Embed Size (px)
Citation preview
Studying Hemagglutin Protein Interactions in Botulinum Neurotoxin (BoNT) complex
PHI LL IP LWI N
L I A N A A B SA M A D
M ELI SSA GUZ M A N M OR R I S
DR . WEI - JEN L I N
What is Botulism?Cause by Clostridium botulinum Gram (+), rod-shaped spore forming bacterium
Strictly anaerobic
Consumption of toxin pre-formed in food
Produces different toxin type A, B, C, E, F, G
BoNT causes muscles paralysis Fatality rate 30-65% if not treated promptly
Lethal dose: ~1 ng/kg
1 gram can kill 20 million people
Currently only anti-toxin is available for treatment
BoNT Mechanism
• SNARE proteins form complex and allow vesicle docking
• neurotransmitter is released• Muscles is contracted
• Botulinum neurotoxins prevents the formation of SNARE complex
• Block neurotransmitter release• Muscle is paralyzed
NTNH -
HA19 -
HA52 -
HA34 -
Complex
HA17 -
Pure toxin
- + - + (reduced)
BoNT -
Type A neurotoxin forms complexes of various sizes with 5 associated non-toxic proteins (ANTPs):• Non-toxic non-hemagglutinins (NTNH)• Hemagglutinins (HA)
BoNT Complex
What are the roles of the complex?The Nontoxic Proteins Stabilize the Neurotoxin in-vitro
1. Protect the neurotoxin from intestinal enzymes and acid digestions
2. Help absorption of neurotoxin by intestinal epithelial cells
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 hr 2 hr 8 hr 24 hr 48 hr 72 hr 120 hr
Time in serum
BoN
T/A
activity (
A450)
Toxin comlex
Toxin alone
(Ito et al. 2011; Matsumura et al. 2008; Sugawara et al. 2010; Fujinaga et al. 2012). Lin et al.
C. botulinum C and D
bont/C1 or D
bont/ A or B
bont/ A or F
bont/G
bont/ E
ntnh
ntnh
ntnh
ntnh
ntnh
ha33ha17ha70botR
botRha34ha17ha70
p47botRorfX1orfX2
botRha17ha70
p47orfX1orfX2
C. botulinum A1 and B
C. botulinum A2 and F
C. argentinense (Type G)
C. botulinum E, C. butyricum
tetxtetRC. tetani
BoNT Complex Gene OrganizationGenes of Different Serotypes
1. BoNT assembly together by measuring the protein-protein interactions between hemagglutinins using Yeast-two-hybrid system
2. Expression and purification of hemagglutinin proteins in-vitro
Objectives
Yeast-Two-Hybrid System
• Strongest interactions between BD-HA17 and AD-HA52
BD-HA in Y2H
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.900
OD
595
day 1 day 2 day 3 day 4
day 1 0.003 0.001 0.003 0.006 0.002 0.006 0.001 0.001 0.003 0.001 0.001 0.000 0.000 0.002 0.016 0.001 0.003
day 2 0.002 0.001 0.003 0.005 0.004 0.040 0.003 0.003 0.003 0.001 0.001 0.000 0.000 0.004 0.507 0.003 0.009
day 3 0.003 0.001 0.004 0.005 0.004 0.722 0.006 0.004 0.005 0.001 0.001 -0.001 0.002 0.008 0.724 0.007 0.009
day 4 0.003 0.001 0.020 0.005 0.004 0.784 0.025 0.005 0.006 0.001 0.001 0.000 0.002 0.010 0.788 0.014 0.011
neg ha33 ha19 ha17 neg ha52 ha33 ha19 ha17 neg ha52 ha33 ha17 neg ha52 ha33 ha19
BD-HA52 BD-HA19BD-HA33 BD-HA17
• HA17 has the highest affinity to C-terminal (fragment 5) of HA52• Protein Combinations: Bont/LC, Bont/Hn, Bont/Hc, NTNH, HA52,
HA33, HA19, HA17
1. Measuring protein-protein interactions between hemagglutinins through Yeast-two-hybrid system
2. Expression and purification of hemagglutinin proteins in-vitro
Objectives
Transforming HA gene into E. coli
https://www.google.com/patents/US20030022179
HA17/52 in pCR vector
Primer Design + PCR
Enzyme Double Digestion
Plasmid Extraction
Ligation into expression pET vector
Transformation into Competent cells
Protein expressioncontrol = HA17/52 in E.coli (in pCR2.1)control = cells from control plate
PCR screening
Future studies • Express and purification of the recombinant protein
• Confirm and detect the interaction of the HA protein
• Mechanism of hemagglutinin role in neurotoxin absorption by intestinal epithelial cells
• Dr. Wei-Jen Lin
• Dr. Junjun Liu
• Grad Students
• Liana Ab Samad
• Melissa Guzman Morris
Lab Members• Justin Lee
• Ann Nasongkla
• Ashley Magin
• Stefan Riedel
• Marina E. De’Leon
• Kevin Costello
• Danielle K. Valencia
• Mahjabeen Ahamed
Acknowledgements
Questions?
