Cancergastrico

BMC Genomics BioMed Central
Research article Open Access

Genomes of Helicobacter pylori from native Peruvians suggest
admixture of ancestral and modern lineages and reveal a western
type cag-pathogenicity island
S Manjulata Devi†1, Irshad Ahmed†2,3, Aleem A Khan2, Syed Asad Rahman4,
Ayesha Alvi1, Leonardo A Sechi5 and Niyaz Ahmed*1,5
Address: 1Pathogen Evolution Group, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India, 2Centre for Liver Research and
Diagnostics, Deccan College of Medical Sciences and allied hospitals, Hyderabad, India, 3Department of Microbiology, Shri Shivaji College of Arts,
Commerce and Science (SGB Amravati University), Akola, MS, India, 4Cologne University Bioinformatics Centre, Cologne, Germany and
5ISOGEM Collaborative Network on Genetics of Helicobacters, The International Society for Genomic and Evolutionary Microbiology, University
of Sassari, Sassari, Italy

Email: S Manjulata Devi - [email protected]; Irshad Ahmed - [email protected]; Aleem A Khan - [email protected];
Syed Asad Rahman - [email protected]; Ayesha Alvi - [email protected]; Leonardo A Sechi - [email protected];
Niyaz Ahmed* - [email protected]
* Corresponding author †Equal contributors
Published: 27 July 2006 Received: 31 March 2006

Accepted: 27 July 2006
BMC Genomics 2006, 7:191 doi:10.1186/1471-2164-7-191
This article is available from: http://www.biomedcentral.com/1471-2164/7/191
© 2006 Devi et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Helicobacter pylori is presumed to be co-evolved with its human host and is a highly
diverse gastric pathogen at genetic levels. Ancient origins of H. pylori in the New World are still
debatable. It is not clear how different waves of human migrations in South America contributed
to the evolution of strain diversity of H. pylori. The objective of our 'phylogeographic' study was to
gain fresh insights into these issues through mapping genetic origins of H. pylori of native Peruvians
(of Amerindian ancestry) and their genomic comparison with isolates from Spain, and Japan.
Results: For this purpose, we attempted to dissect genetic identity of strains by fluorescent
amplified fragment length polymorphism (FAFLP) analysis, multilocus sequence typing (MLST) of
the 7 housekeeping genes (atpA, efp, ureI, ppa, mutY, trpC, yphC) and the sequence analyses of the
babB adhesin and oipA genes. The whole cag pathogenicity-island (cagPAI) from these strains was
analyzed using PCR and the geographic type of cagA phosphorylation motif EPIYA was determined
by gene sequencing. We observed that while European genotype (hp-Europe) predominates in
native Peruvian strains, approximately 20% of these strains represent a sub-population with an
Amerindian ancestry (hsp-Amerind). All of these strains however, irrespective of their ancestral
affiliation harbored a complete, 'western' type cagPAI and the motifs surrounding it. This indicates
a possible acquisition of cagPAI by the hsp-Amerind strains from the European strains, during
decades of co-colonization.
Conclusion: Our observations suggest presence of ancestral H. pylori (hsp-Amerind) in Peruvian
Amerindians which possibly managed to survive and compete against the Spanish strains that
arrived to the New World about 500 years ago. We suggest that this might have happened after
native Peruvian H. pylori strains acquired cagPAI sequences, either by new acquisition in cag-
negative strains or by recombination in cag positive Amerindian strains.
Page 1 of 10
(page number not for citation purposes)
BMC Genomics 2006, 7:191 http://www.biomedcentral.com/1471-2164/7/191
Background We attempted to dissect gene pool diversity of Amerin-

Helicobacter pylori is a Gram-negative bacterium that estab- dian isolates of H. pylori from Peru with an objective to
lished itself in the human stomach possibly thousands of explore ancient and modern features of the Peruvian H.
years ago [1]. This opportunistic pathogen infects over pylori strains corresponding to different waves of human
50% of the worlds' population, causing no harm to most migration. We also looked if it is possible to link some of
colonized people [2]. Only a small subset of infected peo- the native Peruvian strains to their ancestors in Asia.
ple experience H. pylori-associated illnesses such as
chronic gastritis, peptic ulcer disease, gastric carcinoma, Results
and mucosa-associated lymphoid tissue (MALT) lym- FAFLP based genotyping, candidate gene sequence
phoma. Associations of various clinical outcomes with analysis and multi-locus sequencing
disease-specific virulence factors remain dogmatic [3] Phylogenetic relationships assessed by FAFLP genotyping
years after the completion of genome sequences [4]. The of Peruvian isolates (Figure 1A) revealed 2 different line-
debate has been further intensified as some studies have ages corresponding to the hp-Europe and hsp-Amerind.
posed the possibility that H. pylori infection has some pro- Representative isolates from both the lineages obtained
tective effects in esophageal diseases [3]. Also, possible by FAFLP were subjected to reconfirmation by MLST anal-
symbiotic associations have been proposed based on the ysis. We observed that all the isolates corresponding to the
finding that H. pylori harbor protective, bacteriocin like Amerind type FAFLP profile were found to be genetically
effect and may therefore be beneficial to its host [5]. closer to strains from Alaska and were therefore, accepted
as genotype hsp-Amerind. All the other isolates from Peru
Subsequent to the decipherment of the potential of poly- clustered with those from Spain and therefore joined
morphic DNA markers in reconstruction of human migra- MLST genotype hp-Europe.
tion and phylogeography [6,7], pathogen genotypes were
successfully used in tracking and analyzing patterns of We looked at the geographic signatures of oipA gene
human migrations [8-10] in different continents. sequences in Peruvian isolates and found that 7 of the 27
Recently, sequence variation in H. pylori has provided a isolates including the 2 hsp-Amerind isolates had an East-
window into human population migration [11] and also ern type oipA (CT < 6) signature; 4 of these had a non func-
revealed that impact of religions on stratification of tional gene (frame out) (Table 1). Nineteen of the
human ethnic groups can be analyzed based on H. pylori Peruvian isolates had a western type oipA (CT ≥ 6), 15 of
haplotypes [12]. which had a functionally intact coding frame. It appears
that oipA sequence conveys the European ancestry for a
Ancient origins and dissemination of H. pylori are quite majority of these isolates, including some hsp-Amerind,
debatable in the context of the vast South American con- however, oipA is a virulence linked gene and its resolution
tinent that has witnessed many different waves of popula- power for lineage identification is not as robust as that of
tion migration [13], especially in view of the fact that H. the 7 housekeeping genes we used, or the babB gene. It is
pylori has been present in this continent since pre-Colum- likely that oipA might have been exchanged or recom-
bian times [14]. However, evolution of virulence and fit- bined between different lineages (like most of the other
ness in such 'ancient' strains that arrived first in the virulence genes such as the cagPAI) to give hybrid geno-
Americas and then, possibly out-competed by the influx types.
of 'modern' strains from Europe [14] remains largely
unexplored. The babB gene sequences of our hsp-Amerind isolates were
compared to the sequences previously reported for Amer-
A landmark study based on PCR based DNA motif analy- indian isolates from Venezuela by Ghose et al. [16] by
sis proposed that H. pylori jumped recently from animals multiple sequence alignment and phylogenetic analysis
to humans and, therefore, the acquisition of H. pylori by (Figure 1B). These phylogenies, when studied in the light
humans may be a recent phenomenon [15]. This study of MLST based inferences, revealed distinct affinities
has been the basis for the idea of 'H. pylori free New World' between Peruvian Amerindian strains and Venezuelan
[15]. However, two independent studies based on large- Amerind strains, pointing thus to their common Asian
scale analyses of candidate gene polymorphisms con- origins (Figure 1B).
trasted the idea of recent acquisition and suggest that H.
pylori might have co-evolved with humans [11,16]. In In summary, our overall phylogenetic analyses, demon-
view of these intriguing ideas on ancient origin of H. strated that about 20% of the strains from native Peruvian
pylori, additional evidences based on strains from different patients that we examined share common genotypic pat-
geographical regions (especially those with a rich history terns with Asian (Amerind) strains and therefore, Ancient
of multiple waves of human migrations such as the South Asian gene pool must have been the origin of the present
Americas) are clearly needed. day strains inhabiting the native Peruvians.
Page 2 of 10
SJM18
0
A
SJM
80
SJM19
SJM41
SJ
14
M2
M
SJM37
SJ
7
SJ
SJ
M5
M2
3
0
SJ 1 38
SJM M1 M2 SJM
148 SJ
Hp-Europe SJM18
SJM189
3
184 M6 SJM
57
SJ
SJM S
JM SJ
45 M9
3
50
SJ
M
M
SJ
69
SJM
SJM
SJM23
SJ
SJ
10
12
M
M8
92
3
0.0 1
hsp-Amerind
B
Modern branch
VC72
24
VP
95
VC
3
VC9
4
VC9
SJM83
SJM92 V P43
VP
25
VP49
19
VP
0.01
Amerind branch
A.
Figure
FAFLP 1 analysis of H. pylori strains analyzed from native Peruvians (n = 27)
A. FAFLP analysis of H. pylori strains analyzed from native Peruvians (n = 27). The phylogenetic tree was developed based on
various amplitypes generated for individual isolates after allele scoring and generation of similarity profiles in the form of binary
tables. Genetic relationships in the form of a tree were deduced using MEGA 3.0 software using bootstrapping method at
10000 bootstrap trials. Two different lineages observed in the tree are colored as per the previous conventions [11, 20]. Six
isolates from among those represented in the FAFLP tree (highlighted with circles) were also analyzed subsequently by MLST.
B. Phylogenetic analysis of representative hsp-Amerind sequences from our isolates (SJM 83, 92) and those of other Amerind
and non-Amerind (western) H. pylori sequences previously described by Ghose et al. from Caracas and Puerto Ayacucho in
Venezuela [16]. SJM 83 and 92 formed a separate branch (maroon color) only with Amerind isolates (right). Both the Amerin-
dian and western lineages (green color) are differentially colored as per previous conventions [11, 20]. Sequence alignment
(left) of these Amerind sequences revealed significant conservation at the level of nucleotides.
Page 3 of 10
Table 1: Characteristics of 26 Peruvian H. pylori isolates obtained from Amerindians and distribution, presence/absence and or rearrangement of various gene loci in their
genomes; 0 – region not amplified, 1 – region present.
Page 4 of 10
http://www.biomedcentral.com/1471-2164/7/191
Strains Diagnostic PCR Phylogenetic placement oipA status cagA genotype cag-PAI spanning PCR
cagA GlmM FAFLP/MLST babB oipA No. of CT Frame gene EPIYA motif cagAP1 cagAP2 cagA1 cagA2 cagE cagT LEC1 LEC2
A) Native Peruvian Strains
SJM184 1 1 hp Europe Modern Eastern 5 Out 1 EPIYA A, B, C 1 1 1 1 1 1 1 1

SJM80 1 1 hp Europe Modern Western 6 Out 1 EPIYA A, B, C 1 1 0 1 1 1 1 1
SJM83 1 1 hsp Amerind Amerind Western 6 Out 1 EPIYA A, B, C 1 1 1 1 1 1 1 1
SJM92 1 1 hsp Amerind Amerind Eastern 3 In 1 EPIYA A, B, C 1 1 1 1 1 1 1 1
SJM93 1 1 hp Europe Modern Western 6 In 1 EPIYA A, B, C 1 1 0 1 1 1 1 0
SJM10 1 1 hsp Amerind Amerind Western 6 In 1 EPIYA A, B, C 1 1 0 1 1 1 1 1
SJM12 1 1 hsp Amerind Amerind Eastern 4 In 1 EPIYA A, B, C 1 1 0 1 1 1 1 1
SJM18 1 1 hp Europe Modern Eastern 4 In 1 EPIYA A, B, C 1 1 0 1 1 1 1 1
SJM19 1 1 hp Europe Modern Eastern 4 Out 1 EPIYA A, B, C, 1 0 1 1 1 1 1 1
C
SJM23 1 1 hsp Amerind Amerind Western 6 In 1 EPIYA A, B, C 1 1 0 1 1 1 1 1
SJM41 1 1 hp Europe/ Modern Eastern 2 Out 1 EPIYA A, B, C 1 1 1 1 1 1 1 1
WAfrica
SJM45 1 1 hp Europe Modern Western 8 In 1 EPIYA A, B, C, 1 1 1 1 1 1 1 1
C
SJM50 1 1 hp Europe Modern Eastern 5 Out 1 EPIYA A, B, C, 1 1 1 1 0 1 1 1
C
BMC Genomics 2006, 7:191
B) Other Strains
Spain 1 1 hp Europe Modern Western 9 In 1 EPIYA A, B, C, 1 0 1 1 1 0 1 1

(HUPB7 C
6)
Japan 1 1 hp East Asia East Asia Eastern 4 In 1 EPIYA A, B, D 1 1 1 1 1 1 1 1
(HU178)
Africa 1 1 hp S Africa Modern Western 6 In 1 EPIYA A, B, C 0 1 1 1 1 1 1 1
(10)
India 1 1 hp Europe Modern Western 6 In 1 EPIYA A, B, C 1 1 1 1 1 1 0 0
(MS8)
Analysis of the cagPAI and the cagA gene in isolates from in Asia. This supports the hypothesis that H. pylori was
Spain, Peru and Japan associated with its host well before Asian people crossed
Overlapping primer amplification strategy to span entire the Bering strait (20,000 years BP) to colonize America.
cagPAI worked very well with our hsp-Amerind isolates We also report that the cagPAI was acquired by native
(Figure 3A) where all the constituent genes of the PAI were Peruvian strains probably from a European source. This
successfully amplified. This indicates that the 5 Amerind might have occurred as a single import, most probably
strains we looked at represent a 'chimera genome' made during the last 500 years of the spread of H. pylori in the
up of an ancient like core genome component (MLST and South American continent. It is evident from the fact that
babB typing) and a modern type flexible genome compo- the Amerindian Peruvian strains, though of an Asian
nent (cagPAI and its right junction typing). Tyrosine phos- descent, do not share characteristic features of Asian cag-
phorylation of the immunodominant cagA protein is PAIs but show homology to the PAIs of European strains.
known to occur at the EPIYA motifs at the C-terminus by Alternatively, the Amerindian strains might have gained
the SRC family of kinases [17,18]. This EPIYA motif con- the PAI through a series of recombination events over a
sists of 4 distinct EPIYA sites – EPIYA-A, -B, -C and -D, period of time. But this hypothesis appears weak when we
based on the amino acid sequence that neighbor it (Figure take into account the short time span of 500 years within
3B). Based on the presence of these EPIYA motifs, cagA which the European strains spread in the Americas.
can be distinguished into the Western type (W) in case the
-C site is present and the East Asian (EA) type in case We tried to potentate these ideas and to provide evidence
where the -C site is replaced by the -D site. Our data for the in favor of the ancient origin of the pathogen and the pos-
type of EPIYA motifs present in Spanish (n = 6), Peruvian sibility that some extraneous gene cassettes might have
(n = 27), and Japanese (n = 16) strains revealed that the been acquired by otherwise symbiotic H. pylori, some-
western type of cagA was predominant among all the times during its natural history. To support this proposi-
Spanish and Peruvian strains. The Japanese isolates alone tion, our methodology was targeted with a two pronged
revealed East Asian type EPIYA D signatures (Figure 3B). approach to i) further substantiate ancient link of the
pathogen and ii) to prove that the pathogenicity island
Results of the PCR of cagPAI from 26 Peruvian isolates was a 'recent' addition to the genome of H. pylori. Our
studied in the present context have been depicted in Table analyses based on FAFLP and MLST linked about 20% of
1. Briefly, the strains from Peru and Spain did not readily the Peruvian isolates to Amerindian genotype, and con-
amplify regions of the cagPAI as did Japanese isolates, sug- veyed that H. pylori was most probably introduced to the
gesting thereby a distinct allelic diversity at the primer New World by Asian people. We, therefore, disagree with
binding sites in the western PAI of these isolates. This the idea of an 'H. pylori free New World' [15]. This disa-
observation places native Peruvian strains closer to Span- greement of interpretation arose possibly because Kersu-
ish strains and therefore hints that both the Spanish and lyte et al. [15] looked at only a few loci in the genome and
Peruvian strains harbor a similar type of cagPAI. stressed mainly on the motifs surrounding the cagPAI on
its right junction.
The right junction of the cagPAI also revealed similar
acquaintances based on PCR based insertion deletion and We also looked at the cagPAI of such strains and found
substitution analysis of the region spanning cagA right that the Peruvian isolates we tested carried western type
junction to glr, for the 6 Peruvian isolates we analyzed. (EPIYA C) cag islands (Figure 3B). We did not record any
Such genotypes for all the isolates we used from Spain, eastern type signature in the cagPAI (EPIYA D motif) in
Japan and Peru were determined earlier by Kersulyte et al Peruvian isolates, given a distinct presence of ethnic Japa-
[15]. Collectively, this study and the previous observa- nese in Peru. This inference also came from MLST data
tions [15] demonstrated that the cag right junction motif (Figure 2), where none of the Peruvian isolates clustered
types were shared by Spanish and Peruvian isolates and with Japanese genotype hp-EastAsia (Figure 2). Similarly,
that Japanese isolates did not share genetic affinities with the absence of Asian/Amerindian type islands (or their
Peruvian strains. This observation again places native remnants in native Peruvian isolates we analyzed, leads us
Peruvian strains closer to Spanish strains and therefore to speculate that their ancestors in Asia were seemingly
hints that both the Spanish and Peruvian strains share cag- benign due to (natural?) absence of functional cag genes.
PAI insertion sites and the regions flanking them. This finding potentates the idea that cag genes in Peru
mainly originated in Europe and therefore confirms the
Discussion scenario proposed by Kersulyte et al. [15] as far as the cag-
H. pylori is presumed to be an ancient colonizer of the PAI and its right junction is concerned.
human stomach which possibly co-evolved with its host.
We report that at least some of the H. pylori strains found Kauser et al., [19] from our group have previously
in Peru share considerable homology with strains found described PCR analyses of the cagPAI for more than 300
Page 5 of 10
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MLST analysis
2 based on concatenated gene sequences of 7 housekeeping genes of H. pylori (Kimura-2 parameter)
MLST analysis based on concatenated gene sequences of 7 housekeeping genes of H. pylori (Kimura-2 parameter). The phyloge-
netic tree was based on a total of 19 sequence records (concatenated) obtained under this study (SJM, HUPB, HU, CPY) while
incorporating other ~400 sequence records from pubMLST database (pubmlst.org) which were specific to different genotypes
in the world (Courtesy, Daniel Falush). Different genetic populations (Hp) and subpopulations (hsp) or genotypes are named
and differentially colored after previous conventions [11, 20]. All SJM isolates, Amerindians (SJM23 and 92-highlighted) and
non-Amerindians (arrowheads) analyzed by us from Peru are highlighted in bold face black fonts with green twigs indicating
presence of a western type cagPAI.
Page 6 of 10
A
R e p r e s e n ta tiv e c a g P A I p r o f ile o f h s p - A m e r in d , S J M 2 3 /S J M 9 2
33
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H L N F S D IK K E L N A K L G N F N N N N N N G L K N E P IY A K V N K K K T G Q V A S P E E P IY A Q V A K K V T Q K ID R L N Q IA S G L G G V G Q A A G
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Figure
A. (Top)3 PCR based analysis of whole cagPAI of hsp-Amerind isolates from Peru
A. (Top) PCR based analysis of whole cagPAI of hsp-Amerind isolates from Peru. Overlapping primers spanning all of the con-
stituent genes (see methods) amplified all the corresponding PCR products in the expected size range as described previously
[30]. M indicates molecular weight marker (100 bp ladder). (Bottom) Amino acid signature of cagA (phosphorylation motifs –
colored) – characteristic of modern cagA EPIYA (EPIYA C) as observed for all the SJM isolates from Peru. B. Pictorial depic-
tion (right) of different types of cagA-EPIYA motif types prevalent in different H. pylori populations and their distribution in our
isolates (boxes on the left).
Page 7 of 10
strains from different parts of the world. Of these, the and uniform as compared to virulence associated loci
majority of strains from whom complete PAI was ampli- such as the flagellins and vacA [31], recombinant and
fied were from Japan (57%) whereas only 18.6% strains hybrid alleles that blur phylogeographic inferences, could
from Peru and 13.3% strains from Spain could support be a rare occurrence rather than a rule. Nonetheless, it will
amplification of an intact cagPAI, due mainly to allelic be important to ascertain proportions of nearly pure and
diversity present at the primer annealing sites. These hybrid alleles among native Peruvian H. pylori through
observations were further endorsed by our present analy- admixture analyses based on sophisticated population
sis that revealed that all the Peruvian isolates we analyzed genetics tools [32] that reveal contemporary gene flow
carried only the European type PAI. and proportion of different nucleotides inherited from
ancestral populations on an evolutionary time-scale.
It has been recently reported that true Amerind strains
either do not carry a cagPAI or carry only a vestigial, Conclusion
incomplete PAI [20]. It is already well known that H. pylori In summary, our study based on profiling of several gene
can import short stretches of DNA from strains from very loci and neutral markers revealed certain distinctive
different populations when they (presumably co-) infect genetic features of the H. pylori gene pool in Peru. Impor-
individuals in the same location. However, it appears that tant among these features are a demonstrated genetic link
the cagPAI and the region surrounding it had been with Asian (Amerindian) strains and the presence of an
exchanged by the Amerindian strains in Peru, where, the intact cagPAI with western type of gene motifs that point
human population underwent major changes in recent to recent acquisition of this important pathogenicity
history with the arrival of European conquerors and set- island. This indicates possible lateral gene transfers during
tlers. The long isolated Amerindian H. pylori strains thus colonization of Peruvian Amerindians with both 'ancient'
came in contact with the European strains, which har- and 'modern' strains for several generations. Such an
bored a cagPAI. It is established that the cagPAI might give admixed gene pool could be an important source of
selective advantages during host colonization and there- genetic information on pathogen evolution in real time to
fore, "endogenous" Helicobacter, in Peru could be out- possibly understand how gene acquisition and loss on a
competed in a human community with newly arrived cag- population wide scale shape virulence and fitness in dif-
PAI positive strains introduced by the European conquer- ferent pathogens.
ors. The "endogenous" Helicobacter strains could
moreover acquire cagPAI during mixed infection with This could also possibly provide for a reasonable model of
western cagPAI positive strains leading to the observed geographic evolution to understand acquisition of viru-
strains with Eastern-like core genome content and a west- lence in pathogenic bacteria over a period of time.
ern-type cagPAI. So this finding would really be interest-
ing if it is clear that the entire cag-island had been Methods
exchanged. Then, there would be a very interesting ques- Bacterial strains, genomic DNA and diagnostic PCR
tion about the mechanism by which an entire island had Genomic DNA preparations for strains originating from
been transferred; there is no doubt that such exchanges Peru, Spain and Japan were provided by D. E. Berg and
have happened at some point, but if they happened Asish Mukhopadhyay (Washington University, St. Louis,
within the last 500 years, then, there would be much bet- Mo.). These DNA were isolated from patients diagnosed
ter chances of catching it in action. This is probably where with gastric ulcers from Spain (HupB); gastric cancer and
the present data lead us to, and, we suggest that future DU cases from Japan (CPY, Hu); and from gastritis cases
efforts may be directed towards confirmation of this evo- alone from Peru (SJM). However, in the current study, the
lutionary mechanism. clinical background of the individual isolates was not
taken into account. The Peruvian isolates we looked at (n
Finally, it is possible that phylogenetic methods based on = 27) were originally from Native Peruvian people mainly
highly recombining gene loci [21-27] may not be fully of Amerindian ancestry from Lima [15]. PCR based anal-
perfect to predict genetic relationships in terms of inherit- ysis of genes namely glmM, babB [16] and oipA was carried
ance from different ancestral populations, especially out to ascertain the quality of DNA samples we used. Also
when we use tools such as Mega 3.0 [28] which do not these PCR assays served as amplification level controls for
support admixture analysis. Partly in view of this possibil- the analysis of insertion, deletion and substitution in the
ity and to ensure that our conclusions did not represent cagPAI.
shortcomings of a single approach, we adopted an inte-
grated genotyping strategy for the cagPAI [29,30] as well as Integrated genotyping of H. pylori based on chromosomal
the core genome through MLST of less recombining, neu- DNA signatures
tral genes that encode cytoplasmic enzymes. Given the Whole genome fingerprinting based on FAFLP genotyping
fact that these housekeeping genes are selectively neutral was performed as described previously [21-23]. Briefly,
Page 8 of 10
the profiling of whole genome micro-restriction finger- PAI of the representative isolates (SJM92 and SJM23)
prints with EcoRI/MseI enzymes using fluorescence tagged from hspAmerind by PCR using overlapping primers as
primer pairs EcoRI+A/MseI+0 and EcoRI+G or A/MseI+0 described by Blomstergren and colleagues [30]. The 3' end
was performed for all the strains. The PCR amplified frag- of the cagA gene was amplified using primers mentioned
ments for each of the strains were then subjected to elec- elsewhere [17] and the amplified products for strains
trophoretic separation on a 5% acrylamide gel and scoring from Spain, Peru, and Japan were sequenced with forward
of the fluorescent markers was done using an automated and reverse primers. The consensus sequences were then
DNA analysis workstation (ABI Prism 3100 DNA translated into amino acid sequences using GeneDoc soft-
sequencer). Cluster analysis of DNA profiles was con- ware (version 2.6.002) and were then assigned to the
ducted on the basis of fingerprint characteristics. All the Western or the East Asian group based on the C or D
data obtained through molecular genotyping and DNA repeat present respectively in the EPIYA motif [18]. Chro-
profiling were deposited in the genoBASE pylori database mosomal rearrangements are known to give rise to 5 types
[33]. The genoBASE pylori server was queried for compar- of insertion-deletion and substitution motifs in the region
ative analyses. between the 3' end of cagA gene and the 3' end of the
glutamate racemase (glr) gene. Although the statuses of
Genotyping based on candidate genes oipA and babB was these motifs for the Peruvian strains we analyzed were
carried out as described [24-26]. Short stretches of oipA described previously by Kersulyte and colleagues [15], we
gene were analyzed to determine the 'geographic signa- re-assessed 6 of them by PCR exactly as described earlier
ture' based on CT repeat [27]. In addition, 600 bp region [15].
each from the 7 housekeeping genes spread throughout
the genome [atpA, efp, ureI, ppa and mutY, trpC, yphC] was Authors' contributions
amplified and sequenced for all the isolates exactly as SMD and IA performed MLST typing and phylogenetic
described previously [11]. Sequencing was performed analysis. SMD also helped in analysis of babB and oipA
with both forward and reverse primers, using an ABI genotyping. AAK and AA performed PCR based analysis of
Prism 3100 DNA sequencer (Applied Biosystems, USA). the cagPAI genes. SAR extended bioinformatics support.
PCR and direct sequencing were performed at least twice LAS provided expert clinical consultation and contributed
to determine and confirm the DNA sequences for each to manuscript writing. NA performed FAFLP analysis,
isolate. Consensus sequence for each of the samples was planned and supervised the study, wrote the manuscript
generated using Genedoc (version 2.6.002). Multiple and provided overall leadership. All the authors read and
alignments of sequenced nucleotides were carried out approved the final manuscript.
using Clustal X (version 1.81). Neighbor joining trees
were constructed in Mega 3.0 [28], using bootstrapping at Acknowledgements
10000 bootstrap trials (FAFLP and babB) and through We are thankful to Seyed E. Hasnain for his guidance and support. We are
Kimura-2 parameters (for MLST). For construction of phy- highly grateful to Asish Mukhopadhyay and Douglas E. Berg for providing us
logenetic trees based on MLST genotyping procedures, with DNA samples of Peruvian and other strains as a gift. We are thankful
to Daniel Falush for support with international MLST data and advice. We
sequences of 7 housekeeping genes of strains belonging to
are also thankful to the International Society for Genomic and Evolutionary
different established genotypes were obtained from the Microbiology (ISOGEM) for supporting and endorsing the study. Financial
pubMLST database [34] (courtesy, Daniel Falush). support from the Department of Biotechnology, Government of India
(CDFD core grants) is gratefully acknowledged.
The nucleotide sequences of the 7 housekeeping genes for
the 6 hsp-Amerind isolates we analyzed have been depos- References
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to strains from four different continents. J Clin Microbiol 2003, scientist can read your work free of charge
41:5755-5759. "BioMed Central will be the most significant development for
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paired antral and paired antral and corpus Helicobacter pylori Sir Paul Nurse, Cancer Research UK
isolates recovered from individual patients. J Med Microbiol Your research papers will be:
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Ahmed N: Comparing genomes of Helicobacter pylori strains peer reviewed and published immediately upon acceptance
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BMC Genomics BioMed Central

Research article Open Access

of Sassari, Sassari, Italy

Published: 27 July 2006 Received: 31 March 2006

Background We attempted to dissect gene pool diversity of Amerin-

A) Native Peruvian Strains

SJM184 1 1 hp Europe Modern Eastern 5 Out 1 EPIYA A, B, C 1 1 1 1 1 1 1 1

Spain 1 1 hp Europe Modern Western 9 In 1 EPIYA A, B, C, 1 0 1 1 1 0 1 1

fi1 0i112203En1 651303241K K

coH 23bo 8 o3K0 A PHBU53

b o fqi 73 512 404Kuk

GG2 9 CPH UCr1eP7 0

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54092 79 9 X870610 C94

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