Please note:

1. 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

2. Evolution and function of eukaryotic-like proteins in bacterial sponge symbionts Centre for Marine Bio-Innovation & School of Biotechnology and Biomolecular Sciences University of New South Wales Sydney, Australia Ana I. S. Esteves, Mary Nguyen, David Reynolds, Michael Liu, Lu Fan & Torsten Thomas 9th World Sponge Conference 6th November 2013, Fremantle - Australia

3. 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...

4. “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.

5. Metagenomic functional characterisation of planktonic and sponge-associated bacteria The Great Barrier Reef Tropic of Capricorn Sydney n=3 plus seawater 1. Cell fractionation 2. Metagenomic shotgun sequencing (Titanium FLX; ~750K reads per n) 3. Assembly, Filtering and Annotation

6. Sponge symbionts have abundant eukaryotic-like proteins (ELPs) Ankyrin Repeat (ANK) Leucine-Rich-Repeat (LRR) protein motif protein motif Tetratricopeptide Repeat (TPR) Fibronectin type 3 domain (FN3) ** p<0.01; * p<0.05 protein motif protein motif

7. 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

8. Phylogeny of ankyrin-repeat proteins (ARP) from an uncultured γ-proteobacterium of C. concentrica SSA = sponge symbiont ankyrin Some ARPsare more closely related to sponge proteins than to any other known protein Horizontal gene transfer? Nguyen et al. Molecular Ecology 2013

9. Function of ELPs: a recombinant model for phagocytosis Fosmid-clone from γ-proteobacterium of C. concentrica Subcloned individually into pBADin gfp-E. coli(“symbiont”) Expose to amoeba Acanthamoebacastellanii(phagocytic “host”) no SSA with SSA

10. Bacterial persistence in amoeba ssa genes cloned individually Average intracellular bacteria/amoeba % of amoeba containing bacteria Nguyen et al. Molecular Ecology (accepted) Tukey’s test (n=9): ** p<0.01; * p<0.05 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

11. Phylogeny and localisationof γ-proteobacterium in C. concentrica endosymbiont of marine wood-boring bivalves uncultured red: EUB338 yellow: γ-symbiont FISH of C. concentricatissue • symbiont lives in close association with sponge cells

12. Effect of other ELPs classes(from in silicodatasets) Percentage of amoeba containing bacteria neg. control Reynolds et al. unpublished Tukey’s test (n=6): ** p<0.01; * p<0.05

13. Current model of ELP function lysosome phagosome pH ELPs phagocyticcell (e.g. amoebocyte) bacteria/ symbiont

14. 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 transfer of ELP microbial symbiont lineage expression of ELP mutation of acquired gene

15. Torsten Thomas & Group Nicole Webster Rachel Simister Cheers, mateys!