1. Developing aptamer sensors for Bacterial Kidney Disease of salmonids Emily Byrd Berglund Lab

2. Bacterial Kidney Disease Caused by Renibacterium salmoninarum (Rs) Common problem in fish hatcheries and farming industries Disease symptoms: –granulomas and postules on the kidneys –distended belly –exophthalmos –dark coloration of the skin

3. BKD: a problematic disease No vaccine exists; antibiotic treatments have minimal affect Transmitted horizontally and vertically –vertical transmission makes it difficult to prevent the disease Resilient bacteria that can both live and grow in the host’s immune system Bacteria grows slowly, making it difficult to culture and study

4. Goal Current detection methods include ELISA and FAT using kidney tissue Unable to detect non- lethally at subclinical levels Can we develop a nonlethal and noninvasive method that detects the bacteria?

5. How could infections be detected? SELEX is a method that selects for an RNA that binds tightly with the protein. Optical properties of nanoparticles may make it possible to detect bacteria in water samples.

6. MSA Renibacterium salmoninarum produces large quantities of an extracellular protein, p57, also known as Major Soluble Antigen or MSA Protein is acidic and large in size (57 kDa) Native p57 exists as a monomer Does not have a transmembrane domain, but is secreted and then bound to the exterior of the cell In vitro, p57 reassembles onto strains of R. salmoninarum lacking the protein Wiens and Kaattari, 1990

7. MSA and disease MSA is a virulence factor of Rs; it is secreted into the extracellular space and causes agglutination of host leucocytes Causes long term immunosuppression if present in fish eggs Causes agglutination of salmonid spermatozoa Restores cell surface hydrophobicity Forms fimbrial structures and functions as an adhesin for bacterial attachment to cellular receptors –may allow intracellular invasion by the bacteria Also acts to suppress antibody production by the host Wiens and Kaattari, 1990

8. MSA proteins Full Length (27-558) R1a (27-155) R2a (172-356) R2b (172-333) R2c (228-331) R3a (357-558) IPT Domain Protein was sectioned into three regions and constructs formed of varying lengths Regions 1 and 2 have been shown to be exposed on the cell surface when bound to bacteria (Wien and Kaattari, 1990)

9. Cloning MSA

10. Protein purification Two methods for protein purification GST bead purification: –protein bound to beads and eluted with glutathione Anionic exchange Q column: –negatively charged protein binds the positively charged column and is eluted off at high NaCl concentrations

11. R2c protein gel Fraction 4Fraction 5Fraction 6Fraction 7Fraction 8 Protein ladder Cell lysate Wash #1 Bound beads Eluted beads Eluted supernatant Flow through 70 kDa 27 kDa 37 kDa R2c protein

12. SELEX Method that isolates RNA aptamers that bind with high affinity to a protein (MSA) A 90 bp RNA with a 40 bp randomized sequence is created using PCR and transcription RNA is run through several SELEX rounds to isolate the most effective binding species N 40 constant 90 bp RNA aptamer N = random nucleotide

13. Bind RNA to immobilized protein Remove unbound RNA Elute bound RNA RT-PCR and transcribe to amplify RNA Bind RNA pool to protein 10 14 -10 15 Random Sequence RNA’s GST-MSA N 40 constant

14. A potential detection scheme Use colorimetric approach based on gold nanoparticles Gold nanoparticles change color based on their Liu, J., Lu, Y. 2006. Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes. Nat. Protoc.1, 246-252. aggregation state –red - dispersed –blue/purple – aggregated RNA/nano complexes bound to MSA will turn blue in color

15. To be continued… Finish purifying R3a and R2b protein fragments Begin SELEX on the proteins –begin with proteins spanning region 2 since the region appears to be exposed on the cell surface –locate the best RNA aptamer for binding Conjugate RNA aptamer with gold nanoparticles Develop a method to detect color changes in nanoparticles

16. Potential problems Radioactivity: no facilities at EOU –working to find other staining methods Full length construct doesn’t seem to be soluble; how can it be purified? No sensor for detecting color changes in gold nanoparticles

17. Acknowledgements Berglund Lab: –Andy Berglund –Julien Diegel –Amy Mahady –Bryan Warf, Jamie Purcell, Leslie VanOs, Rodger Voelker, Devika Gates, Paul Barber Sarah Servid Anna Cavinato, Eastern Oregon University SPUR Peter O’Day Peter O’Day Chelsie Fish Chelsie Fish