ジャーナルクラブ2013年7月10日中世ヨーロッパのハンセン病菌のゲノム解読

Journal club, 10 July 2013Hiromi MATSUMAE

中世ヨーロッパのハンセン病菌のゲノム解読

published in 13 June 2013

113年7月16日火曜日

development of ancient pathogen genomics

13 June 2013

Neanderthal genome, 2010


development of ancient pathogen genomics

13 June 2013

Neanderthal genome, 2010

中世のペスト（黒死病）菌ゲノム、in 2011


Leprosy (ハンセン病)現代では抗生物質が有効なため、先進国ではほとんど患者がいないantibiotic is availableless infectious, most patients are infected in early childhood

prevalence in developing countries (Brazil, India, etc.)

historically, patients were the objects of discrimination in the world due to damaged skin


Brazil

Republic of South Sudan

Comoros Islands

Republic of BurundiRepublic of Liberia

Micronesia/Melanesia/Polynesia

Downloaded from WHO


Leprosy (ハンセン病)Skin and peripheral nerves lesions (皮膚と末梢神経に病変)

Infection with Mycobacterium leprae (らい菌)

transmitted to human, primates (Chimp, Crab-eating Macaque, Sooty mangabey) and Armadillo by nasal droplets(?)

Mycobacterium (マイコバクテリウム属) or “acid fat bacteria” (抗酸菌)

M. tuberculosis, M. bovis...(TB) (結核菌)

M. avium: rarely infects human, whose immune system has weakened, such as HIV, 動物由来、日和見感染

related to Crohn's disease?

M. ulcerans (“Buruli ulcer” in tropical and subtropic regions) ブルーリ潰瘍, 熱帯・亜熱帯

3.3Mb genome

Cole et al. 2001; Nature IS=Insertion sequence (phage, transposon etc.)


Detection and Strain Typing of Ancient Mycobacteriumleprae from a Medieval Leprosy HospitalG. Michael Taylor1*, Katie Tucker2, Rachel Butler1, Alistair W. G. Pike3, Jamie Lewis4, Simon Roffey2,

Philip Marter2, Oona Y-C Lee5, Houdini H. T. Wu5, David E. Minnikin5, Gurdyal S. Besra5,

Pushpendra Singh6, Stewart T. Cole6, Graham R. Stewart1

1Department of Microbial and Cellular Science, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom, 2Department of

Archaeology, University of Winchester, Winchester, United Kingdom, 3Department of Archaeology, University of Southampton, Highfield, Southampton, United Kingdom,

4 Bristol Isotope Group, School of Earth Sciences, University of Bristol, Bristol, United Kingdom, 5 School of Biosciences, University of Birmingham, Edgbaston, Birmingham,

United Kingdom, 6Global Health Institute, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland

Abstract

Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletalsigns of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemicaland biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from controlskeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae withdetection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, thepreservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locusvariable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestralto contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From theremaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotopeanalysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all threeindividuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as theprecursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains.Thus we show that type 2F had also spread westwards to Britain by the early medieval period.

Citation: Taylor GM, Tucker K, Butler R, Pike AWG, Lewis J, et al. (2013) Detection and Strain Typing of Ancient Mycobacterium leprae from a Medieval LeprosyHospital. PLoS ONE 8(4): e62406. doi:10.1371/journal.pone.0062406

Editor: Stephen V. Gordon, University College Dublin, Ireland

Received January 7, 2013; Accepted March 21, 2013; Published April 30, 2013

Copyright: ! 2013 Taylor et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The osteoarchaeological analysis was funded by a British Academy Small Research Grant (SR). The Radiocarbon dating was funded by the University ofWinchester (SR, PM) and the Hampshire Field Club and Archaeological Society (SR and KT). Work in centre 2 was funded by the Fondation Raoul Follereau (PS,STC). The mycolipid study was supported by Leverhulme Trust Project Grant F/00 094/BL (GSB, DEM, OY-CL). The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Leprosy is a chronic granulomatous disease caused byMycobacterium leprae, first identified as the causative pathogen bythe Norwegian physician Armauer Hansen, in 1873. In leprosythere is a spectrum of clinical presentations dependent upon thecell-mediated immune (CMI) response of the host. Tuberculoidleprosy (TL) is associated with an effective CMI response and fewmycobacteria whereas lepromatous leprosy (LL) is characterisedby poor CMI and widespread lesions with numerous mycobac-teria. A number of intermediate conditions are recognized;borderline tuberculoid leprosy (BT) and borderline lepromatous(BL) leprosy. A recent WHO classification scheme recognises twosimplified categories of either paucibacillary or multibacillaryforms of leprosy [1]. Any form of leprosy may affect the skeleton;even paucibacillary forms resulting in loss of pain sensation withdigital contracture, volar grooving or osteomyelitis complicatingan overlying skin infection [2]. The more typical lesions of thehands, feet and rhino-maxillary area [3] result from multibacillarystates at the severe end of the spectrum.

Mycobacterium leprae is a clonal organism, with limited geneticvariation between strains. Although sequencing and comparativegenomics of four main strains has revealed a total of 215polymorphic sites, primarily of single nucleotide polymorphisms(SNPs) this is against a background of high overall genomeconservation (.99.99% homology). Study of the phylogeneticallyinformative SNPs in M. leprae isolates from different geographicalregions has allowed the original spread of leprosy to be retracedback to a likely origin in East Africa [4]. There remains somedoubt as to how long leprosy has been a human pathogen. Theleprosy bacillus may have moved out of Africa and around theworld with early human migrations around 100 K years BP, ormay have encountered different populations much later inprehistory, with documented spread in the later historical perioddue to military adventures, colonialism and trade. As genetic datafor modern and particularly archaeological strains of leprosy areaccumulated, an increasingly detailed understanding of the riseand fall of leprosy across the globe is being revealed.Leprosy was present in the British Isles by the 4th century AD

with palaeopathological evidence described from the Romano-

PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e62406


from areas adjacent to pathological lesions but on occasion alsofrom uninvolved skeletal elements such as rib or cranium. Theweights of bone fragments, roughly cuboidal (6–8 mm) in shape,

varied from 20–120 mg. Measures to prevent cross-contaminationbetween cases were applied from the outset. To this end, an areaof bench was set aside and cleaned before and between cases.

Figure 1. Schematic plan of the St. Mary Magdalen hill dig site, looking north. The north wall of the medieval chapel is in the foregroundwhilst the remains of the medieval infirmary can be seen to the north. The northern cemetery can be seen depicted in the centre of the plan andunderlies most of the later medieval phases. Numbers on grave cuts identify individual skeletons examined in the present study.doi:10.1371/journal.pone.0062406.g001

Leprosy in Medieval Britain

PLOS ONE | www.plosone.org 3 April 2013 | Volume 8 | Issue 4 | e62406


OverviewSamples:

Ancient: UK, Sweden and Denmark in the Middle Ages (11～17 Centuries)

Modern strains (1982～2012)

Methods: Illumina MiSeq/HiSeq/GAIIx (次世代シーケンサーを使った解析)

Two capture methods (target enrichment) for ancient DNA(1) Beads capture for screening(3) DNA array capture to cover whole genome of M. laprae

constructed in the Black Death project in 2011 (Bos, Schuenemann et al. 2011, Nature)(Schuenemann et al. 2011, PNAS)

Modern strains (S7, S9, S12 and S15)(2) Shotgun sequencing (re-sequencing or de novo assembly)

5 ancient strains (10th-14th century)12 modern strains (including 1 armadillo)

Comparative genomics (比較ゲノム解析)

(not sequenced) + a few published genomesPhylogenetic analysis (系統解析)


27 samples from 24 skeletons (tooth and bones), with osteological lesions

(1) Screened for the M. leprae DNAby Bead Capture for three genomic loci

DNA extraction in five independent laboratories

(Stockholm, Surrey, Lausanne, Kiel, Tübingen)

NGS sequencing MiSeq (HiSeq) in Tübingen(2) shotgun sequencing (genome assembly or re-sequencing) for 5 ancient samples and 11 modern samples(3) Array-based enrichment for 5 samples showing low DNA yields







without target enrichment (DNA rich)


Methods to identify SNPs


Genome Sequencing Methods in BioinformaticsRe-sequencing （リシーケンス解析）human reference genome

(hg19)shotgun sequences (reads)

(35bp～1kb)

Map to a reference

Depth=5

T

ATAAATTT

Depth=8 (a case of diploid genome)

Reference: TThis individual: A/T

ATAAA

Reference: TConsensus of this individual: A

Depth=5

Coverage(50%?)

Re-sequencing method is generally used for aDNA analysis


Genome Sequencing Methods in Bioinformatics

De novo assembly （デノボ, 新規ゲノム決定）

shotgun sequences (reads)(35bp～1kb)

Making “reference” genome

Making contigs(Super-contig, Scaffold)

Assemble


Capture methods (target enrichment) to avoid non-target sequences(1) Beads-bait-based capture (3) Array-based capture

Computationally design probes for a gene

Probeshybridize aDNA

Designed probes on an array

(Bos, Schuenemann et al. 2011, Nature)(Schuenemann et al. 2011, PNAS)(Fu et al. 2013 PNAS)(Taylor et al. 2013 PLoS ONE)

sequencing aDNA

Probes

screened: three loci of M. laprae and human mtDNA covered M. laprae genome with probes

Agilent (in Max-Plank)








1. Bead Capture for screening

gyvA

RLEP

proS

repetitive sequence specific for M. leprae

different from other mycobacteria

common to all mycobacteria (detection of soil-dwelling mycobacteria)

MiSeq, paired-end reads

M. leprae genes

Sweden Winchester Denmark

Prepare multi-indexing libraries

three pools(mixed libraries)

Capture on beads

SK4, 8, 12 ...

②①

②

③

④

③④


Example: damage plots obtained from beads-captured DNA

gyvA RLEPproS

3519Hsin

FI3320

32H

Refshale_32

Jorgen_625

Fig. S1

Deamination patterns

No deamination?


1. Bead Capture for screening

MiSeq, paired-end reads

Sweden Winchester Denmark

Prepare multi-indexing libraries

three pools for each area

(mixed libraries)

Capture on beads

SK4, 8, 12 ...

②①

②

③

④

③④

human mtDNA

human mtDNA


Why are damage patterns different?Mycolic acid in Mycobacteria cells contributes high quality of DNA remains

Fig. S6

Fig. S5

ミコール酸Dsitribution of endogenous M. leprae and human DNA

(M. leprae is longer than human)


Damage Plots in human mtDNA

3519Hsin

FI3320

32H

Refshale_32

Jorgen_625

human mtDNAgyvA

Fig. S1, S2

Deamination

Deamination

DeaminationDeamination

Not deaminated?

Not deaminated?

Not deaminated?

Not deaminated?

Deamination?

less damage in bacteria than human mtDNA


5 samples passed their criteria (bead capture)10-14th century samples






NGS sequencing in Tübingen(2) shotgun sequencing (genome assembly or re-sequencing) for 5 ancient samples and 11 modern samples(3) Array-based enrichment for 5 samples showing low DNA yields

without target enrichment (DNA rich)


only genome sequencing

Armadillo


Rich enough to perform de novo assembly!

(2) Fig .2AResequencing

completely matched to M. leprae reference genome (Fig. 2A)

5 ancients

10 moderns

755 SNPs

57 InDels (< 7bp)


excavated from the same cemetery, but no osteological evidence of leprosy

target enrichment (array-based) GAIIx

shotgun HiSeq

target enrichment (array-based) MiSeq

most reads were highly conserved regions among all mycobacteria (Table S10)

AntillesNew CaledoniaYemenTurkeyNew CaledoniaNew CaledoniaChinaIndia

MaliAntilles

MaliNew Caledonia

> 90% coerage


only genome sequencing

Armadillo


最節約法(サマーセミナーにて) BEAST

shorter branch due to sample sizegrey: bootstrap values

bold: nucleotide substitutions

grey: posterior probabilities

Phylogenetic analysis•Radiocarbon dates were used for calibration in both models•the same tree topology was obtained by NJ, NeighborNet, Maximum Likelhood methods, including TB, M.avium, and M. ulcerans (Buruli ulcer) as an outgroup in Fig. S7.

relatively closed Mycobacterium (rarely

infects in human)



Modern Branch 2: reported only in Iran and Turkey

shorter branch due to sample sizegrey: bootstrap values





infects in human)



Modern Branch 2: reported only in Iran and Turkey

shorter branch due to sample size

Divergence Time for Most Recent Common Ancestor (MRCA) (excluding S15)

relax: BP 2,871 ya(1,350-5,078 ya)strict: BP 3,126 ya (1,975-4,562 ya)

the oldest known osteological evidence for leprosy: 2,000 BC India

grey: bootstrap values





infects in human)


Phylogenetic analysis


•Radiocarbon dates were used for calibration in both models•the same tree topology was obtained by NJ, NeighborNet, Maximum Likelhood methods, including TB, M.avium, and M. ulcerans (Buruli ulcer) as an outgroup in Fig. S7.

shorter branch due to sample size

grey: bootstrap values




infects in human)

S9, S10: New branch (based on SNP typing)S9 & S15: Thr53Ile mutation in folP1 gene,dapsone-resistance (anti-leprosy drug)


Summaryscreened 27 samples (24 skeletons with osteological evidence) by Bead-bait-capture method

damage patterns (deamination) in M. leprae were different from those in human mtDNAlonger reads were obtained from M. leprae than humanstrong lipid cells in Mycobacteria keep better quality of aDNA

five skeletons and modern strains were sequenced by Array-based capture and shotgun

one (Jorgen_625) included rich endogenous DNA, so that was enable to do de novo assembly755 SNPs among reference, 5 ancient and 10 modern strainsindicates large IN/DELs in 5 ancient strainsThree out of five strains in Europe in the Middle Ages belong to the same branch in modern Middle East (Branch2)Two ancient European strains grouped into Branch 3 with Modern USA



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ジャーナルクラブ2013年7月10日 中世ヨーロッパのハンセン病菌のゲノム解読

ジャーナルクラブ2013年7月10日中世ヨーロッパのハンセン病菌のゲノム解読