inducers of heat shock protein 70: a new disease ... 5 - thursday... · ß-glucan + ßgbp...
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larvi 2013
ghent university, belgium, 2-5 september 2013
6th fish & shellfish larviculture symposium
Inducers of heat shock protein 70:A new disease preventive option in
aquaculture production systems
Kartik Baruah
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Inducer of Heat Shock Protein 70: A New Disease PreventiveOption in Aquaculture Production Systems
Dr. Kartik Baruah
Parisa Norouzitallab, Patrick Sorgeloos & Peter Bossier
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Abiotic Stressors Biotic Stressors
Larval Mortality
Diseases
Underdeveloped Immune System
Diseases
Bacterial diseases – Still a Threat to Larviculture
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Emphasis on PREVENTION which is likelyto be more cost effective than the CURE
Antibiotics – The Unsustainable Solution
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Strategies successfully applied inaquaculture
Green watertechnique
Probiotics
Immuno-stimulants Vaccines
Acidifiers
More alternatives required as no anti-infective techniqueseems to be able to solve every problem alone.
Alternative Solutions for Larval Diseases
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Multiple agents cause disease outbreak
Biotic Stressors
Larval Mortality
Shell(fish) health
Disease Outbreak
Abiotic Stressors
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Hsps are ubiquitous and highly conserved protein molecules,
available in all prokaryotic and eukaryotic cells.
Different types – sHsps, Hsp60, Hsp70, Hsp90, Hsp100.
Hsp70s form one of the major Hsp families, most extensivelystudied one.
Synthesized constitutively in the cells (heat shock cognate 70).
Induced after exposure to stressors (cold, O2 deprivation, etc).
What are Heat Shock Proteins (Hsps)?
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Function of Intracellular Hsp
Unfolded
Misfolded
Correctly folded protein
Hsp
Hsp
Inside the cell they act as molecular chaperones - bind to unfolded proteins(nascent polypeptides or denatured ones) - facilitate their refolding to thenative state.
Involved in protein translocation and degradation.
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Function of Extracellular Hsp
Due to necrosis, the Hsp70 gets released from the cell.
Extracellular Hsp70 serves as danger signal and modulates bothinnate and adaptive immune responses.
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Hsp70 Induction in Artemia : Non-Lethal Heat Shock (NLHS)
Gnotobiotic Artemia
Survival scored after 48 h
Challenged with vibrios at 107 cells/ml
NLHS at 37°C for 30 min followedby 6 h recovery period
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Results
• Correlation exists between enhanced protection and Hsp70 accumulation
Hsp70
Hsp70
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Heat shock is not an ideal way to enhance Hsps inaquaculture animals.
Is there any less traumatic approach to manipulate Hspsexpression within (shell)fish?
QUESTION ????
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Compounds extracted from plants
Can they induce Hsp70production in aquaculture
animals???
Can they induce protectionagainst abiotic stressors???
Can they induce protectionagainst pathogenic biotic
stressors???
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Compounds extracted from plants
Compound B
Compound A
Unpublished work in progress
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Axenic hatching
Pretreatment of axenic Artemia with compound
Pretreament of Artemia instar IIwith compound for 2 h (28°C)
Washing of Artemia withsterile sea water (28°C)
Given a recovery period of 2 h(28°C)
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Challenge with abiotic or pathogenic biotic stress
Thermal stress Vibrio challenge
Heat shock at 41°C for 20 min Challenged at 107 cells/mL
Survival scored after 6 h Survival scored after 48 h
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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RESULTS
Compound A
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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A
C C
B
C C
a
c
b
ab
c c
0
20
40
60
80
100
Positive control Negative control Comp A (2.5 ppm) Comp A (5 ppm) Comp A (10 ppm) Comp A (50 ppm)
Surv
ival
(%)
Treatments
V. campbellii V. harveyi
Protective effect of compound A against vibrios
Positive control: No pretreatment, no challenge.
Negative control: No pretreatment, challenged with Vibrio
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Protective effect of compound A against lethal heat shock
*
0
20
40
60
80
100
Control Compound A (5 ppm)
Surv
ival
(%)
Treatments
Control: No pretreatment
2.7-fold
Compound A conferred protection against vibrios and thermal stress coincides withHsp70 production
100
75
Marker Hela cells control Compound A
0
0,25
0,5
0,75
1
Hela cells Control Compound A
Rela
tive
indu
ctio
n of
Hsp
70
Treatments
Survival after thermal shock vs Induction of Hsp70
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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RESULTS
Compound B
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Protective effect of compound B against lethal heat shock
d d
c
b
a
d
0
20
40
60
Control 10 ppm 25 ppm 50 ppm 75 ppm 150 ppm
Surv
ival
(%)
Treatments
Control: No pretreatment
20%
45%
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Protective effect of compound B against V. harveyi
10%
46%
Survival after Vibrio challenge100
75
Marker Hela cells Control Comp B
vs Induction of Hsp70
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Pretreatmentfor 2 h Rinsing Recovery for
2 h
0 h
Experimental design for gene expression by RT-PCR
Challenged with V. harveyi for 24 h
6 h 12 h 24 h
Collection of samples for Hsp70 and prophenoloxidase (proPO) genes
Samples collected
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Effect of Compound B on Hsp70 and proPO genes
**
*
0
1
2
3
4
5
Control Compound B
Rela
tive
expr
essi
on o
f Hsp
70ge
ne in
Art
emia
Treatments
0 h 6 h12 h 24 h
Hsp70 gene proPO gene
*
*
0
1
2
3
4
5
Control Compound B
Rela
tive
expr
essi
on o
f pro
POge
ne inArtemia
Treatments
0 h 6 h12 h 24 h
Control: No pretreatment
0 h - Pretreatment without Vibrio challenge
6, 12 & 24 h: Pretreatment followed by 6, 12 & 24 h of Vibrio challenge
4.3-fold
2.3-fold
2.9-fold
1.9-fold
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Artemia genome
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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CONCLUSIONS
These plant-extracted compounds are potential inducersof Hsp70.
They induced tolerance against subsequent abiotic (thermal)stress.
They generated protective immunity in Artemia againstpathogenic biotic (vibrios) stressor by priming the proPO system.
These compounds represent potential preventive modality forvibriosis in Artemia and possibly in other commerciallyimportant aquaculture species.
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Thank you
BOF-Special Research Fund
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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CLARIFICATIONS
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Natural environment Gnotobiotic environment
Compound
Artemia(endogenous Hsps)
Microbial communities(bacterial Hsp)
V. campbelliichallenge
Filter
feeding
Interaction
Protective effect???
Compound
Artemia(endogenous Hsps)
V. campbelliichallenge
Protective effect dueto Hsps
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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LPS + LBP
Serine proteinase cascade
ß-glucan + ßGBP peptidoglycan + PGBP
Prophenoloxidase Phenoloxidase
Phenols
QuinonesMelanin
Prophenoloxidase Activation system
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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Experimental Model System
Host Environment
Differentmicro organisms
Complex system
Host
Targetmicro organisms
Gnotobiotic system
Artemia
Title: Inducer of Heat Shock Protein 70: A New Disease Preventive Option in Aquaculture Production Systems
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M HC C T C T
Muscle Gill