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Helicobacter pylori: recombination, population structure and human migrations
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International Journal of Medical Microbiology 294 (2004) 133–139 www.elsevier.de/ijmm
REVIEW
Helicobacter pylori: recombination, population structure and human migrations Sebastian Suerbauma,, Mark Achtmanb a
Institut fu¨r Medizinische Mikrobiologie und Krankenhaushygiene, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
b
Abteilung Molekularbiologie, Max-Planck-Institut fu¨r Infektionsbiologie, Schumannstr. 21/22, D-10117 Berlin, Germany
Abstract Helicobacter pylori shows extensive genetic diversity and variability due to frequent intraspecific recombination during mixed infection. In the last years, modern genetic and genomic technology as well as cutting-edge population genetic analysis have been used to investigate the population structure and genetic variability of this pathogen. This review article summarizes recent developments in this rapidly moving field. r 2004 Elsevier GmbH. All rights reserved. Keywords: Helicobacter pylori; Population genetics; Microevolution
Content Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Geographical subdivisions in H. pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Spread together with human migrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Sources of modern diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Genetic changes of H. pylori during chronic colonization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Genome changes during chronic colonization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Corresponding author.
E-mail address: [email protected] (S. Suerbaum). 1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.06.014
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Introduction Helicobacter pylori infects more than one half of the world population, causing chronic gastritis in all infected humans, and clinical disease in 10–15% of those infected. The spectrum of clinical diseases associated with H. pylori is wide, ranging from peptic ulcer disease to gastric adenocarcinoma and MALT lymphoma (Suerbaum and Michetti, 2002). H. pylori has long been known to be genetically highly diverse (Langenberg et al., 1986; Majewski and Goodwin, 1988). In the course of the last years, this genetic diversity and variability have been intensively studied. Using modern population genetics and genomic technologies, several groups have used H. pylori as a model for the coevolution of pathogenic bacteria and humans, and for microevolution of pathogens within a single host. In this article, we will review these recent studies. For reviews of earlier studies of genetic variability of H. pylori, see (Suerbaum, 2000; Suerbaum and Achtman, 2001).
bacteria with identical sequences at multiple loci (Maiden et al., 1996). Instead, the difference reflects the exceedingly high frequency of recombinants in H. pylori (Suerbaum et al., 1998). And yet, multiple genes showed geographic specificity when a global sample of H. pylori was investigated (Achtman et al., 1999a). The simplest explanation of this apparent paradox is that H. pylori from different continents have been genetically isolated from each other until recently due to geographic isolation of their human hosts, i.e. they have not yet had time to recombine and blur the geographic distinctions because transmission largely occurs during childhood, and adults probably carry the same bacteria for decades. Under these conditions, H. pylori behaves like a marker of human descent and reflects the human population in which the host spent his childhood. And those populations in turn reflect the ancient, intercontinental spread of anatomically modern humans from Africa that began approximately 200,000 years ago (Cann et al., 1987; Diamond, 1997). The geographical differences in H. pylori might then, in turn, be markers for ancient human migrations (Achtman et al., 1999a; Covacci et al., 1999).
Geographical subdivisions in H. pylori The population structure of diverse bacteria differs with the species (Achtman, 2002; Spratt et al., 2002). Some bacteria are so young that minimal sequence diversity has yet accumulated (Achtman et al., 1999b). Others possess higher levels of sequence diversity and often recombine relatively frequently (Feil et al., 2001). Despite recombination, clonal descent, epidemic spread and ecological adaptation result in clonally related groups of strains, even in species that undergo frequent recombination (Maiden et al., 1998). Sequence diversity was thought to be high in H. pylori (Garner and Cover, 1995; Kansau et al., 1996) and no clonal groupings could be discerned by multilocus enzyme electrophoresis (Go et al., 1996). However, the cagA gene differed dramatically between isolates from East Asia versus Europe and North America (van der Ende et al., 1998; Azuma et al., 2004), and particular variants of vacA were apparently also restricted to East Asia (van Doorn et al., 1999). These observations seemed to contradict each other and demanded an intensive analysis of the population genetic structure of this species. Sequence analysis of multiple genes has shown, with very few exceptions, that each isolate of H. pylori possesses unique sequences (Achtman et al., 1999a; Falush et al., 2003b; Suerbaum et al., 1998). This is not simply a reflection of high sequence diversity, because the synonymous sequence diversity is comparable between H. pylori and other bacteria, such as Neisseria meningitidis (Suerbaum et al., 1998), but it is readily possible to isolate numerous isolates of these other
Spread together with human migrations If H. pylori were a marker for ancient human migrations, one might expect to find the greatest diversity within the species in Africa, and particularly among the most diverse human ethnic groups in Africa, Khoisans and Pygmies. This prediction remains to be tested. Instead, attention has been focussed on a second prediction, namely, that if H. pylori has accompanied humans for over 50,000 years, then it should have accompanied Siberians during their emigrations that resulted in the colonization of the Americas in the last 13,000 years (Bonatto and Salzano, 1997). It would be expected that 13,000 years of isolation should have left sufficient genetic signatures, that modern isolates from native Americans should be distinct from isolates that were imported from Europe and Africa in the last few hundred years. However, only isolates with European signatures were found in an extensive investigation of the right end of the cag pathogenicity island among 96 isolates from Amerinds in Peru and Ladinos in Guatemala (Kersulyte et al., 2000). The closest relatives of these isolates in a large, global survey were isolated in Spain. Therefore, Kersulyte et al. (2000) suggested that H. pylori colonization of humans is recent, more recent than the colonization of the Americas from Siberia. In more recent work from the same group, they have suggested that H. pylori may have evolved in a different host than humans and only made a species jump about 10,000 years ago (Dailidiene et al., 2004).
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It is dangerous to extrapolate from negative results. Indeed, other groups have now shown that some isolates of H. pylori from native Americans in remote areas of both North and South America are most closely related to isolates from East Asia (Ghose et al., 2002; Yamaoka et al., 2002). In contrast, isolates from Mestizos in South American cities who are a mixture of native Americans, Africans and Europeans were most similar to European isolates. These results show that H. pylori has infected humans for over 13,000 years and accompanied their hosts during the migrations to the Americas from Siberia. Their rare occurrence in cities may reflect a variety of causes, including possible lesser fitness than modern European isolates. These results also indicate that the comparison of additional isolates from Native Americans and from diverse ethnic groups in Siberia might help to resolve some of the ambiguities concerning the details of human migrations to the Americas. If H. pylori accompanied ancient human migrations, then a global analysis of diversity should find traces of those migrations in modern populations. Indeed, the analysis of 370 isolates from 27 geographic, ethnic and/ or linguistic human groups by multilocus sequencing of 3.8 kb from each isolate revealed that modern H. pylori can be assigned to four populations that were designated hpEurope, hpAfrica1, hpAfrica2 and hpEast Asia (Falush et al., 2003b). Almost all isolates from East Asia were hpEastAsia while almost all isolates from Europe were hpEurope. hpAfrica2 were restricted to South Africa while hpAfrica1 was particularly frequent in Africa but was also common in the Americas. Finer analyses revealed that hpEastAsia contained three subpopulations called hspEAsia (common in East Asia), hspAmerind (found in native Americans) and hspMaori (isolated from Polynesians). Similar fine analyses revealed two subpopulations within hpAfrica1, called
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hspWAfrica (common in West Africa) and hspSAfrica (common in South Africa). These results suggest that hspMaori resulted from the dispersion of H. pylori during the Polynesian migrations that began about 5000 years ago (Diamond, 2000; Oppenheimer and Richards, 2001) while hspAmerind resulted from the migrations to the Americas described above. Similarly, the existence of hspWAfrica and hspSAfrica may reflect the Bantu migrations that left West Africa about 4000 years ago and reached South Africa about 1300 years ago (Ehret, 2001). The frequent isolation of hpAfrica1 in the Americas presumable reflects the slave trade, which reached its peaks three centuries ago. An overview of putative migrations of H. pylori is given in Fig. 1.
Sources of modern diversity Given frequent recombination, modern H. pylori should contain genetic signatures of multiple ancestral populations in areas where modern humans have migrated from different sources. Indeed, both hpEurope and hpAfrica1 were found in the Americas, reflecting the combination of European colonization plus the slave trade. Similarly, both groups were found together with hpAfrica2 in South Africa, presumably reflecting the combination of European colonization and admixture between African populations. A novel algorithm, implemented in the Bayesian program STRUCTURE (Falush et al., 2003a) was used to reconstruct the recombinant stretches arising from different ancestral populations (Falush et al., 2003b). The results showed that recombination occurred between all populations in areas where multiple populations were present. Only
Fig. 1. Putative modern and ancient migrations of H. pylori. Arrows indicate specific migrations of humans and H. pylori populations. For details, see text.
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hpEastAsia remains relatively pure and almost all nucleotides are derived directly from an ancestral East Asian population. Interestingly, modern hpEurope derives its nucleotides primarily from two ancestral populations, AE1 and AE2, neither of which exists in pure form today. AE1 is thought to have originated in Central Asia because isolates from Estonia, Finland and Ladakh were particularly enriched in AE1 nucleotides. The AE2 population may have accompanied the Neolithic migrations from the Near East, beginning about 8500 years ago, which introduced agriculture and husbandry to Europe (Piazza et al., 1995) because the highest proportion of AE2 nucleotides was found in isolates from Spain, Sudan and Israel (Falush et al., 2003b). A second analysis of modern admixture was performed with isolates from Muslims and Buddhists in Ladakh, which is an isolated Sino-Tibetan province in northern India (Wirth et al., 2004). Buddhists in Ladakh are thought to have arisen by migrations from Tibet that occurred about 1000 years ago while the Muslim religion accompanied missionaries from the Near East about 500 years ago. The ability to detect ancient admixture was compared between multilocus sequences from H. pylori with sequences of the hyper-variable HVS1 region in mitochondrial DNA and with microsatellites on autosomal chromosomes. Neither of the human markers was capable of distinguishing between Buddhists and Muslims. In contrast, with the exception of three of the 18 Muslim isolates that seem to represent recent migrations from the Near East or Europe, all the nucleotides within H. pylori were derived from AE1 and ancestral East Asia. The proportions of ancestry from AE1 and ancestral East Asia differed between the isolates from Buddhists and Muslims. Fifteen Muslim isolates derived most of their ancestry from AE2 while the isolates from Buddhists showed variable proportions of ancestry from AE2 and ancestral East Asia, indicating extensive admixture between these populations. These results indicate that ancient migrations and social separation can maintain distinct populations of nucleotides within H. pylori over centuries and that this organism possesses great potential for helping to decipher the history of modern human populations (Disotell, 1999).
Genetic changes of H. pylori during chronic colonization H. pylori is usually acquired in childhood and after establishing chronic colonization, persists for decades, unless specific treatment is administered, or the mucosa becomes atrophic. Genetic changes of H. pylori that occur during chronic colonization have been studied
using either sequential strains, isolated from patients during repeated endoscopies, or multiple isolates taken from one or multiple locations in the same stomach. Kersulyte et al. (1999) were the first to demonstrate the presence of multiple recombinant strains of H. pylori in the stomach of a single patient from Lithuania. They showed that some isolates from the patient possessed the cag pathogenicity island (Censini et al., 1996) while others did not. The cag strains were derived from cag+ strains by gene conversion after transformation with DNA from a cag strain (‘‘empty site allele’’). We investigated 24 pairs of sequential isolates from patients in New Orleans, USA, and Narino, Colombia in order to detect genetic changes during in vivo colonization (Falush et al., 2001). For each of these paired isolates, ten gene fragments with a total length of 4658 base pairs were sequenced. Twenty-two out of 24 strains were identical at five or more loci, indicating that the strains were related to each other. Thirteen strains had identical sequences at all ten loci, while the remainder of pairs differed by single nucleotide polymorphisms in a single fragment (three strains) or clusters of polymorphic sequences (mosaics) in one or more fragments. The rate of genetic changes in vivo depends on three parameters: the mutation rate, the recombination rate and the size of the DNA fragments incorporated into the genome during a recombination event. In order to calculate these parameters from the data set, we developed a mathematical model based on Bayesian statistics. The estimated mean length of imported fragments was 417 bp, much shorter than has been observed for other bacteria, where imported fragments are usually much larger. The recombination rate per nucleotide was estimated as 6.9 10 5, indicating that every pair of strains differed on average by 114 recombination events. Finally, the mutation rate was estimated to be equal or lower than 4.1 10 5. Only an upper bound for the mutation rate could be estimated, because the three single nucleotide polymorphisms in the dataset could have arisen by the import of short fragments. Sequence diversity is an important source of information about the age of bacterial species, because bacterial populations will normally accumulate diversity with time. Lack of sequence diversity in a pathogen population, such as Y. pestis, is indicative of very recent evolution of a population (Achtman et al., 1999b). In the case of H. pylori, a first estimate for the minimal age of H. pylori could be calculated from the sequential isolate data set, which were isolated on average 1.8 years apart by using the maximal mutation rate to calculate the time needed for H. pylori to reach its current high level of sequence diversity. Using this approach, the minimal age of H. pylori was calculated to be 2500–11,000 years. This estimate is almost certainly
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too low. However, the analysis showed the power of using sequential isolates for quantitative analysis of in vivo evolution of a chronic pathogen.
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Acknowledgements Our work about H. pylori populations genetics was generously supported by the DFG priority program 1047 ‘‘Ecology of bacterial pathogens: molecular and evolutionary aspects’’ (Grants Su 133/3 and Ac 36/10).
Genome changes during chronic colonization The comparison of two complete H. pylori genome sequences showed that approximately 7% of the genes in each genome are unique to that isolate (Alm et al., 1999; Tomb et al., 1997). Additional evidence for extensive genomic heterogeneity between H. pylori strains was provided by genomic comparisons with comprehensive DNA microarrays (Salama et al., 2000). 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 even more genes that are lacking from individual isolates will be elucidated with larger sets of isolates that represent the various H. pylori populations. The presence or lack of a functional cag island in a strain has profound consequences for its interaction with the host (Guillemin et al., 2002). The role of the other genes that are outside the core gene pool is currently 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. (2001) 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 (Alm et al., 1999) and the carriage of H. pylori 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. 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 come from a recent study, where Solnick et al. (2004) 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 (Ilver et al., 1998), was non-functional, either due to mutations or to gene conversion from babB, a related gene that is present elsewhere on the genome.
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International Journal of Medical Microbiology 294 (2004) 133–139 www.elsevier.de/ijmm
REVIEW
Helicobacter pylori: recombination, population structure and human migrations Sebastian Suerbauma,, Mark Achtmanb a
Institut fu¨r Medizinische Mikrobiologie und Krankenhaushygiene, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
b
Abteilung Molekularbiologie, Max-Planck-Institut fu¨r Infektionsbiologie, Schumannstr. 21/22, D-10117 Berlin, Germany
Abstract Helicobacter pylori shows extensive genetic diversity and variability due to frequent intraspecific recombination during mixed infection. In the last years, modern genetic and genomic technology as well as cutting-edge population genetic analysis have been used to investigate the population structure and genetic variability of this pathogen. This review article summarizes recent developments in this rapidly moving field. r 2004 Elsevier GmbH. All rights reserved. Keywords: Helicobacter pylori; Population genetics; Microevolution
Content Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Geographical subdivisions in H. pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Spread together with human migrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Sources of modern diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Genetic changes of H. pylori during chronic colonization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Genome changes during chronic colonization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Corresponding author.
E-mail address: [email protected] (S. Suerbaum). 1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.06.014
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Introduction Helicobacter pylori infects more than one half of the world population, causing chronic gastritis in all infected humans, and clinical disease in 10–15% of those infected. The spectrum of clinical diseases associated with H. pylori is wide, ranging from peptic ulcer disease to gastric adenocarcinoma and MALT lymphoma (Suerbaum and Michetti, 2002). H. pylori has long been known to be genetically highly diverse (Langenberg et al., 1986; Majewski and Goodwin, 1988). In the course of the last years, this genetic diversity and variability have been intensively studied. Using modern population genetics and genomic technologies, several groups have used H. pylori as a model for the coevolution of pathogenic bacteria and humans, and for microevolution of pathogens within a single host. In this article, we will review these recent studies. For reviews of earlier studies of genetic variability of H. pylori, see (Suerbaum, 2000; Suerbaum and Achtman, 2001).
bacteria with identical sequences at multiple loci (Maiden et al., 1996). Instead, the difference reflects the exceedingly high frequency of recombinants in H. pylori (Suerbaum et al., 1998). And yet, multiple genes showed geographic specificity when a global sample of H. pylori was investigated (Achtman et al., 1999a). The simplest explanation of this apparent paradox is that H. pylori from different continents have been genetically isolated from each other until recently due to geographic isolation of their human hosts, i.e. they have not yet had time to recombine and blur the geographic distinctions because transmission largely occurs during childhood, and adults probably carry the same bacteria for decades. Under these conditions, H. pylori behaves like a marker of human descent and reflects the human population in which the host spent his childhood. And those populations in turn reflect the ancient, intercontinental spread of anatomically modern humans from Africa that began approximately 200,000 years ago (Cann et al., 1987; Diamond, 1997). The geographical differences in H. pylori might then, in turn, be markers for ancient human migrations (Achtman et al., 1999a; Covacci et al., 1999).
Geographical subdivisions in H. pylori The population structure of diverse bacteria differs with the species (Achtman, 2002; Spratt et al., 2002). Some bacteria are so young that minimal sequence diversity has yet accumulated (Achtman et al., 1999b). Others possess higher levels of sequence diversity and often recombine relatively frequently (Feil et al., 2001). Despite recombination, clonal descent, epidemic spread and ecological adaptation result in clonally related groups of strains, even in species that undergo frequent recombination (Maiden et al., 1998). Sequence diversity was thought to be high in H. pylori (Garner and Cover, 1995; Kansau et al., 1996) and no clonal groupings could be discerned by multilocus enzyme electrophoresis (Go et al., 1996). However, the cagA gene differed dramatically between isolates from East Asia versus Europe and North America (van der Ende et al., 1998; Azuma et al., 2004), and particular variants of vacA were apparently also restricted to East Asia (van Doorn et al., 1999). These observations seemed to contradict each other and demanded an intensive analysis of the population genetic structure of this species. Sequence analysis of multiple genes has shown, with very few exceptions, that each isolate of H. pylori possesses unique sequences (Achtman et al., 1999a; Falush et al., 2003b; Suerbaum et al., 1998). This is not simply a reflection of high sequence diversity, because the synonymous sequence diversity is comparable between H. pylori and other bacteria, such as Neisseria meningitidis (Suerbaum et al., 1998), but it is readily possible to isolate numerous isolates of these other
Spread together with human migrations If H. pylori were a marker for ancient human migrations, one might expect to find the greatest diversity within the species in Africa, and particularly among the most diverse human ethnic groups in Africa, Khoisans and Pygmies. This prediction remains to be tested. Instead, attention has been focussed on a second prediction, namely, that if H. pylori has accompanied humans for over 50,000 years, then it should have accompanied Siberians during their emigrations that resulted in the colonization of the Americas in the last 13,000 years (Bonatto and Salzano, 1997). It would be expected that 13,000 years of isolation should have left sufficient genetic signatures, that modern isolates from native Americans should be distinct from isolates that were imported from Europe and Africa in the last few hundred years. However, only isolates with European signatures were found in an extensive investigation of the right end of the cag pathogenicity island among 96 isolates from Amerinds in Peru and Ladinos in Guatemala (Kersulyte et al., 2000). The closest relatives of these isolates in a large, global survey were isolated in Spain. Therefore, Kersulyte et al. (2000) suggested that H. pylori colonization of humans is recent, more recent than the colonization of the Americas from Siberia. In more recent work from the same group, they have suggested that H. pylori may have evolved in a different host than humans and only made a species jump about 10,000 years ago (Dailidiene et al., 2004).
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It is dangerous to extrapolate from negative results. Indeed, other groups have now shown that some isolates of H. pylori from native Americans in remote areas of both North and South America are most closely related to isolates from East Asia (Ghose et al., 2002; Yamaoka et al., 2002). In contrast, isolates from Mestizos in South American cities who are a mixture of native Americans, Africans and Europeans were most similar to European isolates. These results show that H. pylori has infected humans for over 13,000 years and accompanied their hosts during the migrations to the Americas from Siberia. Their rare occurrence in cities may reflect a variety of causes, including possible lesser fitness than modern European isolates. These results also indicate that the comparison of additional isolates from Native Americans and from diverse ethnic groups in Siberia might help to resolve some of the ambiguities concerning the details of human migrations to the Americas. If H. pylori accompanied ancient human migrations, then a global analysis of diversity should find traces of those migrations in modern populations. Indeed, the analysis of 370 isolates from 27 geographic, ethnic and/ or linguistic human groups by multilocus sequencing of 3.8 kb from each isolate revealed that modern H. pylori can be assigned to four populations that were designated hpEurope, hpAfrica1, hpAfrica2 and hpEast Asia (Falush et al., 2003b). Almost all isolates from East Asia were hpEastAsia while almost all isolates from Europe were hpEurope. hpAfrica2 were restricted to South Africa while hpAfrica1 was particularly frequent in Africa but was also common in the Americas. Finer analyses revealed that hpEastAsia contained three subpopulations called hspEAsia (common in East Asia), hspAmerind (found in native Americans) and hspMaori (isolated from Polynesians). Similar fine analyses revealed two subpopulations within hpAfrica1, called
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hspWAfrica (common in West Africa) and hspSAfrica (common in South Africa). These results suggest that hspMaori resulted from the dispersion of H. pylori during the Polynesian migrations that began about 5000 years ago (Diamond, 2000; Oppenheimer and Richards, 2001) while hspAmerind resulted from the migrations to the Americas described above. Similarly, the existence of hspWAfrica and hspSAfrica may reflect the Bantu migrations that left West Africa about 4000 years ago and reached South Africa about 1300 years ago (Ehret, 2001). The frequent isolation of hpAfrica1 in the Americas presumable reflects the slave trade, which reached its peaks three centuries ago. An overview of putative migrations of H. pylori is given in Fig. 1.
Sources of modern diversity Given frequent recombination, modern H. pylori should contain genetic signatures of multiple ancestral populations in areas where modern humans have migrated from different sources. Indeed, both hpEurope and hpAfrica1 were found in the Americas, reflecting the combination of European colonization plus the slave trade. Similarly, both groups were found together with hpAfrica2 in South Africa, presumably reflecting the combination of European colonization and admixture between African populations. A novel algorithm, implemented in the Bayesian program STRUCTURE (Falush et al., 2003a) was used to reconstruct the recombinant stretches arising from different ancestral populations (Falush et al., 2003b). The results showed that recombination occurred between all populations in areas where multiple populations were present. Only
Fig. 1. Putative modern and ancient migrations of H. pylori. Arrows indicate specific migrations of humans and H. pylori populations. For details, see text.
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hpEastAsia remains relatively pure and almost all nucleotides are derived directly from an ancestral East Asian population. Interestingly, modern hpEurope derives its nucleotides primarily from two ancestral populations, AE1 and AE2, neither of which exists in pure form today. AE1 is thought to have originated in Central Asia because isolates from Estonia, Finland and Ladakh were particularly enriched in AE1 nucleotides. The AE2 population may have accompanied the Neolithic migrations from the Near East, beginning about 8500 years ago, which introduced agriculture and husbandry to Europe (Piazza et al., 1995) because the highest proportion of AE2 nucleotides was found in isolates from Spain, Sudan and Israel (Falush et al., 2003b). A second analysis of modern admixture was performed with isolates from Muslims and Buddhists in Ladakh, which is an isolated Sino-Tibetan province in northern India (Wirth et al., 2004). Buddhists in Ladakh are thought to have arisen by migrations from Tibet that occurred about 1000 years ago while the Muslim religion accompanied missionaries from the Near East about 500 years ago. The ability to detect ancient admixture was compared between multilocus sequences from H. pylori with sequences of the hyper-variable HVS1 region in mitochondrial DNA and with microsatellites on autosomal chromosomes. Neither of the human markers was capable of distinguishing between Buddhists and Muslims. In contrast, with the exception of three of the 18 Muslim isolates that seem to represent recent migrations from the Near East or Europe, all the nucleotides within H. pylori were derived from AE1 and ancestral East Asia. The proportions of ancestry from AE1 and ancestral East Asia differed between the isolates from Buddhists and Muslims. Fifteen Muslim isolates derived most of their ancestry from AE2 while the isolates from Buddhists showed variable proportions of ancestry from AE2 and ancestral East Asia, indicating extensive admixture between these populations. These results indicate that ancient migrations and social separation can maintain distinct populations of nucleotides within H. pylori over centuries and that this organism possesses great potential for helping to decipher the history of modern human populations (Disotell, 1999).
Genetic changes of H. pylori during chronic colonization H. pylori is usually acquired in childhood and after establishing chronic colonization, persists for decades, unless specific treatment is administered, or the mucosa becomes atrophic. Genetic changes of H. pylori that occur during chronic colonization have been studied
using either sequential strains, isolated from patients during repeated endoscopies, or multiple isolates taken from one or multiple locations in the same stomach. Kersulyte et al. (1999) were the first to demonstrate the presence of multiple recombinant strains of H. pylori in the stomach of a single patient from Lithuania. They showed that some isolates from the patient possessed the cag pathogenicity island (Censini et al., 1996) while others did not. The cag strains were derived from cag+ strains by gene conversion after transformation with DNA from a cag strain (‘‘empty site allele’’). We investigated 24 pairs of sequential isolates from patients in New Orleans, USA, and Narino, Colombia in order to detect genetic changes during in vivo colonization (Falush et al., 2001). For each of these paired isolates, ten gene fragments with a total length of 4658 base pairs were sequenced. Twenty-two out of 24 strains were identical at five or more loci, indicating that the strains were related to each other. Thirteen strains had identical sequences at all ten loci, while the remainder of pairs differed by single nucleotide polymorphisms in a single fragment (three strains) or clusters of polymorphic sequences (mosaics) in one or more fragments. The rate of genetic changes in vivo depends on three parameters: the mutation rate, the recombination rate and the size of the DNA fragments incorporated into the genome during a recombination event. In order to calculate these parameters from the data set, we developed a mathematical model based on Bayesian statistics. The estimated mean length of imported fragments was 417 bp, much shorter than has been observed for other bacteria, where imported fragments are usually much larger. The recombination rate per nucleotide was estimated as 6.9 10 5, indicating that every pair of strains differed on average by 114 recombination events. Finally, the mutation rate was estimated to be equal or lower than 4.1 10 5. Only an upper bound for the mutation rate could be estimated, because the three single nucleotide polymorphisms in the dataset could have arisen by the import of short fragments. Sequence diversity is an important source of information about the age of bacterial species, because bacterial populations will normally accumulate diversity with time. Lack of sequence diversity in a pathogen population, such as Y. pestis, is indicative of very recent evolution of a population (Achtman et al., 1999b). In the case of H. pylori, a first estimate for the minimal age of H. pylori could be calculated from the sequential isolate data set, which were isolated on average 1.8 years apart by using the maximal mutation rate to calculate the time needed for H. pylori to reach its current high level of sequence diversity. Using this approach, the minimal age of H. pylori was calculated to be 2500–11,000 years. This estimate is almost certainly
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too low. However, the analysis showed the power of using sequential isolates for quantitative analysis of in vivo evolution of a chronic pathogen.
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Acknowledgements Our work about H. pylori populations genetics was generously supported by the DFG priority program 1047 ‘‘Ecology of bacterial pathogens: molecular and evolutionary aspects’’ (Grants Su 133/3 and Ac 36/10).
Genome changes during chronic colonization The comparison of two complete H. pylori genome sequences showed that approximately 7% of the genes in each genome are unique to that isolate (Alm et al., 1999; Tomb et al., 1997). Additional evidence for extensive genomic heterogeneity between H. pylori strains was provided by genomic comparisons with comprehensive DNA microarrays (Salama et al., 2000). 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 even more genes that are lacking from individual isolates will be elucidated with larger sets of isolates that represent the various H. pylori populations. The presence or lack of a functional cag island in a strain has profound consequences for its interaction with the host (Guillemin et al., 2002). The role of the other genes that are outside the core gene pool is currently 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. (2001) 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 (Alm et al., 1999) and the carriage of H. pylori 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. 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 come from a recent study, where Solnick et al. (2004) 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 (Ilver et al., 1998), was non-functional, either due to mutations or to gene conversion from babB, a related gene that is present elsewhere on the genome.
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