WHOLE GENOME SEQUENCING OF MYCOBACTERIUM LEPRAE FROM

LEPROSY SKIN BIOPSIES and skeletons

Pushpendra Singh

Laboratory of Prof. Stewart Cole Global Health Institute School of Life Sciences

Ecole Polytechnique Fédérale de Lausanne, Switzerland.

Mycobacterium leprae

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• First bacterial pathogen to be discovered (Hansen 1873) - still remains uncultured in vitro.

- over 1300 pseudogenes.

• Very long doubling time (2 weeks)

• Very low genetic diversity, very few SNPs: -Challenging for genotyping.

-Optimal for re-sequencing projects and mapping.

= several opportunities

PCR based M. leprae Genotyping

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• SNPs

- Excellent phylogeographic association

- Very limited resolution (only 16 subtypes known)

- Type 1(A,B,C,D), 2(E,F,G,H), 3(I,J,K,L,M) and 4(N,O,P).

• VNTRs

- High resolution but limited phylogeographic association.

- Intra-patient variability at different sites of infection.

Limitations of PCR-based genotyping

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• Limited information

- Targeted information and hence not comprehensive.

- Many loci need to be investigated (Not effort-effective).

• Scarce amounts of gDNA from an unculturable pathogen

- Input of whole genome template for a few kb of information.

- not a renewable approach (Empty tubes phenomenon).

Genome-wide analysis (GENOME-Typing) offers great potential, given the

improvements in sequencing technologies and sample prep methods (1-10 ng gDNA).

Outline of the present study 1. DNA isolation directly from leprosy skin

biopsies from diverse geographical areas (n=12) and medieval leprosy skeletons from Europe, 700-1000

yr old).

2. Library preparation and array-based capture of M. leprae DNA.

3. Sequencing and genome-wide comparison (medieval and modern M. leprae).

* * * *

* 3I strains European

aDNA Jorgen_625 (Den),

SK2 (UK)

4P

3I

N.Caledonia 1B, 3K, 3L

3K 1A, 1D 1A

* 2F strains European aDNA

Refshale_1 (Den) , 3077 (Sweden),

SK8 (UK)

Br4923

NHDP63 v1 ‘Zoonotic’ strain (NEJM 2011)

TN Thai53 4N&4O

*

Geographic origins

of samples

Complete drop

of foot-fingers

Leprosy deformities in hand

fingers (1-3) destruction of joints

(4-6)

Maxillary alveolar bone resorption

Rounding of nasal aperture,

Porosity of mandibular bone

Leprosy deformities

Sources and origins of

the medieval M. leprae

strains

In collaboration with archeologists

Methods: Array-

Capture for

Whole Genome

Sequencing

(WGS)

WGS results

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1. Genome wide coverage of 5 ancient and 7 modern strains (coverage of >80% of genome at > 10X).

2. Comparison with 4 reference strains

3. Detailed & Diverse “Phylo-Geo-Chrono”

comparison: 16 genomes (separated by Evolution, Space, and Time):

• Phylogeny: from all four SNP types.

• Geographic: from all 5 continents.

• Chrono (Time): 1000 yr time span (10th century - 2012).

As few as 755 SNPs, and 57 InDels.

Coverage stats of modern M. leprae strains

after array enrichment (on a single array, in multiplex)

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Patient Biopsy (Yr)

% genome covered

Av. fold coverage

SNP type (Origin)

S2 (1992) 84.38 10.25 1B (N. Caledonia)

S9 (1996) 94.51 14.45 3K (N. Caledonia)

S10 (2006) 96.09 17.03 3K (China)

S11 (1990s) 97.59 161.7 1D (India)

S13 (2012) 93.2 15.12 4N (Mali)

S14 (2012) 90.53 22.4 4O (Mali)

S15 (1992) 91.73 11.86 3L (N. Caledonia)

Over 84% genome coverage at over 10x depth

Coverage stats of ancient M. leprae strains

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Skeleton ID %

genome

covered

Av. fold

coverage

SNP type/Origin Radiodate

3077 83.77 10.13 2F (Sweden) 938 ± 19 yr BP

SK8 96.41 20.01 2F (UK) 932 ± 70 yr BP

Refshale_16 97.48 105.1 2F (Denmark) 909 ± 24 yr BP

SK2 92.88 14.87 3I (UK) 950 ± 70 yr BP

Jorgen_625 98.26 101.2 3I (Denmark) 644 ± 23 yr BP

SNP type 2F strains are reported from patients in Turkey and Iran

and SNP type 3I strains are predominant in Americas.

Phylogenetic relationship of M. leprae genomes &

divergence times

Branch 2 strains (2F) are currently reported from Middle East.

Branch 3 strains (3I) are found in Americas, (almost identical to zoonotic v1 strain from southern US

(Truman et al NEJM 2011).

~3400 yrs

300 yrs

Genome-Typing for revealing strain specific

features of the biology of a pathogen

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• 10 NEW pseudogenes were identified in various strains.

• Premature stop codons by SNPs.

• Frameshift caused by Insertion/Deletion events (InDels).

• Drug resistance mutations

• Thr53Ile in folP1 (Dapsone Resistance mutation in 2 strains).

• High diversity in an immunodominant protein encoded by serine-rich antigen ML0411:

• 10 Non-Synonymous SNPs = immune pressure from the host? (Also reported recently in M. leprae Kyoto-2 genome from Japan (Kai et al, Infect

Genet Evol 2013).

Important findings by genome scale comparisons

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1. No major variations in M. leprae in past 1000 yrs.

– No evidences of change in the virulence/adaptability etc.

– Sudden decline of leprosy in Europe in 14th century was NOT due

to the medieval European strain of M. leprae losing virulence.

2. Confirmation of European origin of Leprosy in Americas.

3. Link between medieval European strains and those

currently reported from the Middle East. (SNP type 2F strains of

Iran & Turkey were present in medieval skeletons from 3 European countries).

Why genome-typing instead of genotyping?

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• Comprehensive information

All in one:

- Genotyping.

- Molecular drug susceptibility testing.

- Detailed phylogeny.

- Identification of any additional pseudogenes in a strain.

- Uniform experimental approach for all strains.

- Genome scale investigations into the biology of a strain.

• Why not performed routinely then?

- Costs and technical difficulties.

- streamlining of bioinformatic analysis needed.

Acknowledgements

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• Stewart Cole

– Andrej Benjak, Philippe Busso, all other members of Prof Cole lab (UPCOL).

• Univ. of Surrey: GM Taylor, GR Stewart, TA Mendum.

• Tuebingen: Johannes Krause Lab (Verena Schuenemann & Kirsten Bos)

• Kay Nieselt Lab (Alexander Herbig, Günter Jäger).

Christos (Swedish aDNA), Ben Kyora (Danish aDNA)

Teams of Dr Samba Sow for samples from Mali, C. Johnson from Benin and collaborators from Niger.

Ongoing studies using array capture approach:

Milton Moraes, Luciana, P Suffys and Dr Samira Buhrer-Sekula and their teams

• All patients, clinicians and researchers for contributing clinical samples & relevant details.

Thank you for your attention

Brazil Swiss Joint Research Project

Thank you for your attention!