Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization

Sophie Sanchez,1 Stéphane Hourdez,1 and François H Lallier1

1Equipe Ecophysiologie: Adaptation et Evolution Moléculaires, UMR 7144 CNRS UPMC, Station Biologique, Place Georges Teissier, BP 74, 29682 Roscoff Cedex, FranceCorresponding author.

Sophie Sanchez: sanchez@sb-roscoff.fr; Stéphane Hourdez: hourdez@sb-roscoff.fr; François H Lallier: lallier@sb-roscoff.fr Received June 8, 2007; Accepted September 24, 2007.

Erin Dougher &

Lauren Hartigan

Background

• Riftia pachyptila live around hydrothermal vents in the East Pacific Rise

• 2600 meters-depth• Gut-tubeworms who form

dense communities that are dependent on chemosynthetic primary production

• Adults lack a mouth, gut and anus

• They make up a major component of biomass in the deep-sea

Background• They have specialized tissue

called a trophosome that contains a symbiotic sulfide oxidizing bacteria

• They acquire bacteria by interacting with the environment via their skin and mouth during the larva stage

• As they mature bacteria become trapped in the trophosome (mesodermal tissue)

Goal of the Experiment

• To determine what molecular components were present in the various tissues of the worm body through SSH and PCR techniques

• They essentially used the equation (Bacteria DNA + Worm DNA) – (Worm DNA) = Bacteria DNA to discern species specific DNA sequences in the variety of bacteria involved in the Riftia, bacteria symbiosis

Table 6• Standard curve equation and efficiency (E) in the different tissues (calculated from one sample each time

• Transcript Branchial plume Trophosome Body wall•

• 18S y = -3.31x + 24.67E = 101 % y = -3.38x + 28.30 E = 98 % y = -3.29x + 24.55 E = 101 %• RpCAbr y = -3.24x + 39.13E = 103 % y = -3.37x + 51.40 E = 98 % y = -3.30x + 44.66 E = 101 %• RpCAtr y = -3.30x + 49.19 E = 101 % y = -3.21x + 38.84 E = 105 % y = -3.25x + 47.08 E =

103 %• MVP y = -3.35x + 39.63 E = 99 % nd y = -3.24x + 42.51 E = 104 %• Cathep y = -3.22x + 43.13 E = 104 % y = -3.33x + 47.41 E = 100 % y = -3.26x + 43.98 E =

102 %• ChPr y = -3.37x + 39.57 E = 98 % nd nd• 16S y = -3.31x + 29.86 E = 101 % y = -3.17x + 38.77 E = 107 % y = -3.43x + 34.80 E = 96 %• ccoxI y = -3.54x + 36.05 E = 92 % y = -3.42x + 40.74 E = 96 % y = -3.46x + 38.51 E = 94 %• ATPF1 y = -3.43x + 41.54 E = 96 % y = -3.41x + 44.95 E = 96 % y = -3.34x + 43.14 E =

99 %• MH nd y = -3.24x + 41.08 E = 103 % nd• TCR nd y = -3.27x + 41.34 E = 102 % nd• TRbwC27 nd y = -3.36x + 39.07 E = 98 % nd

Experimental Interest

• The plume is only organ in direct contact with sea-water

• The trophosome is the only organ in contact with the symbiotic bacteria

• The plume is strongly exposed to hydrogen sulfide and other toxic molecules (heavy metals-abundant in vent environment)

• But the trophosome is the one dealing with the symbiotic relationship not the plume

Methods

• Tissue samples were taken from the plume, body wall, and the trophosome

• The body was used as reference tissue to find specific proteins expressed in the gills (main metabolite exchange organ) and the trophosome (organ that houses the symbiotic bacteria)

Suppression Subtractive Hybridization (SSH)

• Takes PCR-based amplifications of complementary DNA fragments

• It removes double stranded DNA formed by hybridization between a control and test group so that only complementary DNA or genomic DNA is left in similar quantities

• Shows the variability of sequences and expressed by genes and transcripts– For example it allows us to see how many transcripts are

being copied and the genetic differences between species

Figure 3

Polymerase Chain Reaction (PCR)

• It used to amplify or single or a few copies of DNA

• It generates thousands or millions of copies of particular DNA sequences

Protein Degradation and Turnover

• Electron dense organelles structures are common in tissues of sulfide adapting marine annelids

• In order for the symbiosis to work the lysosomes have to maintain mitochondrial integrity in the harsh sulfide environment

Protein Degration and Turnover in Branchial Plume TIssue

• Cathespin transcript was expressed more so in the bronchial plume tissue than the body wall tissue and trophosome tissue because of its direct contact with the sea water

• Sulfide exposure poisons the mitochondria leading to irreversible depolarization and the presence of lysosomes helps degrade damaged mitochondria– Other similar degradation proteins were also found in

the plume tissue (Valosin-Containing Protein (VCP))

Hydroxlamine Reductase Protein

• Plays a role in nitrogen matabolism • Catalyses reduction of hydroylamine to form

ammonia using NADH• Sequences for this protein did not match the

Riftia tissue samples, but it did match the bacteria tissue sequences

• This could be due to contamination because the bacteria live so close to the plume

Major Vault Protein (MVP) Gene Expression

• Major protein component of vaults• These vaultes are involved with nucleocytoplasmic

transport of ribosome and/or mRNA• This showed up in branchial plume tissue and body

wall tissue but it was not detected in the trophosome tissue

• Presence of protein in branchial plume tissue may be used to temporarily stop toxic molecules from being processed

Chitinase Gene Expression

• Branchial plume specific transcript

• Involved in tube growth and tube shape modifications

• Produced in the body wall and the vestimentum

• Potential host defense against pathogens

Tissue-Specific Expression of Carbonic Anhydrase

• Carbonic Anhydrase converts CO2 and water to bicarbonate and protons

• There was a high abundance of RpCAbr transcript in branchial plume tissue compared to the trophosome and body wall tissue

• RpCAtr was found in high abundance in the trophosome tissue in comparison to the branchial plume tissue

Myohemerythrin, T-Cell Receptor and Unidentified Transcripts

• Myohemerythrin is an oxygen-binding protein that participates in the storage of oxygen in muscles– It can deprive bacteria of iron essential for growth

• Cellular recognition by T-Cells helps the trophosome function with the bacteria– TCR transcripts were found that code for T-Cell

receptors

Results• Four cDNA libraries were

produced1. Body wall subtracted

bronchial plume (BR-BW)

2. Bronchial plume subtracted body wall (BW-BR)

3. Body wall subtracted trophosome (BW-TR)

4. Trophosome subtracted body wall (TR-BW)

Results

• PCR analysis was done that showed Major Vault Proteins in use

• Carbonic anhydrase, cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and body wall tissues

• Carbonic anhydrase, myohemerythrin (putative T-Cell receptor), and one non identified transcript were highly specific of the trophosome tissue

Conclusion

• The PCR analysis matched Table 1 library which confirmed the existence of tissue-specific transcripts

• Even with all the research some sequences could not be identified.

• Further research would have to be done for those individual sequences in order to verify expression level transcripts in the different parts of the worm

References• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2175520/• http://invertebrates.si.edu/Features/stories/vestimentifera.html• http://foundation.total.com/environment/processes-and-dynamics-of-marine-

life/ongoing-projects/world-inventory-of-marine-biodiversity-census-of-marine-life-800069.html

• http://bioweb.uwlax.edu/bio203/s2007/rossing_jaco/introduction__the_worm_under_inv.htm

• http://en.wikipedia.org/wiki/Polymerase_chain_reaction• http://www.mbari.org/staff/vrijen/dives/sspring/sspgs/riftia.htm• http://publishing.cdlib.org/ucpressebooks/view?docId=kt167nb66r&chunk.id=

ch17&toc.id=ch17&brand=eschol• http://www.marine-genomics-europe.org/index2.php?rub=b&pid=419• http://en.wikipedia.org/wiki/Carbonic_anhydrase• http://en.wikipedia.org/wiki/Complementary_DNA• http://en.wikipedia.org/wiki/gDNA• http://en.wikipedia.org/wiki/polymerase_chain_reaction