Transcript Developing aptamer sensors for Bacterial Kidney Disease of

Developing aptamer sensors for
Bacterial Kidney Disease of
salmonids
Emily Byrd
Berglund 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 nonlethally 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 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)
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
Fraction 8
Fraction 7
Fraction 6
Fraction 5
Fraction 4
Flow through
Eluted supernatant
Eluted beads
Bound beads
Wash #1
Cell lysate
Protein ladder
R2c protein gel
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
90 bp RNA aptamer
N = random nucleotide
constant
N40
constant
• RNA is run through several SELEX rounds to
isolate the most effective binding species
constant
1014-1015
Random
Sequence
RNA’s
N40
constant
GST-MSA
Bind RNA pool to protein
Bind RNA to immobilized protein
RT-PCR and transcribe
to amplify RNA
Remove unbound RNA
Elute bound RNA
A potential detection scheme
• Use colorimetric approach based on gold
nanoparticles
• Gold nanoparticles change color based on their
aggregation state
– red - dispersed
– blue/purple –
aggregated
• RNA/nano complexes
bound to MSA will turn
blue in color
Liu, J., Lu, Y. 2006. Preparation of aptamer-linked
gold nanoparticle purple aggregates for
colorimetric sensing of analytes. Nat. Protoc.1,
246-252.
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:
–
–
–
–
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
Chelsie Fish