Taplin Capstone

Characterization of a toll-interacting protein (TOLLIP) gene in the black abalone

(Haliotis cracherodii)

Cullen TaplinMay 16, 2008

Presentation Outline

• Background to abalone and withering syndrome• Introduction to the Toll-like Receptor Pathway• My research goal and objectives• Results• Future work and conclusions

Black Abalone

• Single shelled gastropods

• Found in intertidal zone at depths from 0 – 5 m

• Can reach shell sizes of 200 mm

Abalone Distribution in Western U.S.

• Black abalone are confined to the west coast of the U.S., extending from Coos Bay, Oregon to Cabo San Lucas, Baja California.

Black Abalone Declines

• Overharvesting

• Environmental Contaminants

• Climate change

• Disease Withering syndrome

1986

1988

1999

Withering Syndrome

• Caused by a bacterium which infects the post esophagus, followed by a change in tissue morphology of the digestive gland.

• The abalone is forced to rely on glycogen stores from the foot.

J. Moore

Healthy black abalone (left) and one suffering from withering syndrome (right).

Abalones’ Immune Response

• Abalones’ immune response is not well understood.

• Like all invertebrates, abalones only have an innate immune system.

• Several immune components are conserved among invertebrates, including toll-like receptors, lectins in non-self recognition, and the prophenoloxidase system.

Identifying Immune Related Genes

• Used comparative bioinformatic techniques

• Several immune related genes were found in black abalone.

• TOLLIP, an intermediate in the toll-like receptor pathway was chosen for study.

Toll-like Receptor Pathway• Recognizes molecules

and patterns produced exclusively by bacteria and viruses.

• It functions to signal the presence of a pathogen and produce proinflammatory cytokines.

Singh, Bhanu P., R. S. Chauhan, and Kokesh K. Singhal. 2003. Toll-like Receptors And Their Role In Innate Immunity. Current Science. vol. 85, no. 8, pgs. 1156 – 1164.

Research Goal & Objectives

• The goal of my research was to characterize the toll-interacting protein in black abalone.

• Objectives to accomplish the goal were:– Obtain more TOLLIP sequence information– Discover other TLR genes in black abalone.– Compare differential expression of TOLLIP– Correlate TOLLIP expression to bacterial loads

Abalone Used In Research

• Visually healthy black abalone were collected and maintained in Dr. Carolyn Friedman’s lab at the University of Washington.

• Control and bacterium exposed treatments were then constructed.

Research Methods• Tissues were isolated and RNA was

extracted via tri-reagent method.• cDNA was made via reverse

transcription.• qPCR was carried out using an Opticon 2

System and SYBR Green Fluorescent Dye.

• Data was analyzed with Real-time PCR Miner (www.miner.ewindup.info/miner/)

• Bacterial Loads were quantified via qPCR methods.

Partial TOLLIP sequence and comparison to the Pacific abalone (H. discus)Using primers designed from a TOLLIP expressed sequence tag from the Pacific abalone (gene accession #CX726806), partial sequence information was obtained for TOLLIP in black abalone and submitted to GenBank. Comparisons show 97.5% similarity.

Results: Sequence Information

TOLLIP Sequencing Results (cont.)

• Complete genomic sequencing continues.

• Preliminary evidence indicates the gene is over 10,000 bp long.

• Comparisons to the fully sequenced sea urchin genome indicates six introns.

10kbp –

4kbp –

2kbp –

800bp –

Results: Discovering Other TLR Genes

• Classifies genes by their functions.• Can predict the function of genes based on relationships.• Searched for genes within the TLR pathway.• Identified several other TLR genes in black abalone, including toll-like

receptor #9, NFκB and several TRAFs.

Protein Analysis Through Evolutionary Relationshipswww.pantherdb.org/

Results: TOLLIP Expression in Digestive Gland Tissue

Five months after initial exposure. N=8 for control groups, N=9 exposed groups. Means were not significantly different (p > 0.05).

Results: TOLLIP Expression in Gill Tissue

Eight months after initial exposure. N = 6 for both treatment groups. Means were not significantly different (p > 0.05).

Results: Correlation of TOLLIP Expression in and Bacterial Loads

No significant correlations between TOLLIP expression and bacterial loads (p > 0.05).

Summary of Results

1. Obtained TOLLIP coding sequence & isolated genomic DNA

2. Identified other TLR members3.4.

Data suggests the TLR pathway is active in gill tissue.

Future Work

• Explore other intermediates in the toll-like receptor pathway.

• Examine expression in other tissues; in particular, the post-esophagus.

• Compare laboratory results to field results.

Acknowledgements

• Dr. Carolyn Friedman and her lab staff.• Sam White• Dr. Steven Roberts• NOAA• Sea Grant California

Questions?