Relatedness of Helicobacter pylori populations to gastric carcinogenesis

Abstract

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that infects half of the human population. The infection is associated with chronic inflammation of the gastric mucosa and peptic ulcers. It is also a major risk factor for gastric cancer. Phylogenetic analysis of global strains reveals there are seven populations of H. pylori, including hpAfrica1, hpAfrica2, hpEastAsia, hpEurope, hpNEAfrica, hpAsia2 and hpSahul. These populations are consistent with their geographical origins, and possibly result from geographical separation of the bacterium leading to reduced bacterial recombination in some populations. For each population, H. pylori has evolved to possess genomic contents distinguishable from others. The hpEurope population is distinct in that it has the largest genome of 1.65 mbp on average, and the highest number of coding sequences. This confers its competitive advantage over other populations but at the cost of a lower infection rate. The large genomic size could be a cause of the frequent occurrence of the deletion of the cag pathogenicity island in H. pylori strains from hpEurope. The incidence of gastric cancer varies among different geographical regions. This can be attributed in part to different rates of infection of H. pylori. Recent studies found that different populations of H. pylori vary in their carcinogenic potential and contribute to the variation in incidence of gastric cancer among geographical regions. This could be related to the ancestral origin of H. pylori. Further studies are indicated to investigate the bacterial factors contributing to differential virulence and their influence on the clinical features in infected individuals.

Key Words: Helicobacter pylori; Population genetics; Gastric cancer; Virulence; Genome

INTRODUCTION

Helicobacter pylori (H. pylori) is a Gram-negative bacterium which colonizes the human stomach. As a pathogen, H. pylori induces inflammation of the gastric mucosa[1]. It plays a causal role in the ulceration and recurrence of peptic ulcer[2]. Eradication of the bacterium heals ulcers and prevents recurrence of the disease. The infection is also associated with an increased risk of gastric cancer[3,4].

The incidence of gastric cancer shows geographical variation. This is attributed in part to the difference in the prevalence of the H. pylori infection among geographical regions. In Africa and South Asia, however, the incidence of gastric cancer in these areas is much lower than in other countries in spite of the high prevalence of the H. pylori infection[5]. Such a disparity has also been found in other local regions[6]. Analysis of global strains reveals seven populations of H. pylori that are consistent with their geographical origin[7-10]. These current populations derive from six ancestral populations[7]. It appears that the ancestry, genomic contents and carcinogenic potentials are diversified among H. pylori populations. Studies at a population level have improved our understanding of gastric carcinogenesis associated with the H. pylori infection.

GENETIC DIVERSITY AND POPULATIONS OF H. PYLORI

There are three types of bacterial population structure: clonal, panmictic and endemic[11]. If intra-species or inter-species recombination is rare, the genetic diversity of a bacterial species predominantly comes from evolution of the ancestry. This species has a clonal population structure. In a species with high frequency of recombination, introduction of foreign gene fragments into the genome occurs frequently in the evolution history. As foreign genes have a different evolution history, the evolution speed of individual genes is different. In this case, the species possess a panmictic structure. For a bacterial species with a panmictic structure, a temporal clonal structure may occur if it rapidly spread among naïve hosts. In this situation, a bacterial species has an endemic structure.

H. pylori shows great inter-strain variation in genetic content[12]. None of the individual strains is identical as demonstrated by multiple fingerprinting methods[13,14]. Sequence divergence is the main cause of this variation. Comparison of two sequenced genomes revealed occurrence of substantial silent mutation in the genetic loci[15]. A number of mechanisms are involved in the generation of the sequence variation: H. pylori shows a higher mutation rate than Escheria coli[16]. Approximately a quarter of strains possess a mutator-like phenotype. This is attributed to the lack of a functional DNA repair system[16,17] and error-prone DNA polymerase in H. pylori[18]. Recombination in H. pylori is more frequent than in any other organism studied to date[19]. Foreign DNA from the same species or phage has been found in the bacterium[15]. Strand slippage mispairing is another mechanism responsible for genetic diversity. A number of homopolymeric tracts and dinucleotide repeat regions are present in the H. pylori genome[20,21], which may cause replication error and subsequently sequence variation. H. pylori has a specialized type IV system for uptake of foreign DNA from the same species or other species[22]. Foreign DNA fragments are subsequently integrated into the genome by recombination. A high frequency of recombination and a high mutation rate in H. pylori result in a panmictic structure of the bacterium[23].

Recombination is a rare genetic event in house-keeping genes. Phylogenetic analysis of highly conserved housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI and yphC) has then been used to study populations of H. pylori[24]. Examination of global strains of H. pylori reveals that it has seven populations: hpAfrica1, hpAfrica2, hpEastAsia, hpEurope, hpNEAfrica, hpAsia2 and hpSahul[7-10,25,26]. Some populations could be further divided into subpopulations. For hpEastAsia, there are three subpopulations including hspEAsia, hspAmerind and hspMaori[27], while hpAfrica1 is split into hspWAfrica and hspSAfrica[28]. These populations and subpopulations reflect not only their geographical origin but also ethnic groups of their hosts. Spatial separation reduces bacterial recombination between different geographical regions. A weakly clonal population may thus be generated in strains from a particular geographical region[29]. H. pylori spreads mainly through a mode of family transmission[30,31], leading to a reduced chance of recombination between strains from different ethnic groups. Therefore, strains from different ethnic groups could be distinguished in the phylogenetic analysis. This is one of the features that allows phylogenetic analysis of H. pylori to be used to trace the history of human migration.

GENOMIC DIVERGENCE BETWEEN H. PYLORI POPULATIONS

H. pylori has been accompanying human hosts for more than 58 000 years[10]. The genome of H. pylori is thus shaped by its human hosts due to the long co-existence[32]. H. pylori strains differ in their affinity to bind blood group antigens expressed in the gastric mucosa[33]. Strains from Europe bind all three blood group antigens, while Amerindian strains have higher affinity for O blood antigen as this antigen is predominant in Amerindians[32]. Therefore, the genomic content of H. pylori may vary in different populations.

To date, a number of strains of H. pylori have been sequenced[15,34-37]. Of these, the origin and other information of 30 strains are publicly available. These include 14 strains from hpEastAsia (7 from hspEasia and 7 from hspAmerind subpopulations, respectively), 10 from hpEurope, 5 from hpAfrica1 and 1 from hpAfrica2[38-41]. All, except for strain B38 from hpEurope, possess cagA and the cag pathogenicity island (cag PAI). The genomic size of cagA-positive H. pylori ranges from approximately 1.55 mbp to 1.71 mbp with an average of 1.61 mbp. For cagA-negative strains, their genome is generally smaller because of the lack of the cag pathogenicity island of about 40 kbp. We analyzed the average genomic size of cagA-positive H. pylori strains from different populations[42-47]. The average genomic size of strains from hpEurope is approximately 1.65 mbp, which is significantly larger than that from hpEastAsia (1.60 mbp, P < 0.05) or hpAfrica1 (1.60 mbp). Consistent with this, strains of hpEurope have the highest number of coding sequences. There was a statistically significant difference between hpEurope and hpEastAsia in the average genomic size and number of CDS. The size of bacterial genomes is primarily determined by two counteracting processes: the gain of new genes by gene duplication or by horizontal gene transfer; and the decay of non-essential genes[15]. Both of these processes have been observed in H. pylori. Recombination, conjugation, insertion elements, mutation and slipped-strain replication lead to gene gain or loss[34]. They may be involved in the variability of genomic size among H. pylori of different populations.

For bacteria, a larger genome requires more metabolic activities and consumes more energy[48,49]. Therefore bacteria containing a larger genome may have a lowered capacity of growth, reducing its competitive ability with other bacteria in the same ecological niche. This leads to a decreased spread of bacteria. It is well known that H. pylori is less prevalent in Western countries than in other parts of the world[50]. Hygiene, economical incomes and social status have been suggested to contribute to this differential prevalence[51]. It is arguable that a larger genome of H. pylori strains in Western countries may also contribute to the low prevalence of the infection in this region.

Comparison of the genomic content of H. pylori from different populations revealed differences in the compositions of outer membrane proteins and central metabolism[39]. Compared with hpEurope, strains from hpEastAsia tend to have fewer genes of these two categories. There are a total of 12 genes in H. pylori involved in molybdenum metabolism, including those encoding proteins for molybdenum transport and cofactor synthesis and a molybdenum-containing enzyme. A massive decay of molybdenum-related genes occurs in strains from hpEastAsia. At least five genes are fragmented due to mutations. The molybdenum-containing enzyme functions in electron transfer and responses to oxidative and acid stress[52]. It is probable that in hpEastAsia populations, H. pylori use alternative pathways for the purpose[39]. Outer membrane proteins consist of several paralog families interacting with the human host[53,54]. In the hpEastAsia population, there is a tendency for a reduced number of these proteins resulting from mutations and recombination. Therefore it appears that H. pylori from hpEastAsia have evolved to possess a reduced genome.

The cag PAI is a 40-kb DNA fragment which contains 27 to 31 genes flanked by 31-bp direct repeats[55]. It encodes CagA, the major virulence determinant of H. pylori and components of a type IV secretion system[56,57]. The latter translocates CagA into host cells[58]. Once inside the host cells, CagA binds to a number of host cell proteins disrupting intracellular signaling systems via tyrosine phosphorylation-dependent or -independent pathways[59]. This causes elongation and loss of polarity of host cells, promoting proliferation and inflammation. The presence of the cag PAI in H. pylori is associated with increased risk of severe gastritis, atrophic gastritis, and distal gastric cancer compared with strains that lack the cag island[60-62].

A marked difference lies between hpEurope and hpEastAsia in the prevalence of strains possessing the cag PAI. Approximately 60% to 70% of Western H. pylori strains express CagA[61,63], indicating the presence of the cag PAI. In East Asia, however, almost 100% of strains possess the cag PAI irrespective of pathology[64,65]. It is believed that the cag PAI is deleted in Western strains resulted from recombination between the repeats flanking the island[66]. This results in a reduced genomic size by approximately 40 kbp. In addition, it has been demonstrated that the prevalence of strains with an intact cag PAI is the lowest in Western countries[67]. As described above, strains from hpEurope are coincidently 40 kbp larger than the average genomic size of H. pylori. Thus, the occurrence of cagA-negative strains in hpEurope is probably due to the evolution of the bacterium towards a smaller genome.

VARIATION IN THE CARCINOGENIC POTENTIAL OF H. PYLORI POPULATIONS

The incidence of gastric cancer varies in different geographical regions. It is higher in East Asian countries than in any other countries when age-standardized rates are considered[68]. In some countries of West Africa and South America, there is also an increased incidence of gastric cancer[69,70]. The geographic difference in the incidence of gastric cancer can be attributed partially to the difference in the prevalence of H. pylori infection[71]. A high prevalence of virulent strains of H. pylori is another contributing factor to the high incidence of gastric cancer in East Asia. Virulent strains possess the cag PAI and express VacA. There is, however, a disparity between the prevalence of H. pylori or virulent strains and the incidence of gastric cancer. In Linqu County, China, the incidence of gastric cancer is extremely high, while in its neighboring county Cangshan the incidence is very low[72,73]. The rate of H. pylori infection and the proportion of virulent strains in these two counties, however, show no significant difference[74]. Similar results have been found when comparing two regions in Mexico with contrasting incidence of gastric cancer[75]. These results indicate differential incidence of gastric cancer among different geographical regions is attributable to other bacterial factors.

To explore other bacterial factors related to carcinogenesis, the phylogeographical origin of H. pylori has been investigated[76]. In the Andean mountain region of Colombia, habitants have a high incidence of gastric cancer (150 per 100 000 people per year)[77,78], while habitants in the coastal line 200 kilometers away, have a very low incidence of gastric cancer (6/100 000)[77,78]. The prevalence of the infection and virulent strains of H. pylori in these two regions are similar[75]. All H. pylori strains isolated from the Andean region, however, are from hpEurope, in contrast to strains isolated from the coastal line which are mainly from hpAfrica1[76]. Furthermore, strains from the Andean region caused more severe mucosal inflammation and more DNA damage in epithelial cells. This suggests that strains of hpEurope probably have an increased carcinogenic potential compared with those of hpAfrica1[76]. Ancestral origin of the bacterium could be an important factor contributing to gastric carcinogenesis. This conclusion is further supported from a study conducted in Malaysia[7]. There are three ethnic groups in the country: Malay, Indian and Chinese. The infection rate of H. pylori in Malays is lower than that in Indian and Chinese subjects[79]. The incidence of gastric cancer, however, is similar in Malays and Indians, but is much lower than in the Chinese[80]. Analysis of the ancestral origin of H. pylori found that strains isolated from both Malay and Indian subjects belonged to hpAsia2, whereas those isolated from Chinese subjects belonged to hpEastAsia. This suggests a different potential for carcinogenesis between hpAsia2 and hpEastAsia. H. pylori populations generally reflect the geographical regions from which they are isolated. Differences in the incidence of gastric cancer among geographical regions could be in part attributed to different populations of H. pylori. Further study is required to investigate other bacterial factors involved in the carcinogenesis.

CONCLUSION

In summary, geographical separation reduces the frequency of recombination between H. pylori strains from a local area and those from outside. This leads to the formation of a clonal population structure of H. pylori in the local area. Thus, populations of H. pylori could be identified through examination of global strains. For each population, H. pylori have experienced relatively separate evolution processes, resulting in genomic diversity and differential potential for carcinogenesis. Further study to characterize these differences may help elucidate mechanisms involved in the development of gastric cancer induced by H. pylori.