WHOLE GENOME SEQUENCING OF MYCOBACTERIUM LEPRAE FROM LEPROSY SKIN BIOPSIES and skeletons

Size: px

Start display at page:

Download "WHOLE GENOME SEQUENCING OF MYCOBACTERIUM LEPRAE FROM LEPROSY SKIN BIOPSIES and skeletons"

Kellie Bennett
5 years ago
Views:

1 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.

2 Mycobacterium leprae 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 2

3 PCR based M. leprae Genotyping 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. 3

4 Limitations of PCR-based genotyping 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). 4

5 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, yr old). 2. Library preparation and array-based capture of M. leprae DNA. 3. Sequencing and genome-wide comparison (medieval and modern M. leprae).

Br4923 ** * 4N&4O * 2F strains European adna Refshale_1 (Den),

6 Geographic origins of samples NHDP63 3I v1 Zoonotic strain (NEJM 2011) * 3I strains European adna Jorgen_625 (Den), SK2 (UK) 4P Br4923 ** * 4N&4O * 2F strains European adna Refshale_1 (Den), 3077 (Sweden), SK8 (UK) 1A, 1D TN 3K 1A Thai53 N.Caledonia 1B, 3K, 3L

7 Leprosy deformities Leprosy deformities in hand fingers (1-3) destruction of joints (4-6) Complete drop of foot-fingers Maxillary alveolar bone resorption Rounding of nasal aperture, Porosity of mandibular bone

8 Sources and origins of the medieval M. leprae strains In collaboration with archeologists

9 Methods: Array- Capture for Whole Genome Sequencing (WGS)

10 WGS results 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 (10 th century ). As few as 755 SNPs, and 57 InDels. 10

11 Coverage stats of modern M. leprae strains after array enrichment (on a single array, in multiplex) Patient Biopsy (Yr) % genome covered Av. fold coverage SNP type (Origin) S2 (1992) B (N. Caledonia) S9 (1996) K (N. Caledonia) S10 (2006) K (China) S11 (1990s) D (India) S13 (2012) N (Mali) S14 (2012) O (Mali) S15 (1992) L (N. Caledonia) Over 84% genome coverage at over 10x depth 11

12 Coverage stats of ancient M. leprae strains Skeleton ID % genome covered Av. fold coverage SNP type/origin Radiodate F (Sweden) 938 ± 19 yr BP SK F (UK) 932 ± 70 yr BP Refshale_ F (Denmark) 909 ± 24 yr BP SK I (UK) 950 ± 70 yr BP Jorgen_ I (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. 12

13 Phylogenetic relationship of M. leprae genomes & divergence times ~3400 yrs 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). 300 yrs

14 Genome-Typing for revealing strain specific features of the biology of a pathogen 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). 14

15 Important findings by genome scale comparisons 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 14 th 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). 15

16 Why genome-typing instead of genotyping? 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. 16

17 Acknowledgements 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. Brazil Swiss Joint Research Project Thank you for your attention 17

18 Thank you for your attention!

Mycobacterium lepromatosis Infections in Nuevo León, México

Mycobacterium lepromatosis Infections in Nuevo León, México JCM Accepted Manuscript Posted Online 25 March 2015 J. Clin. Microbiol. doi:10.1128/jcm.03667-14 Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 1 2 3 4 5 6 7 8 9 10 11 12 13