Please note:This is a scientific communication for personal

use only. All data is an intellectual property of Ana I. S. Esteves. Please do not copy or use without consent. For more info or to obtain

permission, please email me at aidsesteves@gmail.com

Thank you

Centre for Marine Bio-Innovation & School of Biotechnology and Biomolecular Sciences

University of New South WalesSydney, Australia

Ana I. S. Esteves, Mary Nguyen, David Reynolds, Michael Liu, Lu Fan & Torsten Thomas

9th World Sponge Conference 6 th November 2013, Fremantle - Australia

Evolution and function of eukaryotic-like proteins

in bacterial sponge symbionts

Sponges feed on bacteria

So are Bacteria food...

The bacterial paradox: Sponges establish symbiotic relationships with bacteria

...or are they friends?

Lessons from sponge microbiology:

Wilkinson (1978) Marine Biology 49, 177-185.

“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?”

Metagenomic functional characterisation of planktonic and sponge-associated bacteria

The Great Barrier Reef

Sydney

Tropic of Capricorn

n=3 plus seawater

1. Cell fractionation

3. Assembly, Filtering and Annotation

2. Metagenomic shotgun sequencing (Titanium FLX; ~750K reads per n)

Sponge symbionts have abundant eukaryotic-like proteins (ELPs)

Ankyrin Repeat (ANK) Leucine-Rich-Repeat (LRR)proteinmotif

proteinmotif

proteinmotif

proteinmotif

Fibronectin type 3 domain (FN3)Tetratricopeptide Repeat (TPR)** p<0.01; * p<0.05

A quick guide to ELPs

Michaely et al. EMBO J. 2002

Fan et al. PNAS USA 2012

** p<0.01; * p<0.05

Repeat domains

Involved in protein-protein interactions

Found in eukaryotic transcriptional initiators, cell cycle regulators, cytoskeletal proteins, ion transporters and signal transducers

Function in bacteria largely unknown

Abundance increased in symbiotic bacteria

SSA = sponge symbiont ankyrin

Phylogeny of ankyrin-repeat proteins (ARP) from an uncultured γ-proteobacterium of C. concentrica

Nguyen et al. Molecular Ecology 2013

Some ARPs are more closely related to sponge proteins than to

any other known protein

Horizontal gene transfer?

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% of amoeba containing bacteria

Nguyen et al. Molecular Ecology (accepted)Tukey’s test (n=9): ** p<0.01; * p<0.05

Average intracellular bacteria/amoeba

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

ssa genes cloned individually

ARPs interfere at various steps of phagocytosis

Phylogeny and localisation of γ-proteobacterium in C. concentrica

uncultured

endosymbiont of marinewood-boring bivalves

• symbiont lives in close association with sponge cells

FISH of C. concentrica tissue

red: EUB338yellow: γ-symbiont

neg.

con

trol

Perc

enta

ge o

f am

oeba

con

tain

ing

bact

eria

Tukey’s test (n=6): ** p<0.01; * p<0.05 Reynolds et al. unpublished

Effect of other ELPs classes (from in silico datasets)

Current model of ELP function

phagosome

lysosome

phagocytic cell(e.g. amoebocyte)

bacteria/ symbiont

pHELPs

Function and evolution of ELPs

eukaryotichost lineage

microbial symbiont lineage

horizontal gene transfer

mutation ofacquired gene

expressionof ELP

transfer of ELP

Alteration ofhost phenotype

Model:

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?

Torsten Thomas & Group

Nicole WebsterRachel Simister

Cheers, mateys!