, and peptic ulcer disease.[109] Indeed, CagM is necessary to mediate the activation of the NF-κB required for IL-8 induction.[111] The T4SS also injects peptidoglycan into host-cell cytoplasm and induces the production of proinflammatory cytokines.[112]

The cagPAI can undergo partial deletions or it can be entirely excised from a given strain.[113] Indeed, there are results demonstrating the colonization of patients by the same or very similar strains, but with differing cagPAI status.[114,115,116,117] As discussed earlier, since cagPAI can influence cell metabolism in a number of ways, the existence of a balance between cagPAI-positive and -negative strains colonizing the stomach may modulate the host response to infection, contributing to the persistence of H. pylori in the host.[115]

Vacuolating Cytotoxin & the Cytotoxic Effect Induced by H. Pylori

The vacA gene is universally present in H. pylori and encodes an approximately 88 kDa toxin termed vacuolating cytotoxin (VacA).[118,119] VacA is synthesized as a 140 kDa precursor protein, which undergoes cleavage at both N-terminal signal sequences and in the C-terminal domain to yield a mature toxin. After secretion, part of the VacA mature toxin is released as free molecules, and part of it remains associated with the bacterial cell surface.[120] The toxin molecules associated with the cell surface are fully toxigenic and are transferred to host cells after contact with the bacterium.[121] The free toxin molecules oligomerize into a complex that resembles a flower, but it is not clear if this complex is fully toxigenic.[122]

VacA is significantly associated with gastric diseases and seems to play an important role in the pathogenesis of peptic ulceration and gastric cancer.[123,124]  In vivo experiments also suggest that this toxin contributes to gastric colonization in a mouse model of infection.[125]

The vacA gene is polymorphic and exhibits considerable nucleotide sequence variability, resulting in vacA alleles with different vacuolating activities (Table 1).[1,2,4,123,126] Therefore, the level of toxicity varies between different isolates of H. pylori, as determined by the type of vacA allele expressed by a given bacterium. This variability is especially correlated with the signal sequence (s), intermediate (i) and middle regions (m) of the vacA gene.[123,126] Two types of signal sequence (s1 and s2) as well as two types of middle (m1 or m2) and intermediate (i1 or i2) regions are documented.[123,126] Therefore, the vacA gene of a given strain is composed of any of the possible combinations of s and m region sequence types, although the s2/m1 combination is rare.[123,127] Both s1/m1 and s1/m2 alleles of vacA encode toxic forms of the toxin and are common in patients presenting gastric diseases (reviewed in [4]) but the vacA s1 and vacA s1/m1 genotypes are most frequently associated with ulcers and gastric carcinoma, especially in western populations.[11,42,69,70,71,72,128,129] The s2 form has a short N-terminal peptide extension on the mature protein, which blocks the biological activities of VacA.[130] H. pylori strains that have the s2/m2 or s2/m1 genotype encode proteins of low toxicity and are less frequently associated with peptic ulcers and gastric cancer.[2,4] The vacA polymorphism related to the i region was recently described.[126] This study demonstrated that only the s1/m2 vacA allele diverges with respect to the i type, since s1/m1 and s2/m2 were invariably typed as i1 and i2, respectively. The vacuolating activity of s1/m2 H. pylori strains was found to depend on the i type and vacA i1 was associated with adenocarcinoma. In a recent study of H. pylori strains in western populations, vacA i1 was associated with peptic ulcer.[131] Together, these data point to the i region as an important and interesting virulence marker to be investigated further.

Diverse biological effects can be induced by VacA (Figure 1C).[1,2,4,6,132] It is a toxin that forms pores in host-cell membranes, which are selective to anions and small neutral molecules, including urea.[133,134,135] The endocytosis of VacA and its subsequent processing into late-endosomal compartments induce the formation of large acid vacuoles. This is the most notable effect of this toxin in vitro,[136] although its in vivo role is not yet clear.[4,6] This toxin also increases paracellular permeability, mediating the liberation of various molecules that can be used as nutrients by H. pylori.[137] For instance, the liberation of urea is important since it is the target of urease in the production of ammonia to buffer the acidic environment. In the cytosol of the host cell, VacA targets the mitochondrial inner membrane, inducing the efflux of cytocrome C and the apoptosis of gastric epithelial cells.[138,139] VacA can also induce immunosuppression by blocking antigen presentation to T cells,[140] inhibition of T-lymphocyte activation[141] and interruption of maturation of macrophage phagosomes.[142]

Although the cagPAI and vacA are located separately in the H. pylori genome, the presence of the cagPAI (with the presence of cagA) and expression of an active s1 form of VacA seems to be associated with features of H. pylori strains that are closely related to greater gastric inflammation and the development of peptic ulcers, atrophic gastritis and gastric cancer.[4,6] It is not yet clear why this combination is important, but it is frequently difficult to associate one of these factors independently with clinical symptoms.[1,4]

Induced by Contact with Epithelium A Gene

The induced by contact with epithelium (iceA) gene has two main allelic variants, named iceA1 and iceA2.[143] The iceA1 gene is expressed when H. pylori comes into contact with the gastric epithelial cells, and its presence has been associated with peptic ulceration in some studies[143] but not in others (Table 1).[11,43,144,145,146] The iceA2 gene is generally more prevalent in H. pylori strains isolated from patients with nonulcer dyspepsia, but there are also some exceptions.[43,146] Therefore, the role of these two alleles in pathogenicity, if any, is not certain.

The iceA1 gene, unlike iceA2, is homologous to a restriction endonuclease gene (nlaIIIR) of Neisseria lactamica. The iceA1 and iceA2 alleles are located upstream of hpy1M, a CTAG-specific methyltransferase gene in H. pylori, and the presence of iceA1 leads to greater expression of this methylase than the presence of iceA2.[147] Therefore, iceA1-hpy1M genes form a restriction-modification (RM) unit. RM systems are composed of a methyltransferase, which catalyzes the methylation of a specific DNA sequence and thus protects the DNA from cleavage by the restriction enzyme, which possesses endonuclease activity. Therefore, RM systems pose barriers to gene transfer, as well as protecting the bacterium against phage infections, since heterologous DNA entering the bacterial cell may not be methylated and would then be accessible to the restriction enzyme. It is not known if iceA1- hpy1M genes have a role in pathogenesis, but even if research suggests that the full-length iceA1 gene is a functional endonuclease, many strains of H. pylori contain nonfunctional mutant variants of this gene.[148]

In light of this, a possible explanation for the association between iceA1 and disease is just the higher level of hpy1M expression when associated with iceA1. DNA methylation influences several events in the cell, including gene expression, and thus has an important role in bacterial pathogenicity. For instance, DNA adenine methylase (Dam) plays a part in the virulence of Salmonella enterica Typhimurium. A Dam strain of S. enterica was defective in colonization of deep tissues and avirulent when assayed in a mouse model of infection.[149] Hence, it may be speculated that hpy1M influences the pattern of gene expression and pathogenicity in H. pylori infection. If this is the case, the role of iceA1 in pathogenicity may be indirect and due to its association with a higher level of hpy1M expression. However, since H. pylori contains various RM systems and the association between iceA1 and virulence is not always found, further studies are needed to confirm whether methylation promoted by hpy1M has a direct role in H. pylori virulence.

 jhp940, jhp947, jhp949 & dupA Genes

The so-called plasticity region (PR) is a large sequence of 45 kb in strain J99 and 68 kb in strain 26695, which received this name on account of its variability in nucleotide sequence and gene content. The G-C content of this region is different tos the rest of the H. pylori genome,[14,15] indicating that it may have been acquired by lateral gene transfer from other species of bacteria. This would explain why nearly half the strain-specific genes of H. pylori are located in this region. For instance, the PR is composed of 38 genes in J99, 33 of which are absent in strain 26695.[14,15,150] Moreover, several studies have implicated various CDS of the PR in virulence (Table 1), as we will discuss below.

Occhialini et al. identified two new potential pathogenicity markers in the PR.[151] These genes (jhp0940 and jhp0947) were frequently detected in strains obtained from patients with gastric cancer, suggesting that the product of these genes might be associated with the genesis of this pathology. More recently, another study has confirmed the association between jhp0947 (but not jhp0940) and gastric cancer, and showed that this gene may be implicated in the development of duodenal ulcer.[152] Therefore the jhp0947 gene is a candidate for a virulence marker. In addition, its presence was linked to that of cagAin the same study. However, the association between CagA and jhp0947 is not universal.[69,153] Considering the high genetic variability exhibited by H. pylori, the adaptation of strains to particular human populations is probably mediated by the gain or loss of specific groups of genes. This is also true of genes present in the PR. Thus, the presence of pathogenic genes in this region may depend on the specific human population and/or the geographic region involved.

Jonge et al. reported that jhp0949, another gene in the PR, is significantly associated with duodenal ulcer patients.[153] These results were obtained by comparing the distribution of jhp0949 in 19 patients with gastritis and 26 with duodenal ulcers in Holland. It would be interesting to ascertain whether this association is maintained in other countries.

The VirB4 homolog sequences jhp0917 and jhp0918 are considered a single continuous gene, which has been designated the duodenal ulcer-promoting gene (dupA).[154] This gene is considered a marker for the development of duodenal ulcers, but it is also inversely associated with gastric atrophy, intestinal metaplasia and gastric cancer.[154] The association of dupA with duodenal ulcers was confirmed in another study.[155] However, recent studies performed in Brazil[110,156] and other countries[157,158] have reported a lack of association between dupA and duodenal ulcers. These discrepant results may be explained by genetic differences between the H. pylori strains isolated in different human populations and/or geographic regions. Certainly, it would be very interesting to perform more studies on the dupA status of H. pylori strains in other human populations to clarify its role in the genesis of duodenal ulcers.

Recently, an excellent review on the roles in pathogenicity of genes present in the PR of H. pylori was published.[159]

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