1. Requirements for EGFRvIII-mediated
enhancement of SecA2-dependent
secretion from Listeria monocytogenes
Shane Klumpp EACRI Summer 2015 project
Bahjat Lab

2. Epidermal Growth Factor Receptor
(EGFR) Protein
• EGFR: Epidermal Growth Factor
Receptor
• EGFRvIII: EGFR with 801 bp deletion
• Novel Glycine at junction
EGFRvIII acts as an oncogene. An EGFRvIII-expressing L. monocytogenes clinical
strain has been constructed

3. Efficient intracellularsecretionof 5xEGFRvIIIinJ774
macrophages
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our “gold-standard”
for intracellular protein
secretion...
Hypothesis: EGFRvIII when included in a construct should help secretion

4. Objectives
• Determine the important residues in the EGFRvIII epitope that are relevant for
SecA-dependant secretion.
• Construction of L. monocytogenes strains containing vaccinia and EGFRvIII epitopes
• Test for secretion and presentation of constructs

5. QVACNEW Lm-ΔactA-ΔgcpE Strain
Strain Name Characteristics Strain image
QVACNEW ActAN100, New
vaccinia virus epitopes
(Vac epitopes),
SIINFEKL, EGFRvIII
ActA-N100 Vac epitopes SIINFEKL EGFRvIII
Strain will be a standard we can measure against when testing EGFRvIII
mutations
A3L A23R B16RB2R
VAC
Epitope
Amino Acid
sequence
A3L KSYNYMLL
A23R IGMFNLTFI
B2R YSQVNKRYI
B16R ISVANKIYM

6. Protocol
Construct in Lm ΔactA-ΔgcpE
Macrophage
Infection/Lysis
Western
Blot
B3Z Assay
3) Ligation to pPL2e
2) Digestion with SalI-NotI
Construct in pPL2e
Use of QVACNEW construct as a
template
Construct in Top10

7. B3Z assay
SIINFEKL in QVACNEW is being presented by
dendritic cells
24 hours
Absorbance
C
lone
5
C
lone
6
C
lone
9
+
Q
vac
Lm
ΔactA
-Lm
ΔactA
ΔgcpE
0.00
0.05
0.10
0.15
0.20
0.25
Qvac Lm-ΔactA does not
contain EGFRvIII

8. QVACNEW Lm-ΔactA-ΔgcpE Western
α-ActA
α-P60
α-β-Actin
1 4 5 6 9 + - No
α-ActA: Targets ActA protein
α-P60: Targets secreted Lm protein
α-β-Actin: Targets actin protein
+: QV LmΔactA
-: LmΔactAΔgcpE
The QVACNEW protein is secreted into the
cytoplasm of RAW264.7 macrophages
p60
ActA-QVACNEW
ActA-Qvac
β-Actin
20KDa
25KDa
55KDa

9. Constructed strains
Strain Name Characteristics Strain image
QVACNEW ACTAN100, New
vaccinia virus epitopes
(Vac epitopes),
SIINFEKL, EGFRvIII
QVACNEW-ΔGSC QVACNEW epitopes +
GSC deletion in
EGFRvIII
QVACNEW-GSC-AAA QVACNEW epitopes,
GSC replaced with AAA
QVACNEW-ΔTDH QVACNEW epitopes,
TDH deleted
QVACNEW-ΔEGFR QVACNEW epitopes,
without EGFRvIII
E-QVACNEW-S QVACNEW strain
rearranged to EGFRvIII-
QVAC-SIINFEKL
QVACNEW-E-S QVACNEW strain
rearranged to QVAC-
EGFRvIII-SIINFEKL
ActA-N100 Vac epitopes SIINFEKL EGFRvIII
ActA-N100 Vac epitopes SIINFEKL EGFRvIII-ΔGSC
ActA-N100 Vac epitopes SIINFEKL EGFRvIII-GSC-AAA
ActA-N100 Vac epitopes SIINFEKL EGFRvIII-ΔTDH
ActA-N100 Vac epitopes SIINFEKL
ActA-N100 Vac epitopes SIINFEKLEGFRvIII
ActA-N100 Vac epitopes SIINFEKLEGFRvIII
EGFRvIII epitope: PASRALEEKKGNYVVTDHGSC
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R

10. The different strains showed various levels of SIINFEKL
presentation by B3Z assay
24 hours
Absorbance
G
SC
-A
A
A
Δ
TD
H
Δ
G
SC
Δ
EG
FR
E-Q
VA
C
N
EW
-S
Q
VA
C
N
EW
-E-S
+
-
0.0
0.1
0.2
0.3
GSC-AAA
DTDH
DGSC
DEGFR
E-QVAC-S
QVAC-E-S
+ QVAC Lm DactA
- Lm ΔActAΔgcpE

11. Constructs in Western Blot α-ActA Replicate 1
20KDa
25KDa
37KDa
1 42 3 65 7 8 9 10 11 N
o
M
20KDa
25KDa
37KDa
Lane Strain
1 QVACNEW-ΔEGFR
2 QVACNEW-E-S
3 E-QVACNEW-S
4 QVACNEW
5 QVACNEW-ΔGSC
6 QVACNEW-GSC-AAA
7 QVACNEW-ΔTDH
8 Lm-ΔactA-OVA
9 Lm-ΔactA-Qvac
10 EGFRvIII 5x
11 Lm ΔactA-ΔgcpE
No No Lm
M Marker
α-β-actin
*
*: Unspecific Protein
*
α-P60

12. 1 42 3 65 7 8 9 10 11 N
o
Constructs in Western Blot α-ActA Replicate 2
M
Lane Strain
1 QVACNEW-ΔEGFR
2 QVACNEW-E-S
3 E-QVACNEW-S
4 QVACNEW
5 QVACNEW-ΔGSC
6 QVACNEW-GSC-AAA
7 QVACNEW-ΔTDH
8 Lm-ΔactA-OVA
9 Lm-ΔactA-Qvac
10 EGFRvIII 5x
11 Lm ΔactA-ΔgcpE
No No Lm
M Marker
20KDa
25KDa
37KDa
20KDa
25KDa
37KDa
α-β-actin
*
*
*: Unspecific Protein

13. Conclusions
• Location of EGFRvIII does not make a
difference in secretion
• Deleting EGFRvIII entirely does effect
secretion

14. Further studies
• Lysis of QVAC-ΔEGFR Lm-ΔactAΔgcpE strain
and western blot to confirm construct
translation
• Constructing strains with EGFR5x in different
locations to see effect on expression

15. Acknowledgments
Keith Bahjat
Alejandro Alice
Gwen Kramer
Shelly Bambina
EACRI staff