doi: 10.1038/nature07952 supplementary … · immunoblotting with antibodies against ilk or ospe....

a

bMDDIFTQCREGNAVAVRLWLDNTENDLNQGDDHGFSPLHWACREGRSAVVEMLIMRGARINVMNRGDDTP

LHLAASHGHRDIVQKLLQYKADINAVNEHGNVPLHYACFWGQDQVAEDLVANGALVSICNKYGEMPVDKA

KAPLRELLRERAEKMGQNLNRIPYKDTFWKGTTRTRPRNGTLNKHSGIDFKQLNFLTKLNENHSGELWKG

RWQGNDIVVKVLKVRDWSTRKSRDFNEECPRLRIFSHPNVLPVLGACQSPPAPHPTLITHWMPYGSLYNV

LHEGTNFVVDQSQAVKFALDMARGMAFLHTLEPLIPRHALNSRSVMIDEDMTARISMADVKFSFQCPGRM

YAPAWVAPEALQKKPEDTNRRSADMWSFAVLLWELVTREVPFADLSNMEIGMKVALEGLRTIPPGISPHV

CKLMKICMNEDPAKRPKFDMIVPILEKMQDK

50kDa50

75100150250

37

25

20

15

HeL

a ly

sate

GS

T+ly

sate

Pull-down

GS

T-O

spE

GS

T-O

spE

+lys

ate

GS

T

y12

y13

y11

y4 y7y3 y5 y8 y9 b14 + H2Oy1y6

b8 b9

b10b7

50

70

100

60

30

20

90

80

40

10

Inte

nsity

%

69.0 389.6 710.2 1030.8 1351.4 1672.0

Mass (m/z)

3743.4

G D D T P L H L A A S H G H Rb8

y7 y6 y5 y4 y3y12y13

b9 b10b7

y8y9y11

Supplementary Figure 1. Identification of ILK as an OspE-interacting protein by GST pull-down assay. (a) GST-OspE or GST beads were mixed with HeLa cell lysates. Bound proteins were analyzed by SDS-PAGE. A protein of approximately 50 kDa highlighted by the red box was subjected to nano-flow reverse-phase liquid chromatography followed by MALDI-TOF/TOF (b) Full amino-acid sequence of human ILK. The residues in red belong to the tryptic peptide identified in the MALDI-TOF/TOF spectrum shown below.

SUPPLEMENTARY INFORMATIONdoi: 10.1038/nature07952

www.nature.com/nature 1

c

a

d

b

ILK

OspE

ILK-/-/ WT-ILK

ILK-/-/ K220M-ILK

ILK-/-/ S343D-ILK

OspE

Whole-cell Lysate IP: IgG IP: ILK

- + +

+ +

++ +

- -

- - -

- -+

- --

- - -

+ +- -- - -- --

- + +

+ +

++ +

- -

- - -

- -+

- --

- - -

+ +- -- - -- --

- + +

+ +

++ +

- -

- - -

- -+

- --

- - -

+ +- -- - -- --

ILK

OspE

ILK-/-/ WT-ILK

OspE

MBP

- + +

+ ++ +

-

- + +-

32P-MBPAutoradiogram

Autoradiogram

CBB staining GST-ILK

GST-ILK

MBP

GST

GST-OspE

- + +

- +

+ - -

- -

+

-

- - -+

32P-MBP

GST-OspE

GST

MBP

pAkt(S473)

Akt

pGSK3β(S9)

GSK3β

Moc

k

Moc

k

Moc

k

Moc

k

Osp

E

Osp

E

Osp

E

Osp

E

ILK-/- WT-ILK K220M-ILK S343D-ILK

ILK-/- + - -+

ILK-/- + - - - - - -+ + - - - - - -+ + - - - - - -+

Supplementary Figure 2. OspE has no effect on ILK kinase activity.(a) The binding between OspE and a series of ILK variants was analyzed by immunoprecipitation. Lysates of ILK-/- cells or the same cells expressing WT-ILK, K220M-ILK, or S343D-ILK with or without OspE were immunoprecipitated with control mouse IgG or an anti-ILK antibody. The precipitated proteins were subjected to immunoblotting with antibodies against ILK or OspE. (b) Anti-ILK immunoprecipitates from ILK-/- cells or the same cells expressing WT-ILK with or without OspE cell lysates were measured by an in vitro kinase assay using MBP as a substrate. Samples were separated by SDS-PAGE, followed by immunoblotting with anti-ILK or anti-OspE antibodies or autoradiography to detect γ-32P incorporated into MBP. (c) The kinase activity of GST-ILK for GST-OspE or MBP was measured by an in vitro kinase assay. Samples were analyzed by SDS-PAGE and autoradiography to detect γ-32P incorporation into each substrate. The amounts of purified proteins were confirmed by CBB staining. (d) Phosphorylation levels of cellular ILK substrates were analyzed. Whole-cell lysates of ILK-/- cells or the same cells expressing WT-ILK, K220M-ILK, or S343D-ILK with or without OspE were subjected to immunoblotting with antibodies against phospho-Akt(S473), Akt, phospho-GSK-3β(S9), or GSK-3β.

doi: 10.1038/nature07952 SUPPLEMENTARY INFORMATION


b

c

d

ILK

α-Parvin

β-Parvin

PINCH

OspE

IP:ILK

ILK

-/-/ M

ock

ILK

flox/

flox /

Moc

k

ILK

-/-/ O

spE

ILK

flox/

flox /

Osp

E

Input

ILK

α-Parvin

β-Parvin

PINCH

OspE

ILK

-/-/ M

ock

ILK

flox/

flox /

Moc

k

ILK

-/-/ O

spE

ILK

flox/

flox /

Osp

E

wild

type

N-te

rm

C-te

rm

C-te

rm E

359K

wild

type

N-te

rm

C-te

rm

C-te

rm E

359K

wild

type

N-te

rm

C-te

rm

C-te

rm E

359K

GST-OspE GST

Pull down

Input

IB:MycIB:Myc

KINASEANKILK wide type

ILK N-term

ILK C-term

ILK C-term E359K E359K

OspE binding

+

-

+

+

6 12 180

OspE

ILK

Actin

ILK

Actin

CHX h

Mock

6 12 180CHX h

a

Supplymentary Figure 3. Stability of ILK and IPP complex formation(a) Half-life of ILK. ILKflox/flox cells with or without OspE expression were treated with cyclohexamide (CHX) and harvested at the indicated time points. The cell lysates were subjected to immunoblotting with antibodies against ILK or actin. (b) ILK-PINCH-Parvin (IPP) complex formation. Lysates of ILK-/- and ILKflox/flox cells with or without OspE were immunoprecipitated with an anti-ILK antibody, and the precipitated proteins were subjected to immunoblotting with the antibodies indicated. (c) A schematic representation of ILK constructs used for (d). (d) Identification of the ILK domain involved in OspE binding. 293T cells were transfected to express a series of Myc-ILK constructs, and then the cell lysates were pulled down with GST-OspE or GST. Bound proteins were subjected to immunoblotting with an anti-Myc antibody.



a

b c

15 min

30 min

60 min

Spre

adin

g ce

lls (%

)

100

80

60

40

20

0

30 min15 min

60 min

ILK-/- WT K220M S343D

ILK-/- ILK-/-/ WT-ILK ILK-/-/ K220M-ILK ILK-/-/ S343D-ILK

Osp

E

Moc

k

Moc

k

Moc

k

Moc

k

Osp

E

Osp

E

Osp

E

No.

of F

As a

ssem

bled

(a

vera

ge p

er c

ell)

30 min

60 min

80

60

40

20

0

ILK-/- WT K220M S343D

Osp

E

Moc

k

Moc

k

Moc

k

Moc

k

Osp

E

Osp

E

Osp

Ed e

0

0.05

0.1

0.15

0.2

0.25

0.3

BSA PLL FN Col LN

ILK-/-/MockILK-/-/OspEILKflox/flox/MockILKflox/flox/OspE

n.s.

n.s.n.s.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

BSA Col1 Col2 Col4 FN LN TN

MockOspEOspE(W68A)

Abs

orba

nce

550n

m

Abs

orba

nce

550n

m

Supplymentary Figure 4. Effect of OspE on FA assembly.(a) ILK-/- cells or the same cells expressing WT-ILK, K220M-ILK, or S343D-ILK with or without OspE were seeded on FN-coated coverslips. After incubating for 15, 30, or 60 min to allow adhesion, the cells were stained with an anti-vinculin antibody. Bar, 20 μm. (b) A time course of the percentage of spreading cells in (a) was evaluated (>100 cells, n = 3). Data are means ± S. D. (c) A time course of the number of FAs per cell in (a) was evaluated. Data are means ± S. D. (d) Adhesion assay with ILK-/- and ILKflox/flox cells stably expressing Mock or OspE on defined extracellular matrices. Cells were allowed to adhere to bovine serum albumin (BSA), poly-L-Lysine (PLL), fibronectin (FN), collagen (Col), or laminin (LN) for 15 min. The bar graph is representative of six independent experiments. n.s., not significiant. Data are means ± S. E. (e) HeLa S3 cells stably expressing Mock, OspE or OspE(W68A) were allowed to adhere to BSA, FN, Col1, Col2, Col4, LN or tenascin (TN) for 15 min. The bar graph is representative of three independent experiments. Data are means ± S. E.

Mock OspE Mock OspE Mock OspE Mock OspE



a b

Starved Nocodazole Washout, 15 min Washout, 30 min Starved Nocodazole Washout, 15 min Washout, 30 min

ILK-/-

/Mock

ILK-/-

/OspE

ILK-/-/ WT-ILK

/Mock

ILK-/-/ WT-ILK

/OspE

ILK-/-/ K220M-ILK

/Mock

ILK-/-/ K220M-ILK

/OspE

ILK-/-/ S343D-ILK

/Mock

ILK-/-/ S343D-ILK

/OspE

Supplymentary Figure 5. Effect of OspE on FA disassembly.ILK-/- cells or the same cells expressing WT-ILK, K220M-ILK, or S343D-ILK with or without OspE were serum-starved for 48 h, and then treated with 2.5 μM nocodazole at 37 ºC for 30 min. After washing out the drug, the cells were incubated for additional 15 or 30 min and then stained with antibodies against tubulin (a) or vinculin (b). Bar, 10 μm.



a

5 15 300 5 15 300

Mock OspE

IP: β1 integrin

IB: β1 integrin

IB: Streptavidin-HRP

c

b

0

0.5

1

1.5

2

Mock OspE OspE(W68A)

*

n.s.

β1 in

tegr

in, a

ctiv

e st

ate

leve

l

MockOspE

β1 in

tegr

in, a

ctiv

e st

ate

leve

l

0

0.5

1

1.5

ILKflox/floxILK-/-ILKflox/floxILK-/-

β1 in

tegr

in, t

otal

leve

l

0

0.5

1

1.5

ILKflox/floxILK-/-

β1 in

tegr

in, s

urfa

ce le

vel

0

1

0.5

1.5

0

0.5

1

1.5

2


β1 in

tegr

in, t

otal

leve

l

0

0.5

1

1.5

2


*

n.s.

β1 in

tegr

in, s

urfa

ce le

vel

Supplymentary Figure 6. β1 Integrin internalization was delayed by OspE expression.(a) The expression of β1 integrin in ILK-/- and ILKflox/flox cells stably expressing Mock or OspE was analyzed by FACS with an anti-β1 integrin (Ha2/5) antibody under non-permeabilizing (left) or permeabilizing (middle) conditions. The active state integrin was detected with an anti-β1 integrin (9EG7) antibody (right). The bar graphs are representative of three independent experiments. Data are means ± S. D. (b) The expression of β1 integrin in HeLa S3 cells expressing Mock, OspE, or OspE(W68A) was analyzed by FACS with an anti-β1 integrin antibody under non-permeabilizing (left) or permeabilizing (middle) conditions. The active state integrin was detected with binding of FITC labeled-FN7-10 fragments to HeLa S3 cells (right). The bar graphs are representative of three independent experiments. Data are means ± S. E. *p<0.01. n.s., not significant. (c) Internalization of β1 integrin. The cell surface proteins were labeled with biotin at 4 °С for 30 min. After labelling the cells were incubated at 37 °С for the indicated time points (min), and then the cells were subjected to immunoprecipitation with an anti-β1 integrin antibody. Precipitated proteins were analyzed by probing with peroxidase-conjugated streptavidin or an anti-β1 integrin antibody.



Inpu

t

a b

c

d e

f g

ILK

CBB

GS

T

Osp

E 1

-58

Osp

E 1

-44

Osp

E 2

9-88

Osp

E 4

4-88

Osp

E 2

9-58

Osp

E 1

-65

Osp

E 1

-75

Osp

E 1

-80

Osp

E 4

A

Osp

E Q

67A

Osp

E W

68A

Osp

E L

69A

Osp

E T

70A

Osp

E

Pull down

Mock

OspE

OspE(W68A)

Actin MergeVinculin

GFP OspE 1-58 OspE 1-44 OspE 29-88 OspE 44-88

OspE 29-58 OspE 1-65 OspE 1-75 OspE 1-80 OspE 4A

OspE Q67A OspE W68A OspE L69A OspE T70A OspE

1 88

58

44

65

75

80

29

AAAA

Q67A

W68A

L69A

T70A

OspE

OspE 1-58

OspE 1-44

OspE 29-88

OspE 44-88

OspE 29-58

OspE 1-65

OspE 1-75

OspE 1-80

OspE 4A

OspE Q67A

OspE W68A

OspE L69A

OspE T70A

88

8844

29 58

FA n

umbe

r/cel

l

β1 in

tegr

in, t

otal

leve

l

β1 in

tegr

in, s

urfa

ce le

vel

ILK

OspE

pFAK(Y397)

FAK

Moc

k

Osp

E

Osp

E(W

68A

)

β1 integrin

Actin

**250

200

150

100

50

0

Moc

k

Osp

E

Osp

E(W

68A

)

2.0

1.5

1.0

0.5

0

Moc

k

Osp

E

Osp

E(W

68A

)

* *2.0

1.5

1.0

0.5

0

Moc

k

Osp

E

Osp

E(W

68A

)

Supplymentary Figure 7. Identification of the OspE sequence involved in ILK binding. (a) A schematic representation of OspE constructs. (b) Identification of the OspE sequence involved in ILK binding. A series of GST-OspE constructs was used for a pull-down assay with HeLa cell lysates, and bound proteins were separated by SDS-PAGE, followed by CBB staining or immunoblotting with an anti-ILK antibody. (c) Localization of GFP-OspE constructs in HeLa cells. Bar, 10 μm. (d) NIH3T3 cells stably expressing Mock, OspE or OspE(W68A) were stained with an anti-vinculin antibody (green) and phalloidin (red). Bar, 10 μm. (e) The number of FAs in (d) visualized was quantified (>20 cells, n = 3). Data are means ± S. D. *p<0.001. (f) Lysates of NIH3T3 cells stably expressing Mock, OspE or OspE(W68A) were subjected to immunoblotting with antibodies against ILK, OspE, phospho-FAK (Y397), FAK, β1 integrin or actin. (g) NIH3T3 cells stably expressing Mock, OspE or OspE(W68A) were subjected to FACS analysis with an anti β1 integrin (Ha2/5) antibody under non-permeabilizing (left) or permeabilizing (right) conditions. The bar graphs are representative of three independent experiments. Data are means ± S. D. *p<0.001.

FAlocalization

ILKbinding

+

-

+

+

-

-

-

+

+

-

+

-

+

+

+

-

+

+

-

-

-

+

+

-

+

-

+

+



b

ILK

CBB

Pull down

Inpu

t

GS

T-O

spE

GS

T-O

spO

1-2

GS

T

GS

T-O

spO

1-1

GS

T-O

sp1 S

TYM

c

a

OspE

OspO1-1

OspO1-2

OspO1STYM

GFP Vinculin Actin Merge

S. flexneri OspE1 --MLTQTIFPCLPQKQENIILEVS------NPVLLSSTVTTDGYTVFNKKAAIYELQIP-AASRTKTLKFTATEMQWLTKINEAGIDEKQSQRYSDF

S. flexneri OspE2 --MLTQTIFPCLPQKQENIILEVS------NPVLLSSTVTTDGYTVFNKKAAIYELQIP-AANRTKTLKFTATEMQWLTKINEAGIDEKQSQRYSDF

S. dysenteriae OspE1 --MLTQTIFPCLPQKQENIILEVS------NPVLLSSTVTTDGYTVFNKKAAIYELQIP-AANRTKTLKFTATEMQWLTKINEAGIDEKQSQRYSDF

S. sonnei OspE2 --MLTQTIFPCLPQKQENIILEVS------NPVLLSSTVTTDGYTVFNKKAAIYELQIP-AANRTKTLKFTATEMQWLTKINEVGIVEKQSQRHSNI

S. boydii OspE1 --MLTQTIFPCLPQKQENIILEVS------NPVLLSSTVTTDGYTVFNKKAAIYELQIP-ATNRTKTLKFTATEMQWLTKINEVGIVEKQSQRHSNI

EHEC OspO1-1 MPFSIKNRFSSSQVHYPEISGPIKDKPASKNCILTSTTCNVDSYTVYQKKACSFDMRPPGAGERTPKLKLSVTEMTWLSKTIETEIHNTKE------

EHEC OspO1-2 MPFSIKSIFSGHTWHQPEISRPIADKSSTKNCILDSTTCNVDGFTVFNRRSCSFDMRPPGSADRTPQLRLSISEVAWMSKIIETETNNTNKS-----

S. typhimurium Osp1STYM MPFSIKNICSGPKGHCPEISSPIQDKPVPRNCTLTSTTCDIQSYTVFSRWSCSYEMRPPGAEERTPRLKFSATELSWLSKTIETERRNTKE------

S. enteritidis Osp1SENT MPFSIKNICSGPQGHYPEISRPIQNKPVPRNCTLISTTCKIQEYTVFSRCSCSFEMRPPGTEERTPRLKLSATELSWLSKTIETEMRNTKE------

EHEC EspO1O103:H2 MPLSIGSCFSCHTGRRPEISDPIIDKPSTKNCELISTTCNVDGITVISRSSYGFDMKPPGAGERAPRLKLSASEAQWMAAIIDAEGHNISNT-----

EPEC EspO1EPE22 MPFSIKNIFSNSKGSYPEISGPVQDKPVSKNCTLTSTTCSMNDYTVFSRKSCTFDMRPPGAGDRTPQLKLSASELIWLSKTIDTERNNIKE------

AEPEC EspO1AEPEC MPFSIKNIFSNSKGNYPEISGPVQDKPVSKNCTLTSTTCPMNDYTVFSRKSCTFDMRPPGAGDRTPKLKLAATEMTWLSKTIETEIHNTKE------

C. rodentium EspO1CIROD MPLSIRNIFSRASTHRPEISGPVIDKPIPKNCTLISSTCNLDGIMVINRRTSFYDIKPPGAGERQPSLKISASEAQWMCKIIETEINNSNK------

*

Supplymentary Figure 8. Characterization of OspE homologs.(a) Alignment of OspE homologs. OspE cognate genes are present in other pathogenic bacteria, including EPEC, EHEC, and Salmonella strains. The Trp68 in OspE, noted by the asterisk, is conserved among all OspE homologs. (b) The binding of OspE-homolog proteins with ILK was assessed by a pull-down assay. Bound proteins were subjected to immunoblotting with an anti-ILK antibody. (c) The ability of OspE-homolog proteins to localize at FAs was assessed. The proteins fused to GFP were transiently expressed in HeLa cells, and the cells were stained with an anti-vinculin antibody (blue) and phalloidin (red). Bar, 10 μm.



a b

c

ILK

Actin

Luc ILK

siRNA

0

10

20

30

40

50

60

70

80

siRNALuc Luc ILK ILK

YSH6000 ΔospE YSH6000 ΔospE

*

Rou

ndin

g ce

lls (%

)

*

Rou

ndin

g ce

lls (%

)

0

20

40

60

80

100

10 20 30 45 60 90 120

YSH6000

ΔospE

min

YSH6000 ΔospE ΔospE / ospE ΔospE / ospE(W68A)

Supplymentary Figure 9. OspE suppresses cell detachment in the response to Shigella infection.(a) HeLa S3 cells were infected with the Shigella strains for the indicated time, and the percentage of cells that rounded up was calculated. (b) HeLa S3 cells were transfected with luciferase siRNA (Luc) or ILK siRNA (ILK). The cells were infected with the Shigella strains for 2 h, and the percentage of cells that had rounded up was calculated. Data are means ± S. E. *p<0.001. The bar graph is representative of three independent experiments. The immunoblot shows ILK levels in the cells transfected with Luc or ILK siRNA. Actin serves as a loading control. (c) Intracellular dissemination of Shigella strains was assessed by a plaque forming assay. MDCK cells grown to a confluent monolayer were treated with Hanks' balanced salt solution containing 10 μM EGTA at 37 °С for 1 hr, and then infected with YSH6000, ΔospE, ΔospE / ospE or ΔospE / ospE(W68A) for 2 h. After washing, medium with antibiotics was added, and the cells were incubated at 37 °С for 24 hr. Plaques were observed with a phase-contrast microscope.



Shigella WT

ΔospE mutant

Shigella

Supplymentary Figure 10. A proposed model of Shigella tactics that reinforces the adherence of infected epithelial cells to the basal membrane through OspE-ILK interaction.



doi: 10.1038/nature07952 supplementary … · immunoblotting with antibodies against ilk or ospe....

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