Requirements for EGFRvIII-mediated enhancement of SecA2-dependent

secretion from Listeria monocytogenes

Shane Klumpp EACRI Summer 2015 project

Bahjat Lab

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

Efficient intracellular secretion of 5xEGFRvIII in J774

macrophages

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our “gold-standard”

for intracellular protein

secretion...

Hypothesis: EGFRvIII when included in a construct should help secretion

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

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

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

B3Z assay

SIINFEKL in QVACNEW is being presented by dendritic cells

24 hours

Ab

so

rba

nc

e

Clo

ne 5

Clo

ne 6

Clo

ne 9

+ Qva

c Lm

ΔactA

- Lm

ΔactAΔgcpE

0.00

0.05

0.10

0.15

0.20

0.25

Qvac Lm-ΔactA does not contain EGFRvIII

QVACNEW Lm-ΔactA-ΔgcpE Western

α-ActA

α-P60

α-β-Actin

1 4 5 6 9 + - No

α-ActA: Targets ActA proteinα-P60: Targets secreted Lm proteinα-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

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 strainrearranged 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

The different strains showed various levels of SIINFEKL presentation by B3Z assay

24 hoursA

bso

rban

ce

GSC-A

AA

ΔTD

H

ΔG

SC

ΔEG

FR

E-Q

VACNEW

-S

QVACNEW

-E-S + -

0.0

0.1

0.2

0.3GSC-AAA

DTDH

DGSC

DEGFR

E-QVAC-S

QVAC-E-S

+ QVAC Lm DactA

- Lm ΔActAΔgcpE

Constructs in Western Blot α-ActA Replicate 1

20KDa

25KDa

37KDa

1 42 3 65 7 8 9 10 11 No

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

1 42 3 65 7 8 9 10 11 No

Constructs in Western Blot α-ActA Replicate 2

MLane 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

Conclusions

• Location of EGFRvIII does not make a difference in secretion

• Deleting EGFRvIII entirely does effect secretion

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

Acknowledgments

Keith Bahjat

Alejandro Alice

Gwen Kramer

Shelly Bambina

EACRI staff