Developing aptamer sensors for Developing aptamer sensors for Bacterial Kidney Disease of Bacterial Kidney Disease of

salmonidssalmonids

Emily ByrdEmily Byrd

Berglund LabBerglund Lab

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

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

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?

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.

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

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

MSA proteinsFull 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)

Cloning MSA

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

R2c protein gel

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70 kDa

27 kDa

37 kDa R2c

protein

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

N40constant constant90 bp RNA aptamer N = random nucleotide

Bind RNA to immobilized protein

Remove unbound RNA

Elute bound RNA

RT-PCR and transcribe to amplify RNA

Bind RNA pool to protein

1014-1015

Random Sequence RNA’s GST-MSA

N40constant constant

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

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

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

Acknowledgements

Berglund Lab:Berglund Lab:– Andy BerglundAndy Berglund– Julien DiegelJulien Diegel– Amy MahadyAmy Mahady– Bryan Warf, Jamie Bryan Warf, Jamie

Purcell, Leslie Purcell, Leslie VanOs, Rodger VanOs, Rodger Voelker, Devika Voelker, Devika Gates, Paul BarberGates, Paul Barber

Sarah ServidSarah Servid

Anna Cavinato, Eastern Anna Cavinato, Eastern Oregon UniversityOregon University

SPURSPUR

Peter O’DayPeter O’Day

Chelsie FishChelsie Fish