Since its successful isolation in 1983 by Warren and Marshall, H. pylori has been linked to various pathologies and a strong association with gastric carcinoma and mucosa-associated lymphoid tissue lymphoma 13 has been established. However, although H. pylori is definitely responsible for these diseases, only less than 10% of people colonized with H. pylori portray disease symptoms. This suggests that specific H. pylori strains may be responsible for virulence in different hosts. Many studies have shown that certain allotypes of the vacA gene and the presence a functional cagA gene are associated with an increased risk of peptic ulceration and gastric cancer, respectively 202122. However, these correlations vary based on the host population studied and efforts to correlate other H. pylori alleles with clinical diseases have failed.

So how do a few H. pylori strains trigger higher virulence as compared to other strains? Current approaches in functional genomics based on protein- protein interaction and microarray based transcription profiling are helping to decode this mystery. Functional genomics often uses the gene chip based expression profiling to provide a condition-dependent and time-specific genome wide profile of an organism's transcriptome 2324. Whereas, comparative genomics juxtaposes two or more genome sequences at the level of gene content and organization 2526. Both the approaches harness extensive computer algorithms and in silico modelling to summarise gene encoded (or putative) functions. H. pylori has been the first prokaryote wherein full genome sequence of two different patient isolates [J99 and 26995] were characterized and compared 2728. As H. pylori is a freely recombining or panmictic organism 11 the question of whether the two genome sequences would accurately represent the myriad of genetic diversity found among the strains was posed. Since the 2 sequenced strains were obtained a decade apart from the two different continents and cultured from the lesions of different gastric disorders, it has been widely assumed that their genome sequences will more likely portray the genetic diversity exhibited by clinical isolates. Comparative genomic analysis of the two completely sequenced strains revealed a significant amount of genetic variation between their genomes. For instance, the J99 genome is shorter (1,643,831 bp) than that of strain 26695 (1,667,867 bp) and has 57 less predicted ORF's 2728. Strains 26695 and J99 contain 110 and 52 strain specific genes, respectively 2930, in which more than a half reside within a locus termed the plasticity cluster. A recent approach helped revised annotation and comparison of the two sequenced H. pylori genomes 31 and reclassified the coding sequences. Based on this study the total number of hypothetical proteins was reduced from 40% to 33%. A large amount of size variation was also discovered between orthologous genes mostly due to natural polymorphisms arising as a result of natural transformation and free recombination within H. pylori chromosome. Recombinational events including the presence of insertion elements, pathogenicity islands, horizontally acquired genes (restriction recombination genes), mosaics and chromosomal rearrangements were frequently annotated in subsequent bioinformatics based attempts. It has been argued that such diversity is a result of a lack of direct competition between strains, even when resident within different individuals within the same community 32. However, a recent study has demonstrated that integration of foreign gene fragments acquired via natural transformation is often prevented by the well-developed restriction-modification systems in H. pylori genome 33. It has been demonstrated that H. pylori has extensive, non-randomly distributed repetitive chromosomal sequences, and that recombination between identical repeats contributes to the variation within individual hosts 34. That H. pylori is representative of prokaryotes, especially those with smaller (<2 megabases) genomes, that have similarly extensive direct repeats, suggests that recombination between such direct DNA repeats is a widely conserved mechanism to promote genome diversification 33. In addition, although H. pylori has been termed as a panmictic organism 1135, it is surprising that clonal lineages within H. pylori populations exist 36373839. Recent reports demonstrated that H. pylori in some populations shows a clonal descent and suggest that a large array of H. pylori clonal lineages co-exist, which evolve in isolation from on another 14. Moreover, in certain parts of the world H. pylori isolates have been shown to exhibit little genetic heterogeneity, based on fingerprint profiles 1740. Functional genomics, utilising microarray technology, has provided researchers with a powerful tool to investigate the genetic diversity of clinical isolates 41, the transcriptional profiles of isolates grown under different conditions 42, the identification of strain-specific and species-specific genes 2930 and the diversity between strains giving rise to differing clinical illnesses 43. One of the interesting findings using microarray based genotyping has been the discovery that H. pylori isolates undergo 'microevolution' and give rise to sub-species during prolonged colonisation of a single host 444546. The presence of stable sub-species within a single individual suggests an adaptation of a H. pylori population to specific host niches, facilitated by unknown advantages conferred to them by select plasticity region genes. Bjorkholm et al. 45 demonstrated that several loci differed within two genetically related isolates from the same host, one major difference being the presence of the cag pathogenicity island (cag PAI) in one isolate and not the other. As the cagA gene and cag PAI are principle virulence factors within the strains, the excision or abrogation of the cag PAI within a strain may indicate that attenuating the virulence of a strain could be a favourable adaptation.

Within the bacterial populations, genome content may not be fixed, as changing selective forces favour particular phenotypes; however, organisms well adapted to particular niches may have evolved mechanisms to facilitate such plasticity. The highly diverse H. pylori is a model for studying genome plasticity in the colonization of individual hosts. For H. pylori, neither point mutation, nor intergenic recombination requiring the presence of multiple colonizing strains, is sufficient to fully explain the observed diversity.

The two H. pylori genomes sequenced to date are each from ethnic Europeans, and genomic comparisons modelled on these data are sufficient to identify novel loci from new strains, especially from understudied Asian populations. However, these genome sequences may not be fully representative of the entire diversity of the gene pool. Identification and characterization of such loci which are more abundant in the Asian gene pool may lead to newer insights into the mechanisms of H. pylori colonization, carriage and virulence in the countries of Asia which are more seriously under threat from H. pylori. Therefore, future high throughput efforts involving a large number of strains are clearly needed. Taking the Indian example for instance, according to the Ethnologue database http://www.ethnologue.com, there are about 1683 languages and dialects ('mother tongues') in this country and H. pylori diversity therefore can be assumed to coincide with this figure. So one has to roughly look at the inter-strain genomic diversity contributed by approximately 1683 different strains representing each dialect and or a community.

Nonetheless, genotypic data from each geographic area or a community is extremely vital and might constitute a missing piece of a large, biologic jigsaw puzzle.

Molecular analysis of bacterial transport has been attempted in several bacterial pathogens. Among such transport systems, Type IV secretion systems have been described in greater detail in diverse bacteria. In H. pylori, 3 different kinds of type IV secretion apparatuses have been identified. The first such secretion system identified in H. pylori was the one comprised of 29 genes encoding the cag pathogenicity island (cag-PAI). One of the principal virulence factors of H. pylori, the cagA antigen is contained in the 40 kb cag-PAI. The tyrosine-phosphorylated cagA protein is translocated to the epithelial cells by the type IV secretion system (forming a sort of syringe like structure) 515253. Upon tyrosine phosphorylation, the cagA protein elicits growth factor like stimuli in epithelial cells (hummingbird phenotype) coupled with interleukin-8 induction for the recruitment of neutrophils. Mutations in several genes of the cag-PAI interfere with tyrosine phosphorylation and induction of interleukin-8 secretion 54. In recent studies, in order to analyse which genes of the cag-PAI are essential for cagA translocation and/or interleukin 8 induction, a complete mutagenesis of the cag-PAI was performed 55. In general, it appears that most of the cag genes are involved in assembly and arrangement of the secretory apparatus. Five of these genes namely HP0524 (virD4), HP0525 (virB11), HP0527 (virB10), HP0528 (virB9) and HP0544 (virB4/cagE) constitute the main apparatus of the type IV secretory system of H. pylori 56. All these genes except HP0524 are associated with IL8 production 55. However, the presence of strains eliciting IL 8 responses irrespective of intactness of the cag-PAI underlines the fact that it's 'not' the only factor linked to IL8 secretion 57.

Very recently, and for the first time, ultrastructure analysis of the surface of H. pylori 26695 has revealed a sheathed, surface organelle, coded by the cag-PAI genes, HP0527 (forms sheath around the pilus needle) and HP0532/ cagT (forms the base of the pilus) 58. This structure, although uncommon, could be the special adaptation of H. pylori to the host niches and this might mediate biological as well as transport functions of the cag-PAI encoded proteins. Computational analyses to predict the macromolecular assemblies of such apparatuses are needed to have a more simplified understanding of the entire model of the H. pylori type IV secretion mechanism

A large-scale prospective study revealed that the risk for development of gastric carcinoma was much greater in the H. pylori-infected population than in the H. pylori-uninfected population 59. The cagA gene of H. pylori is assumed as partially responsible for eliciting signaling mechanisms that lead to the development of gastric adenocarcinoma. Based on the carriage of a functional cagA as a marker for the cag PAI, the H. pylori species is divided into cagA-positive and cagA-negative strains. The cagA-positive strains are associated with higher grades of gastric or duodenal ulceration and are more virulent than the cagA-negative strains 60. Some epidemiological studies have demonstrated roles of cagA positive H. pylori in the development of atrophic gastritis, peptic-ulcer disease and gastric carcinoma 6162. The cagA gene product, cagA, is translocated to the gastric epithelial cells to undergo tyrosine phosphorylation by SRC family kinases 63. Tyrosine phosphorylation is known to occur at the EPIYA motifs on the cagA. The cagA protein upon phosphorylation binds and activates a SHP2 phosphatase that acts as a human oncoprotein. As SHP2 transmits positive signals for cell growth and motility, deregulation of SHP2 by cagA is an important mechanism by which cagA-positive H. pylori promotes gastric carcinogenesis. Cag A is noted for its variation at the SHP2 binding site and, based on the sequence variation, it is sub- classified into two main types – East-Asian cagA and Western cagA. East-Asian cagA shows stronger SHP2 binding and greater biological activity than Western cagA. In East-Asian countries, endemic circulation of H. pylori strains that carry biologically active forms of cagA might underlie the high incidence of gastric carcinoma. One puzzling attribute of H. pylori infection is why some populations with high incidences of H. pylori infection, such as those in Japan and Korea, have high incidences of gastric carcinoma, whereas other highly infected populations, such as populations in central Africa, do not. Possible reasons could be the differences in genetic susceptibility among populations, environmental factors such as dietary habits, and strain differences of H. pylori. Among these, diversity of cagA in H. pylori strains might be involved in determination of the type and severity of disease. As discussed above, East-Asian and Western forms of cagA possess the distinctly structured tyrosine phosphorylation/ SHP2-binding sites – EPIYA-D and EPIYA-C, respectively 64. Notably, the grades of inflammation, activity of gastritis, and atrophy are significantly higher in patients with gastritis who were infected with the East-Asian cagA-positive strain than in patients infected with the cagA-negative or Western cagA-positive strain 65. Furthermore, the prevalence of the East-Asian cagA-positive strain is associated with the mortality rate of gastric cancer in Asia. Therefore, populations infected with East-Asian cagA positive H. pylori are at greater risk for gastric cancer than those infected with Western cagA-positive strains.

Among Western CagA species, the number of EPIYA-C sites directly correlates with levels of tyrosine phosphorylation, SHP2-binding activity and morphological transformation 64. Furthermore, molecular epidemiological studies have shown that the number of EPIYA-C sites is associated with the severity of atrophic gastritis and gastric carcinoma in patients infected with Western CagA-positive strains of H. pylori 66.

The plasticity region as a whole displays certain characteristics of pathogenicity islands 71 with relatively low G+C content (35%) compared to the rest of the genome (39%). This region is about 45 kb long in J99 and 68 kb long in strain 26695. Genomic analysis revealed the region to be highly mosaic with a majority of the genes being transcribed suggesting their functional role. They also express protein level homology to various other recombinases, integrases and topoisomerases 72 accounting to natural transformation and recombination. In addition to these, many ORFs were identified as differentially expressed (JHP0927-JHP0928-JHP0931 and JHP-042-JHP0944-JHP0945-JHP0947-JHP0960). They share same chromosomal orientation and therefore they potentially represent a bacterial operon. It is interesting to study the expression or suppression of these ORFs in strains linked to different clinical conditions. Recent studies have posed a possibility to explore the presence of any new pathogenicity markers in the plasticity zone, although the functions of most of the putatively encoded proteins in this cluster are unknown. But they are thought to play a role in increasing the virulence capacity of H. pylori strains either directly or by encoding factors that could lead to variance in the clinical out come of the infection. More interestingly, it is also noted that some of the genes of the plasticity regions were co-inherited along with cagA. However their co-association with the disease status or with the severity of gastric inflammation was not established either due to small sample size or lack of clinical information 73. Interestingly, a novel pathogenicity marker, JHP947 has been detected within the plasticity zone 74. Many genes putatively linked to the development of gastric cancer have been assigned to the plasticity zone 72. Researchers have looked for genetic markers in H pylori strains isolated from patients with gastric extranodal marginal zone B cell lymphoma (MZBL) of the mucosa-associated lymphoid tissue (MALT)-type and strains from age matched patients with gastritis only 75. Two ORFs were significantly linked with gastric MZBL over gastritis strains: JHP950 (74% v 49%) and JHP1462 (26% v 3%). JHP950 proved specific for gastric MZBL when tested against a group of strains from patients with duodenal ulcer and patients with adenocarcinoma, with significant prevalence (49% and 39%, respectively), and is therefore the candidate marker for gastric MZBL. Interestingly, the candidate ORF JHP950 is located in the plasticity region of the J99 genome 7576. In view of such findings it can be speculated that some members of the plasticity region cluster provide selective advantage to some of the strains to adapt to changing host niches and become more and more invasive. In what way such advantage is gained? This needs to be discovered.