Transcript Document

Phylogenetic reconstruction using secondary structures
and sequence motifs of ITS2 rDNA of Paragonimus
westermani (Kerbert, 1878) Braun, 1899 (Digenea:
Paragonimidae) and related species
P. K. Prasad1, V. Tandon1, D. K. Biswal2, L. M. Goswami1 and A. Chatterjee3
1Departments
of Zoology, 3Biotechnology & Bioinformatics and
2Bioinformatics Centre, North-Eastern Hill University, Shillong, 793022
Email: - [email protected]
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Paragonimus
•
•
•
Zoonotic lung fluke having diversified effect on its final host.
Over 40 species infect lungs of different mammalian hosts.
~15 species known to infect humans.
Egg
80-110×48-60µ
•
•
Encysted metacercaria
~300- 400µ
Pre-adult
Adult: 7.5-12 × 4-6×1-3mm
(l: w = 2:1)
P. westermani (Kerbert, 1878) - Best known species, human
parasite that can undergo development in >16 different snail
species and 50 crustacean species.
Status of prevalence and host range in Indianot well
documented.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Distribution of human paragonimiasis
Species of Paragonimus are encountered in Asia, the Americas, and
Africa.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Distribution in India
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Paragonimus species: India
1. P. westermani (Kerbert,1878)- Bengal tiger, Amsterdam
Zoo collection from India, Indonesia, China
[ Syn. P. edwardsi Gulati, 1926- civet]
2. P. compactus (Cobbold, 1859)- Herpestes edwardsii India
(Vevers,1923; Ravikumar et al.,1979); Sri Lanka (Dissanaike
and Paramananthan, 1962)
3. P. heterotremus Chen and Hsia, 1964- China, Thailand,
Laos, Vietnam
•
Arunachal Pradesh (Narain et al. 2003)
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Manipur (Singh,1996)
4. P. hueitungensis Chung et al.,1975- China
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Manipur (Singh, 2002)
5. P. mungoi, P. pantheri – Orissa (Mishra et al.,1976)-
nomen nudem
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Life Cycle
1. Infective stage: metacercaria
2. Infective mode: eating raw fresh water
crabs and crayfish with metacercariae
3. Infective route: by mouth
4. Site of inhabitation: lungs
5. Intermediate hosts: 1st – snail; 2nd
crab, crayfish
6. Reservoir hosts: carnivores (tiger, lion, wolf,
fox, dog, leopard, cat etc.)
7. Life span: 5-6 years
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Paragonimus westermani is the best-known
species. Diploid & triploid forms in N.E.
Asia: only diploids elsewhere.
Pleurocerid
snail hosts
?
?
?
?
Thiarid snail
hosts
?
Diploid
Triploid
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Many other species described.
Nearly 50 species, mostly in Asia:
over half of these in China alone.
This prompted a molecular
taxonomy approach.
Chengdu
Beijing
Shanghai
(P. westermani not shown here)
Guangzhou
Euparagonimus
cenocopiosus
E. hongzesiensis
P. asymmetricus
P. bangkokensis
P. cheni
P. divergens
P. fukienensis
P. harinasutai
P. heterorchis
P. heterotremus
P. hokuoensis
P. heuitungensis
P. iloktsuenensis
P. jiangsuensis
P. macrorchis
P. menglaensis
P. microrchis
P. minqinensis
P. ohirai
P. paishuihoensis
P. proliferus
P. szechuanensis
P. tuanshanensis
P. veocularis
P. xiangshanensis
P. yunnanensis
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Molecular Taxonomy
• Molecular tools- allow quick and accurate identification of
genetically distinct but morphologically similar species
• Genetic markers in nuclear ribosomal DNA (rDNA)
- to resolve taxonomic issues related to various animal groups
- to find phenotypic variants, geographical isolates
• ITS rDNA– widely used region, to explore species
boundaries in at least 19 digenean (helminth parasites’)
families
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
The ribosomal DNA gene cluster
• Ribosomal genes and their associated spacers are among the most versatile
sequences for phylogenetic analysis.
• Useful for diagnostic purposes at the level of species.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Bioinformatic Tools
• Similarity search - BLAST
http://www.ncbi.nlm.nih.gov/blast
• Phylogenetic prediction - ClustalW/X
http://www.ebi.ac.uk/clustalw
• Phylogenetic trees construction - MEGA (Molecular
Evolutionary Genetics Analysis) format,
- Distance methods (Neighbour-Joining, Minimum
Evolution, UPGMA)
- Character- state method (Maximum Parsimony)
• Bayesian Analysis - Mrbayes 3.1.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
• ITS sequence motifs- useful for development of DNA bar
coding; short DNA sequences from a standardized region of
genome- diagnostic “biomarker” for species identification
(http://www.barcoding.si.edu/)
• Molecular morphometrics- traditional morphological and molecular sequence
comparisons by measuring structural parameters.
- Geometrical features, bond energies, base composition etc.
of secondary structure to study phylogenetic relationships of
species
(http://www.bioinfo.rpi.edu/applications/mfold)
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Materials & Methods
Parasite:
• Collected from mountain stream crabs of suspected focal
area (Changlang Dist. in Arunachal Pradesh).
• Metacercariae
isolated
from
muscles
by
digestion
technique.
DNA isolation:
• DNA extracted in FTA card (Whatman’s), punching
sample discs
• Sample discs- washed with FTA Purification reagent and
TE Buffer; used for PCR.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
DNA amplification and sequencing
• ITS2 primers used :
3S: 5’- GGTACCGGTGGATCACTCGGCTCGTG-3’ (forward)
A28:5’-GGGATCCTGGTTAGTTTCTTTTCCTCCGC-3’ (reverse)
[Designed based on conserved sequences of the 5.8S and
28S genes of Schistosoma spp]
• Marker- Phi X 174 DNA/ Hae III Digest in agarose gel
• PCR products- purified by Genei Quick PCR Purification Kit;
- sequenced in both directions using
primer set 3S-A28
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Molecular Phylogenetic Analysis
• Only unique sequences were used in tree construction.
• ITS sequences entered in MEGA for phylogenetic trees construction.
• Tree building methods- Maximum Parsimony, Neighbor-Joining, UPGMA,
Minimum Evolution.
• Branch support given using 1000 bootstrap replicates in MEGA
Bayesian Phylogenetic Analysis
• Sequences aligned using Clustal W 2.0.7 and converted to NEXUS file.
• Analysis carried out with combined datasets using Mrbayes 3.1.
• Cladogram and phylogram with mean branch lengths generated, and read
by Tree view V1.6.6.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Motif identification, testing and validation
•
Sequences motifs identified from aligned sequences of the data set using
PRATT software.
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Motifs were expressed using DNA alphabet (A,T,C,G) in PROSITE language.
•
Validation of motifs were performed for each species using a ‘PATTERN
MATCHING’ web application.
Evaluation through BLAST analysis
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Sequences motifs subjected to BLAST algorithms against nonredundant
GenBank database of NCBI (nr).
•
BLAST outputs analyzed to find perfect pair-wise matches in terms of percent
identity and E-values for each species.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Predicted ITS2 RNA secondary structures and analyses
• Secondary structures of ITS2 sequences of various
paragonimid species were reconstructed by aligning their
sequences using Bioedit (Hall, 1999).
• The acquired structures with restrictions and constrains
submitted in MFOLD (Zuker, 2003).
• RNA structure chosen from different output files with
highest negative free energy for various similar
structures obtained.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Results
PCR amplification and analysis
M 1 2 3 4 5 6
1b
PCR products of Paragonimus metacercaria DNA using primer set 3S - A28 for ITS2
Amplification conditions:
Product size: ~ 500 bp
Final reaction volume = 50μl
1.6% agarose gel electrophoresis
Marker = Ø x 174 DNA/ HaeIII Digest
Initial denaturation at 94ºC
Denaturation at 94ºC
Annealing at 55ºC
Extension at 72ºC
Final extension at 72ºC
=1 min
= 30 sec
= 38 sec
= 42 sec
= 10 min
}
26 cycles
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Sequence motif in PROSITE format (from 5’ to 3’ ends)
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
NJ tree
MP tree
Phylogenetic trees of ITS2 sequences of Paragonimid species. (*) = Query sequence
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
BLAST outputs of Paragonimus ITS sequence motifs against NCBI GenBank database
Species motif patterns (5’-3’ ends)
Length (bp) No. of best hits Identity
(%)
E-value
>Pattern1
G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-C-GC-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T-A-A-A-C
50
100
100
8e-19
>Pattern2
C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-A-CG-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T-A-A-A
50
100
100
8e-19
>Pattern3
G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-C-AC-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T-A-A
50
100
100
8e-19
>Pattern4
T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-C-CA-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T-A
50
100
100
8e-19
>Pattern5
T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-G-CC-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A-T
50
100
100
8e-19
>Pattern6
A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-G-GC-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T-A
50
100
100
8e-19
>Pattern7
T-A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-T-GG-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T-T
50
100
100
8e-19
>Pattern8
A-T-A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-G-TG-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C-T
50
100
100
8e-19
>Pattern9
C-A-T-A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-T-GT-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G-C
>Pattern10
G-C-A-T-A-T-T-G-C-G-G-C-C-A-C-G-G-G-T-T-A-G-C-C-TG-T-G-G-C-C-A-C-G-C-C-T-G-T-C-C-G-A-G-G-G-T-C-G-G
50
100
100
8e-19
50
100
100
8e-19
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Predicted ITS2 RNA secondary structures with enthalpies: Indian isolates
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Predicted ITS2 RNA secondary structures: Neighbouring countries isolates
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Family Paragonimidae: Hypothetical Bayesian analysis phylogeny based upon
secondary structure alignment data of ITS2
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Discussion
• ITS sequences- high species-specific homogeneity.
• Primary sequence analysis close relationship between query sequence (P. westermani
from India) and isolates of related species from
neighbouring countries.
• Secondary structure analysis provided additional information for correct identification of
the species.
 confirmed the results from primary sequence analysis.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
• ITS sequence motifs
 All the sequence motifs were available in all the
Paragonimus sequences of different geographical isolates
under study.
 Validation of motifs showed high percent identity and low
E-value scores.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Conclusion
• The Paragonimus species prevalent in the region is in fact
Paragonimus westermani, the most common lung fluke
throughout the globe.
• ITS2 sequences:-
- reliable tool to identify
relationships
species and
phylogenetic
- potential as species markers.
• Different geographical isolates of Paragonimus spp need
further study with additional molecular markers and barcoding
to ascertain intra-specific strain variations, if any.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore
Acknowledgements
 DST, DBT, CSIR (GOI)- for Travel Fellowship for InCoB2009.
 DIT Project to VT.
 AICOPTAX programme (MoE&F, GOI) to VT
 DBT Project to VT & AC.
 DSA programme (UGC-SAP) in Zoology;
 UPE (Biosciences) programme in School of Life Sciences,
NEHU, Shillong.
 Co-ordinator, Bioinformatics Centre, NEHU.
8th International Conference 0n Bioinformatics, 7 – 11 September 2009, Biopolis, Singapore