1. Introduction
Aims
Methods
Results
Conclusion
 Successfully cloned multimerised repeats of the dual Toll/IMD and Toll-specific
reporters.
 Demonstrated specificity of the five repeat Toll-specific reporter.
 Validated stimuli with the dual Toll/IMD reporter.
 No Toll induction observed.
 Extensive optimization in progress for the Toll-specific reporters.
Developing Luciferase Reporter Assays To Study Immune Responses In Mosquitoes
Andalus Ayaz, Kevin Maringer1, PhD, Ana Fernandez-Sesma1, PhD
1 Department of Microbiology, Icahn School of Medicine at Mount Sinai
• Each reporter transfected with transcription factors Rel1A, Rel2 or STAT
• Cells were harvested after 48 hours
• Western blots are currently in progress to confirm protein expression in cells.
1xWT: Toll/IMD dual reporter (1 repeat)
1xRel: Toll-specific reporter (1 repeat)
5xRel: Toll-specific reporter (5 repeats)
Future Directions
0
0.5
1
1.5
2
2.5
3
3.5
20% 10% 2% 20% 10% 2%
1x WT 1xRel1a
Foldinduction
Amounts of FBS in media
 Continue optimizing experimental conditions to see induction of the Toll-specific
reporters with bacterial and fungal stimuli
 Test Toll responses to viruses like dengue virus.
 Increase the sensitivity of the Toll-specific reporter by cloning multiple (5) repeats of the
promoter.
 Clone multiple (5) repeats of the dual Toll/IMD reporter as a control
 Verify the specificity of the Toll-specific promoter by over-expressing Rel1A and Rel2 in
Aag2 mosquito cells
 Validate the Toll-specific and the dual Toll/IMD reporter with well-characterized bacterial
and fungal stimuli.
*Gene Blocks (gBlocks) obtained from New England Biotechnologies
• Dual Toll/IMD or Toll-specific promoter fused
to firefly luciferase gene
• Upon promoter activation, the luciferase enzyme
gene is transcribed.
• Cells harvested and lysed in lysis buffer.
• A light signal is emitted when the cells are
exposed to the luciferase substrate.
• Renilla luciferase plasmid is used for normalization.
• Successfully cloned 5 repeats of the Toll-specific
reporter (Fig.5 and 6)
• Successfully cloned 4 repeats of the dual Toll/ IMD reporter.
• The 5-repeat dual Toll/IMD reporter is still in progress.
Demonstrated Specificity Of The Toll-specific Reporter
• C.Striatum, S.aureus and L.cytomonogenes are Gram positive bacteria
• E.coli and Salmonella are Gram negative bacteria
• Zymosan is the fungal mimic
0
0.5
1
1.5
2
2.5
3
Rel1A Rel2 STAT
5xrel
Foldinduction
Transcription factors
0
0.5
1
1.5
2
2.5
Rel1A Rel2 STAT
1Xrel
Foldinduction
Transcription factors
0
0.5
1
1.5
2
2.5
3
3.5
4
Rel1A Rel2 STAT
1xWT
Foldinduction
Transcription factors
Rel1A: Transcription factor
for the Toll pathway
Rel2: Transcription factor
for the IMD pathway
STAT: Control for protein
over-expression
Arthropod borne (arboviruses) like dengue virus require invertebrate vectors like
mosquitoes for transmission to humans (Fig.1) (1). We know the mosquito’s immune
system acts as a strong barrier for viral transmission (2). However, not much is known
about arbovirus-immune interactions in mosquitoes due to a lack of tools.
The Sesma lab has been studying two NF-kB signaling pathways in mosquitoes, the
Toll and IMD pathways. The stimulation of these pathways by bacteria and fungi is well-
characterized (Fig.2) (2). These pathways are also believed to act in an anti-viral
capacity against viruses like dengue (3). During previous lab work, a Toll and IMD dual
reporter, which responds to both Toll and IMD signaling, and a Toll-specific reporter were
developed. The experiments showed that the reporters were specific and inducible by
bacterial and fungal stimuli. However, the Toll-specific reporter was weakly induced.
Fig. 2: The Toll
(purple) and IMD
(orange) pathways.
Rel1A is the
transcription factor
required for Toll
pathway induction.
Rel2 is the
transcription factor
required for IMD
pathway induction.
gBlock
Digested plasmid
Fig. 6: DNA gel showing the
multimerized Toll-specific reporter
fragments. The digests were loaded
proportionally to the fragment size.
DNA ladder is in b.p. on left
Vector
backbone
5 repeat
4 repeat
3 repeat
2 repeat
1 repeat
Panel A Panel B Panel C
6000
2000
1650
1200
850
400
gBlock
Fig.8: Toll/IMD dual reporter is inducible by all stimuli. The 1-repeat and 5-repeat Toll-specific
reporters did not show any induction.
0
5
10
15
20
25
C.Striatum
S.aureus
L.mono
E.coli
Salmon
Zymosan
C.Striatum
S.aureus
L.mono
E.coli
Salmon
Zymosan
C.Striatum
S.aureus
L.mono
E.coli
Salmon
Zymosan
1xWT 1xRel1A 5xRel1A
Foldinduction
Reporters under different stimuli
• Used single repeat of dual Toll/IMD and Toll-specific reporters.
• Used same procedure as Fig. 8. Used the fungal mimic, Zymosan, as stimuli.
Fig. 1:The arbovirus
transmission cycle.
References
In-Fusion
cloning with
synthetic
gBlocks*
Transformed
E.Coli with
reporter
plasmids
Picked
colonies
Purified
plasmids
Screened
by
digesting
plasmids
Ran diagnostic
DNA gel
Fig.3: Cloning
work flow (4)
Restriction sites for
enzymes, Sac-I and
Hind-III
Fig.7: Induction of different reporters with transcription factors. The dual Toll/IMD (1 repeat) reporter
is induced by the Toll and IMD pathways (Panel A). The Toll-specific (1 repeat) reporter responds to
Toll signaling only (Panel B). Toll specificity is retained in the 5-repeat Toll-specific reporter (Panel C).
Fig.5: Schematic of cloned reporters
Fig 9: Dual reporter shows increased
induction with increasing FBS while
Toll-specific reporter shows no
induction
Impact of modified experimental set-up on Toll induction
• Used same procedure as Fig. 8.
• Seeded different cell densities
0
5
10
15
20
25
30
2x10^5 1x10^5 5x10^4 2x10^5 1x10^5 5x10^4
1x WT 1x Rel
Foldinduction
Cell densities (cells/well)
0
1
2
3
4
5
6
2x10^5
1x10^5
5x10^4
2x10^5
1x10^5
5x10^4
1x WT 1x Rel1A
Foldinduction
Cell densities (cells/well)
Fig.11: Dual reporter shows increased
induction with increasing cell density. While
Toll-specific reporter shows no induction
Fig.12: Dual reporter shows increased
induction with increasing cell density while
the Toll-specific reporter shows no induction.
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
1xWT 1xRel1A 5xRel1a 1xWT 1xRel1A 5xRel1a
L-glutamine w/o L-glutamine
Foldinduction
Reporters with and without L-glutamine
Fig10: The dual reporter shows
decreased induction without L-
glutamine. The toll-specific reporters
show no induction in either condition
1. Weaver, S. & Barrett. A. (2004) Nat Rev Micro 2:789
2. Ruckert, C Bell-Sakyi, L Fazakerley, J.K. Fragkoudis, R. Antiviral reponses of
arthropod vectors: an update on recent advances Virus Disease 25(3):249-260
Surrey U.K.
3. Xi,Zhiyong Ramirez,J.L. Dinopoulos, G. The Aedes aegypti Toll Pathway Controls
Dengue Virus Infection PLoS Pathogens Pathog 4(7) e1000098 Baltimore,
Maryland, USA
4. Clontech Laboratories, Inc. Protocol No. TT5162-1 Version No. 072012
Cloning of multimerized luciferase reporters
Induction of reporters with different stimuli
Impact of modified media composition on Toll induction
Generating Multimerized Reporters
Luciferase Reporter Assay
Fig.4: How the luciferase reporter assay works
Transfect cells
(Day 1)
Add different stimuli
(Day 2)
Harvest (Day 3)
• Used same procedure as Fig.8
• Serum starved cells for 48 hours
prior to Zymosan treatment