The 5300-year-old Helicobacter pylori genome of the Iceman

Abstract

Stomach ache for a European mummy
Five thousand years ago in the European Alps, a man was shot by an arrow, then clubbed to death. His body was subsequently mummified by ice until glacier retreat exhumed him in 1991. Subsequently, this ancient corpse has provided a trove of intriguing information about copper-age Europeans. Now, Maixner et al. have identified the human pathogen Helicobacter pylori within the mummy's stomach contents. The strain the “Iceman” hosted appears to most closely resemble pathogenic Asian strains found today in Central and Southern Asia.
Science , this issue p. 162

Full text

microbial function spanning terrestrial ecosys-
tems, and though plant inputs are the dominant
source of organic matter, vertebrate corpse inputs
can be important resources (5,6). For example,
one rain forest in Panama was estimated to receive
750 kg in mammal corpses annually per square
kilometer (12). Although this represents less than
1% of the mass of plant litter received by another
Panamanian rain forest (13), corpse nutrient
sources can be an order of magnitude more con-
centrated than plant litter (5), and direct com-
parisons between plant and animal decomposition
resources are rare (14). Thus, much is still unclear
about the role of corpse inputs in larger-scale
biogeochemical cycling (e.g., global carbon and
nitrogen cycling) and in supporting specific com-
munities and microbial diversity (14), and our
results provide an important microbial perspective.
Asocietalimpactoftheseresultsisthevalueof
microbial data as physical evidence in medico-
legal death investigation. We show that decom-
poser microbial communities could potentially
serve as temporal (succession-based) and spatial
(origin-based) (supplementary text) forms of
physical evidence, such as the time elapsed since
death (PMI) and the location of death. Our obser-
vation that postmortem microbial communities
changed in a clock-like manner that provided
an estimate of absolute PMI is similar to using
the development of fly larvae to estimate PMI.
However, the fly larvae PMI proxy is limited by
corpse accessibility and season, resulting in PMI
estimates in the range of weeks, months, and
even years (15). Taken together, our findings dem-
onstrate that postmortem microorganisms can
provide both spatial and temporal insight into
the events surrounding death.
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ACKNOWL EDGME NTS
The data reported in this paper are available in the Qiita database
(http://qiita.ucsd.edu/) (accession numbers 10141 to 10143 and
10321) and the European Bioinformatics Institute European
Nucleotide Archive (www.ebi.ac.uk/ena) (accession numbers
ERP012866, ERP012879, ERP012880, and ERP012894). We thank
the donors and their families for their contribution to scientific
research; the STAFS Facility at SHSU and the Molecular, Cellular,
and Developmental Biology Transgenic Facility at the University of
Colorado, Boulder, for providing the space and opportunity for this
research; N. Fierer, J. Zelikova, and J. Leff for assistance with
project logistics and data processing; and the Mountain Research
Station and Shortgrass Steppe Long Term Ecological Research for
permission to collect soils. Mice were euthanized humanely under
approved protocol no. 08-04-ACK-01 (principal investigator G.A.).
This research was funded by the Office of Justice Programs
National Institute of Justice Awards NIJ-2011-DN-BX-K533 (J.L.M.,
D.O.C., R.K.) and NIJ-2012-DN-BX-K023 (S.R.B. and A.M.L.).
Research capacity and infrastructure at Chaminade University of
Honolulu is supported by NIH Building Research Infrastructure and
Capacity Program P789097-876. W.V.T. and S.W. were supported
by the National Human Genome Research Institute grant 3 R01
HG004872-03S2, and NIH grant 5 U01 HG004866-04. J.L.M. was
partially supported by a Templeton Foundation grant (R.K. and
V. McKenzie). Use of trade, product, or firm names is for
informational purposes only and does not constitute an
endorsement by the U.S. government. J.F.P. is Chief Scientific
Officer and Founder of Diversigen; C.N. is an employee of miRagen
Therapeutics; and R.K. is Chief Science Officer and employee of
Biota Technology, a member of the Scientific Advisory Panel at
Temasek Life Sciences Laboratory, and a speaker at Nestec, Nestle
Research Center.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/351/6269/158/suppl/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S19
Tables S1 to S20
References (16–29)
19 August 2015; accepted 25 November 2015
Published online 10 December 2015
10.1126/science.aad2646
ANCIENT MICROBIOME
The 5300-year-old Helicobacter pylori
genome of the Iceman
Frank Maixner,
1
*†Ben Krause-Kyora,
2
†Dmitrij Turaev,
3
†Alexander Herbig,
4,5
†
Michael R. Hoopmann,
6
Janice L. Hallows,
6
Ulrike Kusebauch,
6
Eduard Egarter Vigl,
7
Peter Malfertheiner,
8
Francis Megraud,
9
Niall O’Sullivan,
1
Giovanna Cipollini,
1
Valentina Coia,
1
Marco Samadelli,
1
Lars Engstrand,
10
Bodo Linz,
11
Robert L. Moritz,
6
Rudolf Grimm,
12
Johannes Krause,
4,5
‡Almut Nebel,
2
‡Yoshan Moodley,
13,14
‡
Thomas Rattei,
3
‡Albert Zink
1
*‡
The stomach bacterium Helicobacter pylori is one of the most prevalent human pathogens.
It has dispersed globally with its human host, resulting in a distinct phylogeographic pattern
that can be used to reconstruct both recent and ancient human migrations. The extant
European population of H. pylori is known to be a hybrid between Asian and African bacteria,
but there exist different hypotheses about when and where the hybridization took place,
reflecting the complex demographic history of Europeans. Here, we present a 5300-year-old
H. pylori genome from a European Copper Age glacier mummy. The “Iceman”H. pylori is a
nearly pure representative of the bacterial population of Asian origin that existed in Europe
before hybridization, suggesting that the African population arrived in Europe within
the past few thousand years.
The highly recombinant pathogen Helicobacter
pylori has evolved to live in the acidic en-
vironment of the human stomach (1). Today,
this Gram-negative bacterium is found in
approximately half the world’s human pop-
ulation, but fewer than 10% of carriers develop
disease that manifests as stomach ulcers or gas-
tric carcinoma (2,3). Predominant intrafamilial
transmission of H. pylori and the long-term
association with humans has resulted in a phylo-
geographic distribution pattern of H. pylori that
is shared with its host (4,5). This observation
suggests that the pathogen not only accompa-
nied modern humans out of Africa (6), but that
it has also been associated with its host for at
least 100,000 years (7). Thus, the bacterium has
been used as a marker for tracing complex demo-
graphic events in human prehistory (4,8,9). Mod-
ern H. pylori strains have been assigned to distinct
populations according to their geographic ori-
gin (hpEurope, hpSahul, hpEastAsia, hpAsia2,
hpNEAfrica, hpAfrica1, and hpAfrica2) that are
derived from at least six ancestral sources (4,5,8).
The modern H. pylori strain found in most Eu-
ropeans (hpEurope) putatively originated from
recombination of the two ancestral populations
Ancestral Europe 1 and 2 (AE1 and AE2) (6). It
has been suggested that AE1 originated in Cen-
tral Asia, where it evolved into hpAsia2, which
is commonly found in South Asia. On the other
hand, AE2 appears to have evolved in northeast
Africa and hybridized with AE1 to become hpEurope
(4). However, the precise hybridization zone of
the parental populations and the true origin of
hpEurope are controversial. Early studies observed
a south-to-north cline in AE2/AE1 frequency in
Europe (4,6). This finding has been attributed to
independent peopling events that introduced these
ancestral H. pylori components, which eventually
recombined in Europe since the Neolithic period.
More recently, it has been suggested that the
AE1/AE2 admixture might have occurred in the
Middle East or Western Asia between 10,000 and
52,000 years ago and that recombinant strains
were introduced into Europe with the first human
recolonizers after the last glacial maximum (7).
162 8JANUARY2016•VOL 351 ISSUE 6269 sciencemag.org SCIENCE
RESEARCH |REPORTS

In this study, we screened 12 biopsy samples
from the gastrointestinal tract of the Iceman, a
5300-year-old Copper Age mummy, for the pres-
ence of H. pylori. Stable isotope analyses showed
that the Iceman originated and lived in Southern
Europe,intheEasternItalianAlps(10). Genet-
ically, he most closely resembles early European
farmers (11–13). The Iceman’sstomachwasdis-
covered in a reappraisal of radiological data and
containsthefoodheingestedshortlybeforehis
death (Fig. 1) (14). The study material included
stomach content, mucosa tissue, and content of
the small and large intestines (table S1). By using
direct polymerase chain reaction (PCR), meta-
genomic diagnostics, and targeted genome cap-
ture(figs.S1andS2),wedeterminedthepresence
of H. pylori and reconstructed its complete
genome.
Metagenomic analysis yielded endogenous an-
cient H. pylori DNA (15,350 reads) in all gastro-
intestinal tract contents (Fig. 1 and table S4). A
control data set derived from Iceman’smuscletissue
was negative.The distribution of the observed read
counts throughout the Iceman’s intestinal tract
is similar to that in modern H. pylori–positive
humans, with abundance decreasing from the
stomach toward the lower intestinal tract (15,16).
The retrieved unambiguous reads were aligned
to a modern H. pylori reference genome (strain
26695) and showed damage patterns indicative of
ancient DNA (fig. S7) (17). After DNA repair, the H.
pylori DNA was enriched up to 216-fold by using
in-solution hybridization capture (Agilent) (fig. S5).
From this data set, 499,245 nonredundant reads
mapped to 92.2% of the 1.6-Mb H. pylori reference
genome with an 18.9-fold average coverage (Fig. 2).
In comparison with the reference, the Iceman’s
ancient H. pylori genome had ~43,000 single-
nucleotide polymorphisms (SNPs) and 39 dele-
tions that range from 95 base pair (bp) to 17 kb
and mainly comprise complete coding regions.
Owing to deletions, the number of genomic
variants is slightly below the range of what can
be observed between modern H. pylori strains
(table S13). The analysis of SNP allele frequen-
cies does not indicate an infection by more than
one strain (supplementary materials S6). In ad-
dition, as expected for this highly recombinant
bacterium, we found evidence for gene insertions
from H. pylori strains that differ from the refer-
ence genome (details about the InDels are pro-
vided in supplementary materials S8).
SCIENCE sciencemag.org 8JANUARY2016•VOL 351 ISSUE 6269 163
1
Institute for Mummies and the Iceman, European Academy
of Bozen/Bolzano (EURAC), Viale Druso 1, 39100 Bolzano,
Italy.
2
Institute of Clinical Molecular Biology, Kiel University,
Schittenhelmstrasse 12, 24105 Kiel, Germany.
3
CUBE–
Division of Computational Systems Biology, Department of
Microbiology and Ecosystem Science, University of Vienna,
Althanstrasse 14, 1090 Vienna, Austria.
4
Institute for
Archaeological Sciences, University of Tübingen,
Rümelinstrasse 23, 72072 Tübingen, Germany.
5
Max Planck
Institute for the Science of Human History, Kahlaische
Strasse 10, 07745 Jena, Germany.
6
Institute for Systems
Biology, 401 Terry Avenue North, Seattle, WA 98109, USA.
7
Scuola Superiore Sanitaria Provinciale “Claudiana,”Via
Lorenz Böhler 13, 39100 Bolzano, Italy.
8
Department of
Gastroenterology, Hepatology, and Infectious Diseases, Otto-
von-Guericke University, Leipziger Strasse 44, 39120
Magdeburg, Germany.
9
Université de Bordeaux, Centre
National de Référence des Helicobacters et Campylobacters
and INSERM U853, 146 rue Léo Saignat, 33076 Bordeaux,
France.
10
Department of Microbiology, Tumor and Cell
Biology, Karolinska Institutet, 141 83 Stockholm, Sweden.
11
Department of Veterinary and Biomedical Sciences,
Pennsylvania State University, University Park, PA 16802,
USA.
12
Robert Mondavi Institute for Food Science, University
of California, Davis, CA 95616, USA.
13
Department of
Zoology, University of Venda, Private Bag X5050,
Thohoyandou 0950, Republic of South Africa.
14
Department
of Integrative Biology and Evolution, Konrad Lorenz Institute
for Ethology, University of Veterinary Medicine Vienna,
Savoyenstrasse 1a, 1160 Vienna, Austria.
*Corresponding author. E-mail: frank.maixner@eurac.edu (F.M.);
albert.zink@eurac.edu (A.Z.) †These authors contributed equally
to this work. ‡These authors contributed equally to this work.
Fig. 1. H. pylori–specific reads detected in the metagenomic data
sets of the Iceman’s intestine content samples.The color gradient dis-
plays the number of unambiguous H. pylori reads per million meta-
genomic reads. Control metagenomic data sets of the Iceman’s muscle tissue and of the extraction blank
were included in the analysis.The different intestinal content sampling sites are marked in the radiographic
image by the following symbols: asterisk, stomach content; circle, small intestine; square, upper large
intestine; triangle, lower large intestine.The sampling site of the muscle control sample is highlighted in the
Iceman overview picture (diamond).
Fig. 2. Gene coverage
and distribution of the
enriched and validated
Iceman H. pylori reads
mapped onto the 1.6 Mb
large reference genome
H. pylori 26695.The
coverage plot displayed in
black is superimposed onto
the genomic plot. The bar
on the right-hand side indi-
cates a coverage of up to
50×. The gene cod ing
sequences are shown in
blue (positive strand) and
yellow (negative strand)
bars in the genomic plot.
The loci of the ribosomal
RNA genes, of two virulence genes (vacA and cagA), and of seven genes used for MLSTanalysis are highlighted in the genome plot.
RESEARCH |REPORTS

Subsequent sequence analysis classified the an-
cient H. pylori as a cagA-positive vacA s1a/i1/m1
type strain that is now associated with inflammation
of the gastric mucosa (fig. S11) (18). Using multistep
solubilization and fractionation proteomics, we
identified 115 human proteins in the stomach meta-
proteome, of which six were either highly expressed
in the stomach mucosa (trefoil factor 2) (19)or
present in the gastrointestinal tract and involved in
digestion (supplementary materials S10). The ma-
jority of human proteins were enriched in extracel-
lular matrix organizing proteins (P=3.35×10
–14
)
andproteinsofimmuneprocesses(P=2×10
–3
)
(fig. S13). In total, 22 proteins observed in the Iceman
stomach proteome are primarily expressed in neu-
trophils and are involved in the inflammatory host
response. The two subunits S100A8 and S100A9 of
calprotectin (CP) were detected with the highest
number of distinct peptide hits in both analyzed
samples. Inflamed gastric tissues of modern
H. pylori–infected patients also show high levels
of CP subunit S100A8 and S100A9 expression
(20,21). Thus, the Iceman’sstomachwascol-
onized by a cytotoxic H. pylori–type strain that
triggered CP release as a result of host inflamma-
tory immune responses. However, whether the
Iceman suffered from gastric disease cannot be
determined from our analysis owing to the poor
preservation of the stomach mucosa (fig. S3).
Comparative analysis of seven housekeeping
gene fragments with a global multilocus sequence
typing (MLST) database of 1603 H. pylori strains
with the STRUCTURE (22)no-admixturemodel
assigned the 5300-year-old bacterium to the modern
population hpAsia2, commonly found in Central
164 8JANUARY2016•VOL 351 ISSUE 6269 sciencemag.org SCIENCE
Fig. 3. Multilocus sequence analyses. (A) Bayes-
ian cluster analysis performed in STRUCTURE
displays the population partitioning of hpEurope,
hpAsia2, and hpNEAfrica and the Iceman’sH. pylori
strain (details about the worldwide population
partitioning of 1603 reference H. pylori strains
are available in fig. S14). (B) STRUCTURE linkage
model analysis showing the proportion of Ances-
tral Europe 1 (from Central Asia) and Ancestral
Europe 2 (from northeast Africa) nucleotides
among strains assigned to populations hpNEAfrica,
hpEurope, and hpAsia2 and the Iceman’sH. pylori
strain on the extreme right. The black arrows indi-
cate the position of the three extant European
hpAsia2 strains. (C) Principal component analysis
of contemporary hpNEAfrica, hpEurope, and hpAsia2
strains and the Iceman’sH. pylori strain.
Fig. 4. Comparative whole-genome analysis. Co-ancestry matrix showing H. pylori population structure
and genetic flux.The color in the heat map corresponds to the number of genomic motifs imported from a
donor genome (column) to a recipient genome (row).The inferred tree and the H. pylori strain names are
displayed on the top and left of the heat map. Strain names are colored according to the H. pylori pop-
ulation assignment provided in the legend below the heat map. Signs for population ancestry are high-
lighted in the heat map with green, blue, black, and white boxes.
RESEARCH |REPORTS

and South Asia (Fig. 3A and fig. S14). The detection
of an hpAsia2 strain in the Iceman’s stomach is
rather surprising because despite intensive sampling,
only three hpAsia2 strains have ever been detected
in modern Europeans. Stomachs of modern Eu-
ropeans are predominantly colonized by recom-
binant hpEurope strains. Further ana lysis with
the STRUCTURE linkage model (23), used to detect
ancestral structure from admixture linkage d is-
equilibrium, revealed that the ancient H. pylori
strain contained only 6.5% [95% probability in-
tervals (PI) 1.5 to 13.5%] of the northeast African
(AE2) ancestral component of hpEurope (Fig. 3B).
Among European strains, this low proportion of
AE2isdistinctandhasthusfaronly beenobserved
in hpAsia2 strains from India and Southeast Asia.
In contrast, the three European hpAsia2 strains
(Fig. 3B, black arrows) contained considerably
higher AE2 ancestries than that of the H. pylori
strain of the Iceman (Finland 13.0%, PI 5.9 to 21.7;
Estonia 13.2%, PI 6.2 to 22.3; and the Nethe rlands
20.8%, PI 11.5 to 31.7), although 95% pr oba bil ity
intervals did overlap. A principal component
analysis (PCA) of the MLST sequences of the
hpAsia2, hpEurope, and hpNEAfrica populations
revealed a continuum along PC1 that correlates with
the proportion of AE2 ancestry versus AE1 ancestry
of the isolates (Fig. 3C). The Iceman’s ancient H.
pylori was separated from modern hpEurope
strains, and its position along PC1 was close to
modern hpAsia2 strains from India, reflecting its
almost pure AE1 and very low AE2 ancestry.
Comparative whole-genome analyses (neighbor
joining, STRUCTURE, and principle component
analyses) with publicly available genomes (n=45)
confirmed the MLST result by showing that the
Iceman’s ancient H. pylori genome has highest
similarity to three hpAsia2 genomes from India
(figs. S15 to S17). Although the Iceman’sH. pylori
strain appears genetically similar to the extant
strains from northern India, slight differences
were observed alongPC2 in both MLST (Fig. 3C)
and genome PCAs (fig. S17) and in the neighbor
joining tree (fig. S15). To further study genomic-scale
introgression, we performed a high-resolution
analysis of ancestral motifs using fineSTRUCTURE
(24). The resulting linked co-ancestry matrix (Fig. 4)
showed that the ancient H. pylori genome shares
high levels of ancestry with Indian hpAsia2 strains
(Fig. 4, green boxes), but even higher co-ancestry
with most European hpEurope strains (Fig. 4, blue
boxes). In contrast, the Iceman’sH. pylori shares
low ancestry with the hpNEAfrica strain, a modern
representative of AE2 (Fig. 4, black box), and with
European strains originating from the Iberian
Peninsula, where the proportion of AE2 ancestry
is relatively high (Fig. 4, white box) (4). Our sample
size (n= 1) does not allow further conclusions
about the prevalence of AE1 in ancient Europe and
thecourseorrateofAE2introgression.However,
the ancient H. pylori strain provides the first evi-
dence that AE2 was already present in Central
Europe during the Copper Age, albeit at a low level.
If the Iceman H. pylori strain is representative of
its time, the low level of AE2 admixture suggests
that most of the AE2 ancestry observed in hpEu-
rope today is a result of AE2 introgression into
Europe after the Copper Age, which is later than
previously proposed (4,6). Furthermore, our
co-ancestry results indicate that the Iceman’s
strain belonged to a prehistoric European branch
of hpAsia2 that is different from the modern
hpAsia2 population from northern India. The
high genetic similarity of the ancient strain to
bacteria from Europe implies that much of the
diversity present in Copper Age Europe is still
retained within the extant hpEurope population,
despite millennia of subsequent AE2 introgression.
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ACKNO WLED GME NTS
We acknowledge the following funding sources: the South Tyrolean
grant legge 14 (F.M., N.O.S., G.C., V.C., M.S., and A.Z.), the
Ernst Ludwig Ehrlich Studienwerk, dissertation completion
fellowship of the University of Vienna (D.T.), the Graduate School
Human Development in Landscapes and the Excellence Cluster
Inflammation at Interfaces (B.K. and A.N.), the European Research
Council (ERC) starting grant APGREID (J.K. and A.H.), the National
Institutes of Health from the National Institute of General
Medical Sciences under grants R01 GM087221 (R.M.), S10
RR027584 (R.M.), and 2P50 GM076547/Center for Systems
Biology (R.M). E. Leproust and O. Hardy are highly acknowledged
for their help in the RNA bait design. We thank the sequencing
team of the Institute of Clini cal Molecular Biology at Kiel University f or
supportand expertise. We are grateful to E. Hüttenfor proofreading of
the main text. We are grateful to Olympus, Italy, for providing us with
equipment for endoscopy. F.M. and A.Z. conceived the investigation.
F.M., B.K., D.T., R.G., J.K., A.N., Y.M., T.R., and A.Z. designed
experiments. P.M., L.E., E.E.V., M.S.,F.M., and A.Z. were involved in the
sampling campaign. F.M., B.K., M.R.H., J.H., U.K., and G.C. performed
laboratory work. F.M., B.K., D.T., A.H., M.R.H., N.O.S., B.L., R.L.M., R.G.,
J.K.,Y.M.,andT.R.performedanalyses.F.M.wrotethemanuscript
with contributions from B.K., D.T., A.H., M.R.H., U.K., N.O.S., V.C., B.L.,
R.L.M., R.G., J.K., Y.M., A.N., T.R., and A.Z. Data are available from the
European Nucleotide Archive under accession no. ERP012908. The
authors declare no competing interes ts.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/351/6269/162/suppl/DC1
Materials and Methods
Figs. S1 to S17
Tables S1 to S13
References (25–93)
15 August 2015; accepted 20 November 2015
10.1126/science.aad2545
PAL EOC L IMAT E
Reconciliation of the Devils
Hole climate record with
orbital forcing
Gina E. Moseley,
1
*R. Lawrence Edwards,
2
Kathleen A. Wendt,
1,2
Hai Cheng,
2,3
Yuri Dublyansky,
1
Yanbin Lu,
2
Ronny Boch,
1
†Christoph Spötl
1
The driving force behind Quaternary glacial-interglacial cycles and much associated climate
change is widely considered to be orbital forcing. However, previous versions of the iconic Devils
Hole (Nevada) subaqueous calcite record exhibit shifts to interglacial values ~10,000 years
before orbitally forced ice age terminations, and interglacial durations ~10,000 years longer
than other estimates. Our measurements from Devils Hole 2 replicate virtually all aspects
of the past 204,000 years of earlier records, except for the timing during terminations,
and they lower the age of the record near Termination II by ~8000 years, removing both
~10,000-year anomalies.The shift to interglacial values now broadly coincides with the rise in
boreal summer insolation, the marine termination, and the rise in atmospheric CO
2
,whichis
consistent with mechanisms ultimately tied to orbital forcing.
Changes to Earth’s orbital configuration rel-
ative to the Sun, known as the Milanko-
vitch hypothesis, astronomical theory, or
orbital forcing, have long been considered
the leading theory for the primary mech-
anism driving Quaternary glacial-interglacial
cycles (1–3) and associated climate change.
The hypothesis is supported by a huge array
of evidence from paleoclimate records across
the globe, which show that major shifts in
SCIENCE sciencemag.org 8JANUARY2016•VOL 351 ISSUE 6269 165
RESEARCH |REPORTS