Key Features of the H. pylori Genome Related to Pathogenesis Colonization Factors: Urease and Motility

Urease is essential for the ability of H. pylori to colonize the gastric mucosa. More than 10 genes are required for the synthesis of active urease enzyme, the acquisition of sufficient amounts of nickel, the pH-dependent uptake of the urease substrate, urea, and the regulation of the transcription of all these genes [11]. An even larger number of genes (more than 50) are required for the assembly and operation of the polar bundle of flagella, which confer on H. pylori its exceptional ability to move in viscous environments [12]. Motility and chemotaxis are essential for colonization of the mucosa by H. pylori [13-16], and H. pylori uses the transmucus pH gradient to find its ecological niche in the deep layer of the mucus, close to the gastric epithelial surface [17, 18]. Phase Variation

One of the most unusual features of the H. pylori genome is the large number of homopolymeric tracts and dinucleotide repeats which can change their length due to slipped strand mispairing. More than 45 H. pylori genes are predicted to be phase-variable due to such hypermutatable repeats, and length variation has actually been observed for 30 of these [19]. Genes switched on or off by this mechanism encode proteins involved in flagellar motility [20], DNA restriction and modification, and lipopolysaccharide biosynthesis [21, 22], as well as many proteins of unknown function. Many pathogens that are phylogenetically related to H. pylori, such as Campylobacter jejuni and H. hepaticus, have since been shown to share this mechanism of gene switching [4, 6]. The H. pylori Outer Membrane Protein Family

The H. pylori genomes contain a large family of 33 (26695) or 32 (J99) paralogous genes encoding putative outer membrane proteins [2, 3]. This group of proteins was initially termed the Hop family; a phylogenetic analysis based on the C-termi-nal sequences has led to subcategorization of the family into the subfamilies of Hop and Hor proteins [23]. While the functions of most of these proteins are still unknown, the family comprises several proteins that have been shown to be involved in the adherence of H. pylori to gastric epithelial cells, including the Lewis b blood group antigen binding adhesin BabA [24], the sialyl-Lewis x binding adhesin SabA [25], and the putative adhesins AlpA, AlpB [26], and HopZ [27], whose cellular ligands have not yet been identified and whose role in adhesion is thus less clear. Intraspecies Variation of H. pylori Genomes

The comparison of two complete H. pylori genome sequences showed that approx. 7% of genes were unique to each of the two strains [3]. Additional evidence for extensive genomic heterogeneity between H. pylori strains was provided by geno-mic comparisons with comprehensive DNA microarrays [28]. The comparison of 15 isolates (all of which were isolated in the USA) showed that 1281 genes were present in all strains (core genes), while 362 (22%) were absent from at least one isolate. It is likely that studies that include larger sets of isolates that represent the various H. pylori populations will lead to a more precise (and significantly lower) estimate of the number of core genes. Most of the variable genes are located in two regions of the chromosome that have been termed "plasticity regions" [3].

With the exception of the cag pathogenicity island (see next paragraph), the role of the other genes that are outside of the core gene pool is currently largely unknown, but some may well contribute to the adaptation of H. pylori to the individual human host, or to specific niches within the stomach. It is therefore of interest to study the variability of the gene content in sequential and multiple isolates from individual patients. Israel et al. [29] have analyzed a collection of 36 strains isolated 6 years later from the patient from whom J99 had originally been cultured in 1994. Because the genome of J99 has been sequenced [3], and the H. pylori infection in this patient was never eradicated by medical treatment, this provided a superb opportunity to detect changes with time. These recent isolates were compared with 12 isolates cultured in 1994, using microarray hybridizations and RAPD-PCR (random amplification of polymorphic DNA polymerase chain reaction). The data showed that each of the 36 recent isolates was unique, although all were closely related to J99. Many recent isolates lack genes that are present in J99. In some cases, the recent isolates contained additional genes, which have either been acquired recently, or had been deleted during the microevolution of J99. The functional relevance of these genomic changes is still entirely unclear. First indications that genomic changes may indeed be involved in host adaptation comes from a recent study, where Solnick et al. [30] studied serial H. pylori isolates from an experimentally infected rhesus macaque. In all the passaged strains, babA, the gene encoding the Lewis b blood group antigen binding adhesin [24], was nonfunctional, due either to mutations or to gene conversion from babB, a related gene that is present elsewhere on the genome. The cag Pathogenicity Island

The cag pathogenicity island (cag PAI) is an approx. 37-kbp section of the H. pylori chromosome flanked by 31-bp direct repeats [31, 32]. It is now well established that the presence of an intact cag PAI enables an H. pylori strain to interact with the gastric epithelial cells much more closely than a strain lacking the island, resulting in stronger inflammation and a higher risk that the infected person will develop clinical disease [33, 34]. One key activity of the cag PAI is the assembly of a machinery capable of delivering the 128-kDa protein CagA into epithelial cells via a type IV secretion system [35, 36]. Contact of epithelial cells with cag PAI-car-rying strains also leads to the delivery of peptidoglycan fragments into the host cell cytosol. These fragments stimulate the intracellular pattern receptor Nodi, leading to activation of the nuclear transcription factor NF-jB and subsequently to interleukin 8 (IL-8) release [37] (for reviews of cag PAI-dependent signaling and of the interaction of H. pylori with the innate immune system, see Refs. [38, 39]).

A typical cag PAI encodes 27 genes, although islands with up to 33 genes have been reported [40]. At least six of these code for proteins with homology to type IV secretion system components. A systematic mutagenesis study has shown that 17 of the cag island genes are essential for translocation of CagA, and 14 are essential for the ability of H. pylori to strongly induce IL-8 secretion [41]. Filamentous organelles that may represent the structural correlate of the H. pylori type IV secretion system have been observed on the surface of cag PAI-carrying strains (and were not observed in cag island deletion mutants), but the precise structure and role of these organelles have yet to be determined [42, 43]. Due to the 31-bp flanking repeats, the cag PAI is genetically unstable. It can be lost from the chromosome, either by RecA-dependent precise excision [44], or by replacement of the cag PAI-containing region of the H. pylori chromosome with an "empty site allele" from a non-cag PAI-carrying H. pylori strain that simultaneously colonizes the stomach of a patient [45]. In addition, many strains harbor incomplete islands lacking a variable number of genes [28, 46]. The dynamics of cag PAI loss and possible acquisition in vivo are still poorly understood. Nucleotide Sequence Variation in H. pylori

Even more striking than the variation of genome content between strains of H. pylori is the degree of nucleotide sequence variation (allelic variation), in which H. pylori appears to surpass all other known bacterial pathogens [47]. Allelic diversity is so high that almost every clinical H. pylori isolate will differ from any other isolate even when only a few hundred base pairs from a random gene are sequenced [47, 48]. A relatively high mutation rate [49], and the frequent occurrence of interstrain recombination events leading to the uptake of small pieces (average fragment size 417 bp) of DNA into the chromosome of the recipient strain, contribute to this diversity and the rapid change of sequences even during chronic colonization of a single individual [50]. The mechanisms generating genetic variation in H. pylori and their relevance to host adaptation have been reviewed recently [51, 52]. Since H.pylori is transmitted vertically in families and its diversity is more than 50-fold higher than that of human DNA, H. pylori sequences could be shown to reflect ancient and more recent human migrations, such as the peopling ofthe Americas, the colonization ofthe Polynesian islands, or the slave trade between Africa and the Americas [5], and first evidence has recently been presented that H. pylori genotypes can actually be more informative about human migrations than human genetic markers [53].

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