Role of Helicobacter pylori CagL in modulating gastrin expression

Helicobacter pylori is a Gram-negative bacterium that is highly adapted for persistent colonization of the human stomach. Although most H. pylori-infected persons remain asymptomatic, the presence of this organism is a risk factor for gastric adenocarcinoma, peptic ulcer disease, and gastric lymphoma. There is a high level of genetic heterogeneity among H. pylori strains, and the risk of gastric disease is determined in part by characteristics of the H. pylori strain(s) with which a person is infected ¹. One of the most extensively studied genetic features of disease-associated H. pylori strains is the cag pathogenicity island (PAI). This 40-kb region of chromosomal DNA, comprising about 27 genes, may be present, incomplete, or absent in H. pylori strains.

CagA, the first cag PAI-encoded protein to be studied in detail, is a highly antigenic protein that is translocated into gastric epithelial cells. Within gastric epithelial cells, CagA undergoes phosphorylation by host cell tyrosine kinases and interacts with multiple host proteins, leading to cytoskeletal rearrangements, disruption of cellular junctions, altered cellular adhesion and polarity, and increased cell proliferation. Experiments in animal models indicate that CagA has an important role in the pathogenesis of gastric cancer, and therefore CagA has been termed a bacterial oncoprotein ². CagA is translocated into host cells by a process that requires about 17 genes within the cag PAI, several of which are homologous to genes encoding components of type IV secretion systems (T4SS) in other Gram-negative bacterial species ³. T4SSs are complex molecular machines utilized by a wide variety of bacteria for delivery of effector proteins or DNA-protein complexes into recipient cells. Thus far, CagA is the only effector protein known to be translocated into host cells by the H. pylori cag T4SS.

Several recent papers have highlighted functions of a cag PAI-encoded protein known as CagL ^4–6. Early work identified cagL as a gene that was required for translocation of CagA into host cells, and therefore, CagL is considered to be a component of the cag T4SS. More recently, CagL was detected as a component of pilus structures that form at the interface between H. pylori and gastric epithelial cells ⁴, and was shown to be required for the formation of these cag PAI-associated pili ⁶. Importantly, CagL has a role in targeting of the cag T4SS to the α5β1 integrin receptor on gastric epithelial cells ⁴. Several additional Cag proteins, including CagY, CagI, and CagA, can also bind β1 integrin ⁷. By binding to integrin receptors, CagL can cause a variety of cellular alterations, including the stimulation of cell spreading, focal adhesion formation, and activation of tyrosine kinases such as focal adhesion kinase and the epidermal growth factor receptor (EGFR) ^{4, 5}. The occurrence of these cellular changes in response to purified recombinant CagL alone indicates that CagL is not simply a component of the T4SS, but is sufficient to trigger cellular signaling events ^{4, 5}.

A paper published in this issue of Gut analyzes a new function attributed to CagL ⁸. Previous work had shown that serum gastrin levels are typically higher in H. pylori-infected persons than in uninfected persons, and H. pylori-induced hypergastrinemia had also been detected in animal models ⁹. Therefore, the authors set out to identify H. pylori constituents that could stimulate gastrin gene expression. To do this, they used gastric epithelial cells that were stably transfected with a luciferase reporter regulated by the human gastric gene promoter. Co-culture of these cells with cag PAI-positive H. pylori strains induced significantly greater gastrin promoter activation than did co-culture with cag PAI-negative strains. A cagL mutant strain failed to induce gastrin promoter activation, whereas a complemented cagL mutant strain had a phenotype similar to the wild-type strain. Since H. pylori contact with gastric epithelial cells was necessary for gastrin promoter activation, the observed effect was presumed to be mediated by CagL on the surface of H. pylori. Although activation of the gastrin promoter by purified recombinant CagL alone indicated that CagL was sufficient for this effect, stimulation of the gastrin promoter by intact H. pylori requires additional cag PAI-encoded proteins ^{8, 9}.

Experiments with anti-integrin blocking antibodies suggested that β5 integrin, but not β1 integrin, was required for CagL-induced gastrin promoter activation. Consistent with this result, immunoprecipitation analyses indicated that, upon co-culture of H. pylori with gastric epithelial cells, both CagL and β5 integrin were constituents of a protein complex, and in vitro binding studies showed that CagL bound to αvβ5 integrin with high affinity. siRNA experiments indicated that integrin linked kinase (a component of focal adhesions) had a role in CagL-induced gastrin promoter activation, and further studies provided evidence that epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) signaling were involved in the process. These results indicate that CagL can target multiple integrins (rather than only β1 integrin), and provide a view of the signaling pathways through which CagL can stimulate gastrin expression.

What is the relevance of these findings in vivo? The current study showed that in a Mongolian gerbil model, a wild-type H. pylori strain caused inflammation in both the gastric antrum and corpus after 8 weeks of infection, whereas an isogenic cagL mutant strain caused inflammation in the antrum but not the corpus ⁸. This result indicates that CagL has an important functional role in vivo, but further studies will be required to elucidate the effects of CagL (as well as other products of the cag PAI) on gastrin expression and gastric acid production in vivo. H. pylori can potentially regulate gastric acid production in vivo not only by altering gastrin expression, but also through direct effects on parietal cells ¹⁰ and through the actions of host factors such as IL-1β. At present, the relative contributions of these different pathways to H. pylori-induced alterations in gastric acid production are unclear. Temporal factors also are relevant. For example, H. pylori infection in humans is often associated with an initial period of hypochlorhydria and hypergastrinemia, followed by a long-term equilibrium similar to pre-infection conditions, and then a later stage involving atrophic gastritis, hypochlorhydria and hypergastrinemia. Further studies are warranted to investigate the multiple potential mechanisms by which H. pylori can alter gastric acid production, including in vivo analyses of gastrin secretion and gastric pH at multiple timepoints after H. pylori colonizes the stomach.

By showing that CagL can activate the gastrin promoter, the current study highlights the ability of H. pylori to perturb gastric physiology ⁸. Alterations in gastrin expression are of interest because, in addition to its role in stimulating gastric acid secretion, gastrin can regulate the growth and differentiation of gastric epithelial cells; changes in these parameters might be relevant in the pathogenesis of gastric cancer. The findings in the current study add to a large body of evidence indicating that there are striking differences in the interactions of cag-positive and cag-negative H. pylori strains with human hosts. Variation among H. pylori strains in the ability to alter gastrin expression may be a factor that helps to determine the risk of H. pylori-associated gastric disease.

Abbreviations

PAI

pathogenicity island

T4SS

type IV secretion system

EGFR

epidermal growth factor receptor

MAPK

mitogen-activated protein kinase