Evolution and pathogenicity in the
deadly chytrid pathogen of amphibians
Erica Bree Rosenblum
UC Berkeley
Emerging infectious disease
“EID events have risen
significantly over time
after controlling for
reporting bias…”
# EID events
Emerging infectious disease
100
50
0
1940
Decade
- Jones et al. Nature 2008
Host
1970
Pathogen
Environment
Why are EID events on the rise?
2000
EIDs can be due to changes in…
Disease outcome depends on
host-pathogen-environment interaction
The Pathogen
The Host
The Environment
Novelty in the microbial pathogen world
But what is novelty?
Novelty in the microbial pathogen world
* Novel pathogens don’t just appear - they evolve
Importance of studying a pathogen’s
evolutionary history
Novelty in the microbial pathogen world
* Novel pathogens can be novel in multiple ways
(changes in host range, changes in virulence, etc)
Importance of studying novelty at
multiple levels
Novelty in the microbial pathogen world
Evolutionary and functional novelty in an
emerging fungal pathogen of amphibians
Bd and amphibian declines
Bd and amphibian declines
Bd and amphibian declines
Frog photo: Joel Sartore
Bd and amphibian declines
Bd occurs on every continent with
amphibians, infecting >500 species
Published occurrence
Unpublished occurrence
Map courtesy of www.spatialepidemiology.net
Bd and amphibian declines
Genetic and spatio-temporal data demonstrate
that Bd is a novel, emerging pathogen, which has
spread around the world quickly
Bd and amphibian declines
Chytrids are basal fungi and are mostly sapbrobes
Bd is the only chytrid that infects vertebrates
Ascomycota
Basidiomycota
Zygomycota
Chytridiomycota
Metazoa
Bd and amphibian declines
Bd kills frogs by disrupting the structure
and function of their skin
Bd may also have an immune evasion/
suppression strategy
Rosenblum et al. 2009 PLoS ONE
Rosenblum et al. 2012. Molecular Ecology
Novelty in the microbial pathogen world
But there are persistent unanswered questions
about the origin and spread of Bd and the
interaction between Bd and its amphibian hosts
Where did Bd come from and
what makes it so deadly?
Genomics in non-model species
Many of our questions cannot be answered
using ecological approaches, and we cannot
manipulate Bd to use cellular approaches
So we are using genomics approaches to
understand Bd evolution and pathogenicity
Genomics in non-model species
Genomics and EIDs
With technological advances we can
conduct genomic studies even for timecritical studies in non-model species
Genomics and EIDs
AGTCGTAGCCGCTATC!
Comparative genomics
AGTCGTAGCCGCTATC!
AGCCGTAGCCGCTATG!
AGCCGTAACTGCTTTG!
CGCTGCAACTGCTTTG!
Functional genomics
CGCTTCAACTGCTTTG!
DNA
transcription
RNA
Gene expression:
(microarrays, RNAseq)
Using genomics to understand novelty
Understanding evolutionary and
functional novelty in Bd
Using genomics to understand novelty
Understanding evolutionary and
functional novelty in Bd
Novelty at the phylogenetic level
“It” vs “They”?
Bd has a single species name, but
there may be important genetic
and/or functional variation
Novelty at the phylogenetic level
Sequence genomes of 28 Bd isolates from
around the world and the genome of the
closest known non-pathogenic chytrid
7
6
1
4
1
8
1
JGI genome project backbone, Illumina resequencing ~30x coverage per isolate,
~25Mb genomes
Novelty at the phylogenetic level
Dummy Data: Expectations given Bd’s
recent discovery and spread
*
*
*
*
N. America (West)
N. America (East)
Latin America
Asia
Africa
* Bullfrog isolate
Novelty at the phylogenetic level
LFT001_10
UM142
LOH
1: 0.6
Rooted tree
based on
>100,000 SNPs
LOH
5: 1.0
0.07
* * JEL627
CLFT021
CLFT023
LOH
1: 2.4
N. America (West)
N. America
disomy (East)
Latin America
trisomy
Asia
tetrasomy
Africa
* Bullfrog isolate
CJB5.2
CJB7
JEL271
* JEL275
JEL433
CLFT024
EV001
JEL408
NCRC106979
LBAbercrom
JEL310
JEL427
JEL429
JEL289
SRS812
MexMkt
* Bd’s
evolutionary
history is more
complicated
than expected
No clear pointsource for
origin or linear
history of
spread
CLFT026
LOH
5: 1.3
JEL267
JEL359
JEL238
TST75
MLA1
CJB4
chromosome
1 2 3 9 1 1 1 1 5 8 7 1 6 1 4
4
1 2 0 5
3
Novelty at the phylogenetic level
LFT001_10
UM142
LOH
1: 0.6
Rooted tree
based on
>100,000 SNPs
LOH
5: 1.0
0.07
* * JEL627
CLFT021
CLFT023
LOH
1: 2.4
N. America (West)
N. America
disomy (East)
Latin America
trisomy
Asia
tetrasomy
Africa
* Bullfrog isolate
CJB5.2
CJB7
JEL271
* JEL275
JEL433
CLFT024
EV001
JEL408
NCRC106979
LBAbercrom
JEL310
JEL427
JEL429
JEL289
SRS812
MexMkt
No geographic
or host specific
population
structure
Confirms rapid
spread and
broad host
range
* CLFT026
LOH
5: 1.3
JEL267
JEL359
JEL238
TST75
MLA1
CJB4
chromosome
1 2 3 9 1 1 1 1 5 8 7 1 6 1 4
4
1 2 0 5
3
Novelty at the phylogenetic level
LFT001_10
UM142
LOH
1: 0.6
Rooted tree
based on
>100,000 SNPs
LOH
5: 1.0
0.07
* * JEL627
CLFT021
CLFT023
LOH
1: 2.4
N. America (West)
N. America
disomy (East)
Latin America
trisomy
Asia
tetrasomy
Africa
* Bullfrog isolate
CJB5.2
CJB7
JEL271
* There are 2
highly
divergent Bd
lineages
JEL275
JEL433
CLFT024
EV001
JEL408
NCRC106979
LBAbercrom
JEL310
JEL427
JEL429
JEL289
SRS812
MexMkt
Tree has more
structure than
expected
* CLFT026
LOH
5: 1.3
JEL267
JEL359
JEL238
TST75
MLA1
CJB4
chromosome
1 2 3 9 1 1 1 1 5 8 7 1 6 1 4
4
1 2 0 5
3
Novelty at the phylogenetic level
LFT001_10
UM142
LOH
1: 0.6
Rooted tree
based on
>100,000 SNPs
LOH
5: 1.0
0.07
* * JEL627
CLFT021
CLFT023
LOH
1: 2.4
N. America (West)
N. America
disomy (East)
Latin America
trisomy
Asia
tetrasomy
Africa
* Bullfrog isolate
CJB5.2
CJB7
JEL271
JEL275
JEL433
CLFT024
EV001
JEL408
NCRC106979
LBAbercrom
JEL310
JEL427
JEL429
JEL289
SRS812
MexMkt
CLFT026
LOH
5: 1.3
* JEL267
JEL359
JEL238
TST75
MLA1
CJB4
chromosome
Basal lineage
with isolates
from Latin
America
Large clade
with most of
the global
diversity
* Likely more
diversity to be
discovered with
more sampling
1 2 3 9 1 1 1 1 5 8 7 1 6 1 4
4
1 2 0 5
3
Using genomics to understand Bd novelty
Understanding evolutionary and
functional novelty in Bd
Chytrid comparative genomics
Massive expansions of gene families in Bd
Ascomycota
Basidiomycota
Zygomycota
Chytridiomycota
Metazoa
Bd is a unique
chytrid with
functions no other
chytrid has acquired
Chytrid comparative genomics
Massive expansions of protease gene families in Bd
Fungalysin metallopeptidase
Serine protease
Rosenblum et al. 2008 PNAS
Chytrid functional genomics
vs
Zoospores
Sporangia
vs
Lab broth
Frog skin
Chytrid functional genomics
Many proteases are induced by exposure to host tissue
Fungalysin metallopeptidase
Serine protease
zoospore
sporangia
frog skin
Chytrid comparative genomics
When did the gene family expansions occur?
Ascomycota
Basidiomycota
Zygomycota
Chytridiomycota
Metazoa
Chytrid comparative genomics
Chytrid comparative genomics
Confirmed that Hp does not degrade host tissue
Negative control
Hp treatment
Bd treatment
Joneson, Stajich, Shiu, Rosenblum. 2010. PLoS Pathogens
Chytrid comparative genomics
Demonstrated that the dramatic protease
gene family expansions are recent and
mostly Bd-specific
Fungalysin Serine Aspartyl
Crinkler
peptidase protease protease
Joneson, Stajich, Shiu, Rosenblum. 2010. PLoS Pathogens
Using genomics to understand Bd novelty
Genomics approaches have been key for:
Understanding Bd’s complex history &
identifying key evolutionary transition points
Identifying candidate Bd pathogenicity
factors & understanding Bd’s novel functions
Understanding evolution of pathogens and
mechanisms of pathogenesis can inform
strategies for addressing microbial threats
Acknowledgements
Key Rosenblum Lab Personnel
Thomas Poorten
Suzanne Joneson
Jamie Voyles
Lydia Gentry
Karen Pohl
Image: Tom Poorten
NSF-NIH EID Program (EF-0723871)
NIH COBRE Program (P20 RR016448-07S2)
NSF CAREER Program (DEB-1054062)
Additional Collaborators
Jason Stajich
Shin-Han Shiu
Timothy James
Kelly Zamudio
Katy Richards-Hrdlicka
Dan Ilut
David Rodriguez
Michael Eisen
Matt Settles
Joint Genome Institute