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Evolution and function
of eukaryotic-like proteins
in bacterial sponge symbionts
Ana I. S. Esteves, Mary Nguyen, David Reynolds, Michael Liu, Lu Fan & Torsten Thomas
Centre for Marine Bio-Innovation &
School of Biotechnology and Biomolecular Sciences
University of New South Wales
Sydney, Australia
9th World Sponge Conference
6th November 2013, Fremantle - Australia
Lessons from sponge microbiology:
 Sponges feed on bacteria
 Sponges establish symbiotic relationships with bacteria
The bacterial paradox:
...or are they friends?
So are Bacteria food...
“Thus, the question is raised: are sponges able to recognise their
microbial symbionts or is recognition as food material and
phagocytosis prevented by protective extracellular layers around
the microorganisms?”
Wilkinson (1978) Marine Biology 49, 177-185.
Metagenomic functional characterisation of
planktonic and sponge-associated bacteria
Tropic of Capricorn
The Great
Barrier Reef
Sydney
n=3 plus seawater
1. Cell fractionation
2. Metagenomic
shotgun sequencing
(Titanium FLX; ~750K reads per n)
3. Assembly,
Filtering and
Annotation
Sponge symbionts have abundant
eukaryotic-like proteins (ELPs)
Ankyrin Repeat (ANK)
protein
motif
Leucine-Rich-Repeat (LRR)
protein
motif
** p<0.01; * p<0.05
Tetratricopeptide Repeat (TPR)
protein
motif
Fibronectin type 3 domain (FN3)
protein
motif
A quick guide to ELPs
 Repeat domains
Found in eukaryotic transcriptional initiators,
cell cycle regulators, cytoskeletal proteins,
ion transporters and signal transducers
Michaely et al. EMBO J. 2002
Involved in protein-protein interactions
 Function in bacteria
largely unknown
 Abundance increased
in symbiotic bacteria
** p<0.01; * p<0.05
Fan et al. PNAS USA 2012
Phylogeny of ankyrin-repeat proteins (ARP) from an
uncultured γ-proteobacterium of C. concentrica
SSA = sponge symbiont ankyrin
Some ARPs are more closely
related to sponge proteins than to
any other known protein
Horizontal gene transfer?
Nguyen et al. Molecular Ecology 2013
Function of ELPs:
a recombinant model for phagocytosis
Fosmid-clone from γ-proteobacterium
of C. concentrica
Subcloned individually into pBAD in gfp-E. coli (“symbiont”)
Expose to amoeba Acanthamoeba castellanii (phagocytic “host”)
no SSA
with SSA
Bacterial persistence in amoeba
ssa genes cloned individually
% of amoeba containing bacteria
Tukey’s test (n=9): ** p<0.01; * p<0.05
Average intracellular bacteria/amoeba
Nguyen et al. Molecular Ecology (accepted)
Number of extracellular bacteria is not significantly different between control and pBAD-SSA clones
High number of amoeba with many intracellular bacteria due to
prolonged persistence and survival
ARPs interfere at various steps of phagocytosis
Phylogeny and localisation of γ-proteobacterium in C. concentrica
endosymbiont of marine
wood-boring bivalves
uncultured
red: EUB338
yellow: γ-symbiont
FISH of C. concentrica tissue
• symbiont lives in close association with sponge cells
Effect of other ELPs classes
neg. control
Percentage of amoeba containing bacteria
(from in silico datasets)
Tukey’s test (n=6): ** p<0.01; * p<0.05
Reynolds et al. unpublished
Current model of ELP function
lysosome
phagosome
pH
ELPs
phagocytic cell
(e.g. amoebocyte)
bacteria/ symbiont
Function and evolution of ELPs
 Bacterial symbionts from sponges contain high abundance and diversity of ELPs
 ELPs have likely been acquired through HGT, possibly from the sponge host
 ELPs from sponge symbionts modulate phagocytosis
Importance for survival and proliferation in sponge?
Model:
Alteration of
host phenotype
eukaryotic
host lineage
horizontal
gene transfer
microbial
symbiont lineage
mutation of
acquired gene
transfer of ELP
expression
of ELP
Torsten Thomas & Group
Nicole Webster
Rachel Simister
Cheers, mateys!