Helicobacter pylori, a bacterial pathogen of the human stomach, infects an estimated 50% of the population worldwide. Infection by H. pylori causes gastritis initially and, if allowed to persist, can induce a range of pathologies. It is the causative agent of most peptic ulcers, and other serious outcomes such as atrophic gastritis, intestinal metaplasia, and gastric cancer are correlated with long-term infections. It is not yet known whether these outcomes are due to specific factors produced by the organism or whether they result from chronic inflammation due to efficient and persistent colonization of the gastric mucosa. Thus, colonization and persistence factors may themselves constitute virulence factors for this organism.
While mechanisms of virulence with respect to pathogen-host interactions are poorly understood, several important mechanisms of survival required to initiate the infection have been characterized (42
). H. pylori
absolutely requires a family of genes involved in the production of urease, an enzyme critical for neutralizing the pH around the organism during exposure to the acidic lumen of the stomach. H. pylori
also requires a large set of motility genes related to the successful production and operation of its flagellum. Without motility, H. pylori
cannot penetrate the mucous layer which protects the stomach epithelium from the acid it produces. While the bacteria can persist in the mucous layer, they also attach tightly to gastric epithelial cells via a number of adhesins. Particularly important are two outer membrane proteins, BabA and SabA, that bind oligosaccharide antigens present on cellular subpopulations of the gastric epithelium (43
). Successful colonization thus requires intimate interaction with the epithelium itself.
Several virulence factors are thought to be important once contact with the host cell epithelium is established. VacA, a secreted protein, has a vast array of activities linked to it, including membrane insertion, anion-conducting channel activity, alteration of transepithelial resistance, inhibition of antigen processing, and induction of apoptosis (50
). Presumably, the loss of membrane integrity in the host cell increases nutrient availability at the host cell surface. The cag
pathogenicity island (PAI) is composed of 27 genes. The cag
PAI contains homologues to type IV secretion system (T4SS) components in other gram-negative organisms such as Agrobacterium tumefaciens
and Legionella pneumophila
(plant and human pathogens, respectively). The presence of the cag
PAI correlates with more-serious disease outcomes, implying that when functioning, it is important in pathogenesis. The CagA protein (also encoded in the island) is translocated to the host cell cytoplasm by the T4SS (7
). Once CagA is translocated into epithelial cells, it remains associated with the host membrane and becomes tyrosine phosphorylated on C-terminal repeat motifs, Glu-Pro-Ile-Tyr-Ala (EPIYA motifs), by proteins of the Src family of tyrosine kinases. Both phosphorylated and unphosphorylated CagA proteins have been reported to induce cell signaling pathways resulting in altered spreading, migration, and adhesion of epithelial cells. The Ras/mitogen-activated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and Src homology 2 (SH2) domain containing tyrosine phosphatase (SHP-2) pathways are some of the pathways reported to be activated by cagA
-positive strains, explaining observations of increased cell proliferation during H. pylori
infection, a hallmark for a precancerous state.
Several screens for colonization or virulence factors of H. pylori
have been described using libraries of insertional mutants made either by the transposition of H. pylori
DNA clones propagated in Escherichia coli
(shuttle mutagenesis) (26
) or by the integration of plasmids containing random small pieces of H. pylori
). Both these techniques take advantage of H. pylori
bacteria's ability to integrate homologous DNA by recombination. So far, genetic screens have focused mostly on in vitro phenotypes thought to be important for infection of the human stomach, such as motility, acid survival, urease activity, adherence to gastric epithelial cells, and the ability to take up exogenous DNA (reviewed in reference 23
). Recently, the first in vivo screen using a gerbil model of infection was reported (31
). That study queried 960 mutants, giving 252 candidate mutants corresponding to 47 genes (due to redundancy of the original 960-mutant pool). Both the previously characterized colonization genes (such as those that encode motility factors) and the novel components (such as collagenase) were identified. None of the screens to date has been saturating, and none has produced either novel T4SS-dependent genes or genes required for the regulation of virulence gene expression.
We recently developed a genome-saturating library of transposon mutants and a method to track pools of these transposon mutants (MATT), using a whole-genome microarray (54
). We have initiated a screen for H. pylori
colonization factors by using these tools in a mouse infection model (32
). Here we present the results of our initial screen in two different mouse-adapted strain backgrounds. Our goal was to identify genes contributing to bacterial colonization and/or persistence in the stomach.