Beyond Phylogeny: Evolutionary analysis of a mosaic pathogen Dr Rosalind Harding Departments of Zoology and Statistics, Oxford University,UK
Research Collaborators • Naiel Bisharat • Dept of Epidemiology and Preventative Medicine, Tel Aviv University, Israel • Derrick Crook • Nuffield Dept of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, UK • Martin Maiden • Dept of Zoology, University of Oxford Bisharat et al. (2005) Hybrid Vibrio vulnificusEmergInfectDis 11:30-35
Population Genetics • Interplay of micro-evolutionary processes • Mutation and recombination • Population structure and demography • Natural selection • Questions and strategy concern: • Understanding steady-state patterns of diversity • Learning about ancestral history (genealogy) • Understanding dynamics: emergence of new strains • Major technical problem • Trees don’t show recombination events
Vibrio vulnificus • Globally wide-spread inhabitant of marine and estuarine environments • Dangerous waterborne pathogen: case fatality rate for V. vulnificus septicemia may reach 50% • Typically, cases of V. vulnificus infection are sporadic • Human infection acquired through eating contaminated raw or undercooked sea food, or via contamination of wounds by seawater or marine animals
Disease Outbreak in Israel • Major outbreak of systemic V. vulnificus infection among fish market workers and fish consumers • Epidemiology • 1995: first case • 1996: 32 patients • 1997: 30 patients • all handled fresh Tilapia fish cultivated in inland fish farms • 1998: marketing policy changed to prevent sale & handling of live Tilapia fish • New biotype identified • Distinctive biochemistry, eg salicin-negative, lactose-negative (5 atypical characteristics for the species).
V. vulnificus diversity • Biotype 1: sampled from environment, healthy fish, shellfish etc; associated with sporadic human infection • Biotype 2: associated with disease in eels • Biotype 3: new cause of human disease outbreak in Israel. • Where did Biotype 3 come from? Biotypes have been defined based on biochemical tests of phenotype.
Initial genetic analysis • MLST: multi-locus sequence typing • Sequences of fragments of ‘housekeeping’ genes (dN/dS ratios < 1.0) • 10 genes, 5 from each of the two chromosomes, each fragment ~400 bp • Concatenated sequence of 4,326 bp defines sequence types (STs) • Isolates: • Biotype 1: n=82 isolates (39 from human disease, 43 from environment • Biotype 2: n=15 isolates (13 from eels) • Biotype 3: n=61 isolates (60 from human disease, 1 from fish-pond water)
11- Environment (Denmark) 76 65- Environment (Germany) 13- Environment (Denmark) 19- Human (USA) 59- Healthy fish (Israel) 17- Oyster (USA) 49- Environment (Germany) 44- Environment (Germany) 6- Diseased eels (Spain, Japan, Sweden, Taiwan),a 12- Environment (USA) 97 66- Environment (Germany) 9- Diseased eels (Denmark),b 47- Sea water (Japan) 41- Oyster (USA) 62- Oyster (USA) 35- Oyster (USA) 15- 1(Environment), 1(human) (USA) 51- Oyster (USA) 24- Oyster (USA) 29- Human (USA) 43- Human (Germany) 26- Oyster (USA) 78 28- Oyster (USA) 39- Oyster (USA) 48- Diseased eel (Denmark)b 53- Oyster (USA) 63- Oyster (USA) 10- Diseased eel (Denmark)b, healthy fish (Israel) 30- Oyster (USA) 38- Oyster (USA) 31- Oyster (USA) 94 52- Human (USA) 99 27- Oyster (USA) 34- Oyster (USA) 23- Oyster (USA) 54- Oyster (USA) 25- Oyster (USA) 4- Environment (USA) 3- Human (USA) 80 16- Human (USA) 22- Oyster (USA) 8- Human (61), healthy fish (1) (Israel) 45-Environment (Germany) 84 57- Human (Spain) II 88 14- Environment (Denmark) 61- Human (Sweden) 69- Shrimp (Indonesia) 70- Human (Sweden) 1- Human (USA) 70 72 2- Human (USA) 84 56- Human (South Korea) 77 68- Human (Sweden) 55- Human (Singapore) 46- Human (Japan) YJ016- Human (China) 18- Human (USA) 67- Human (Japan) 40- Human (USA) 32- Human (4), oyster (1) (USA) 100 82 42- Human (USA) 5- Environment (Spain) 58- Healthy fish (Israel) 20- Human (USA) 50- Human (Singapore) 7- Shrimp (Thailand) CMCP6- Human (South Korea) 60- Oyster (USA) 21- Human (USA) 64- Human (USA) 36- Human (USA) 98 33- Human (USA) 99 37- Human (USA) Vibrio parahemolyticus 0.002 UPGMA tree of concatenated sequences of 10 genes: two major groups: I & II, plus ST8 I ST8=Biotype 3 All Biotype 3 isolates were identical at level of MLST resolution.
Genetic differentiation into two ‘populations’ is not explained by geographic location of isolates Output from STRUCTURE analysis, assuming K= 3 populations
Genetic differentiation into two ‘populations’ is not explained by biotype distribution. Biotype 1 occurs in both populations However, Biotype 3 does have a distinctive intermediate genetic identity between the populations. Biotype 3 Output from STRUCTURE analysis, assuming K= 3 populations
Two populations: different disease associations Population B is associated with disease in humans Population A is associated with eel disease Output from STRUCTURE analysis, assuming K= 3 populations UPGMA Group II UPGMA Group I
Biotype 3 is a hybrid between parents from Population A and Population B Inferred ancestry
Biotype 3 is a mosaic genome A I II B
Clonal expansion of Biotype 3 Maynard Smith, J et al (2000) BioEssays 22:1115-1122 Disease outbreak clones emerge from a background of low frequency variation connected by mutation and recombination.
Progress summary • The disease outbreak in Israel (Biotype 3) was caused by a clonal expansion of Sequence Type 8 • ST 8 is a mosaic sequence created by recombination between parents from Populations A and B • Next questions • How much recombination? • How did the genetic differentiation between Populations A and B arise? • Population A = UPGMA Group I = Eel disease associated • Population B = UPGMA Group II = Human disease associated
Splits graph of concatenated sequences from 10 genes Cluster I = Population A Association with eel disease (biotype 2) ST8 = Biotype 3 Cluster II = Population B Association with human disease
Recombination exchange between groups I & II is rare Splits graph of allelic sequences from glp gene I ST8 (Biotype 3) has a glp allele from Population B/group II II Alleles 12 and 38 from Cluster II STs are more closely related to Cluster I
Recombination rates within genes within groups are high • Evidence of recombination from Beagle: www.stats.ox.ac.uk/~lyngsoe/beagle Ancestral history is not as simple as a tree. Minimum of 9 recombination events Splits graph of alleles from dtdS gene II I
Next Question. Polymorphism for a complex trait? • Is the genetic differentiation related to pathogenicity phenotype? • higher odds for causing either human or eel disease
Isolation in a metapopulation? Is the genetic differentiation caused by isolation between populations?
Any clues from diversity in individual genes? • If polymorphism, perhaps expect differentiation to localise to one or a subset of genes? • If differentiation is due to isolation between populations, expect all genes to show the same patterns.
USA-Env USA-ENV Denmark-EEL Israel-Env Denmark-Env Baltic Sea USA-Env USA-clinical USA-Env USA-Env USA-Clinical USA-Env Balticsea Balticsea Japan-EEL Denmark-eel Denmark-Env USA-Env Japan-Env USA-Env Germany-Clinical USA-Env USA-Env USA-Env USA-Env Denmark-EEL USA-Env USA-Env USA-Clinical USA-Env USA-Env USA-Env USA-Env USA-Env Baltic sea USA-Env USA-Env USA-eNV USA-Clinical USA-Clinical USA-Env Israel-Clinical Denmark-Env Spain-Clinical Baltic sea Sweden-Clinical USA-Env -S.Korea-Clinical Japan-Clinical Indonesia-Env USA-Clinical Israel-Env Spain-eel farm Singapore-Clinical USA-Clinical Sweden-Clinical USA-Clinical USA-Clinical USA-Clinical USA-Clinical Thailand-Env USA-Clinical USA-Clinical USA-Clinical Japan-Clinical Taiwan-Clinical USA-Clinical Singapore-Clinical Sweden-Clinical S. Korea-Clinical USA-Clinical USA-Clinical 0.005 UPGMA group I (Population A) Biotype 3 In Biotype 3, genes 1, 2, 4, & 10 are from group II, i.e. human disease associated. UPGMA group II (Population B)
The same split is preserved across genes 1, 2, 4 & 10 1. Large chromosome: glp 4. Large chromosome: metG 2. Large chromosome: gyrB 10. Small chromosome: tnaA
But the same split is also preserved across the other 6 genes, e.g. 5. Large chromosome: purM 8. Small chromosome: pntA 9. Small chromosome: pyrC 6. Small chromosome: dtdS
Conclusions • Differentiation between populations is evident across all 10 genes. Recombination exchange between populations is rare across all genes. • Within populations: Large numbers of alleles related through recombination as well as mutation history • Isolation by distance? Polymorphism? • Recombination is key to generating diversity in Vibrio vulnificus
Clonal Expansion In expansions of clonal complexes, new mutations are evident before recombination. (Linkage disequilibrium due to selective sweep.) Differentiation is shaped by selection: clonal complexes emerge as new adaptations Meta-population structure Old population diversity generated by mutation and recombination is sustained. Differentiation is shaped by isolation: outbreaks emerge as new recombinants