Beyond Phylogeny: Evolutionary analysis of a mosaic pathogen

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Beyond Phylogeny Evolutionary analysis of a
mosaic pathogen

• Dr Rosalind Harding
• Departments of Zoology and Statistics, Oxford
  University,UK

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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 vulnificus
Emerg Infect Dis 1130-35
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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 dont show recombination events

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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

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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).

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Severe soft tissue infections/ Necrotizing
fasciitis
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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.
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Initial genetic analysis

• MLST multi-locus sequence typing
• Sequences of fragments of housekeeping genes
  (dN/dS ratios lt 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 n82 isolates (39 from human disease,
  43 from environment
• Biotype 2 n15 isolates (13 from eels)
• Biotype 3 n61 isolates (60 from human disease,
  1 from fish-pond water)

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UPGMA tree of concatenated sequences of 10 genes
two major groups I II, plus ST8
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ST8Biotype 3
All Biotype 3 isolates were identical at level of
MLST resolution.
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Genetic differentiation into two populations is
not explained by geographic location of isolates
Output from STRUCTURE analysis, assuming K 3
populations
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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
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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
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Biotype 3 is a hybrid between parents from
Population A and Population B
Inferred ancestry
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Biotype 3 is a mosaic genome
A
I
II
B
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Clonal expansion of Biotype 3
Maynard Smith, J et al (2000) BioEssays
221115-1122
Disease outbreak clones emerge from a background
of low frequency variation connected by mutation
and recombination.
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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

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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
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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
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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
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Polymorphism for a complex trait?
Next Question.

• Is the genetic differentiation related to
  pathogenicity phenotype?
• higher odds for causing either human or eel
  disease

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Isolation in a metapopulation?
Is the genetic differentiation caused by
isolation between populations?
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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.

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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)
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The same split is preserved across genes 1, 2, 4
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1. Large chromosome glp
4. Large chromosome metG
2. Large chromosome gyrB
10. Small chromosome tnaA
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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
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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

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• Meta-population structure
• Old population diversity generated by mutation
  and recombination is sustained.
• Differentiation is shaped by isolation outbreaks
  emerge as new recombinants

• 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