Supplementary Figures

Autophagic control of Listeria through intracellular innate immune recognition in

drosophila

Tamaki Yano1, Shizuka Mita1, Hiroko Ohmori2, Yoshiteru Oshima1, Yukari Fujimoto3,

Ryu Ueda4, Haruhiko Takada5, William E. Goldman6, Koichi Fukase3, Neal Silverman7,

Tamotsu Yoshimori2 & Shoichiro Kurata1*

1 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578,

Japan

2 Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan,

CREST, Japan Science and Technology Agency, Tokyo 103-0027, Japan

3 Graduate School of Science, Osaka University, Osaka 560-0043, Japan

4 National Institute of Genetics, Mishima, 411-8540, Japan

5 Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan

6 Washington University School of Medicine, St. Louis, Missouri 63110, USA

7 Department of Medicine, University of Massachusetts Medical School, Worcester, MA

01605, USA

Correspondence should be addressed to S.K. (kurata@mail.pharm.tohoku.ac.jp)

1

Co

py

num

ber

(PG

RP

-LE

/rp

49)

0.0002

0.0001

0 0

0.002

0.004

0.006hm

l>At

g5 R

NAi

hml>

LE R

NAi

hml>

GFP W

TPG

RP-

LE11

2

Figure S1

hml>

Atg5

RN

Aihm

l>LE

RN

Aihm

l>G

FP

Co

py

num

ber

(Atg

5/rp

49)

WT

PGR

P-LE

112

2

Supplementary Figure 1

RNA interference against PGRP-LE or Atg5 using an hml-GAL4 driver efficiently

reduces the expression of the target genes in hemocytes. Quantification of the

expression of PGRP-LE and Atg5 in hemocytes from third instar larvae of each

genotype by real-time reverse transcription-polymerase chain reaction. rp49 was used

as an internal control. Bars indicate the variance of duplicate measurements.

Genotypes: UAS-Atg5IR/+;;hml-GAL4/+ (hml>Atg5 RNAi), UAS-PGRP-LE IR/+;

hml-GAL4/+ (hml>LE RNAi), UAS-GFP/+; hml-GAL4/+ (hml>GFP), Oregon R

(wild-type), PGRP-LE112 (LE112)

3

Bac

teri

a/fly

Time after injection (h)

a

Bac

teri

a/fly

400

300

200

100

0 2 4 60

Time after injection (h)

b200

100

0 2 4 60

50

150

Figure S2

PGRP-LE112

PGRP-LC7454

Oregon R

4

Supplementary Figure 2

Differences in bacterial growth in humoral and cellular fractions of PGRP-LE and

PGRP-LC mutant adult flies. After injection of approximately 50 wild-type L.

monocytogenes per fly, wild-type (Oregon R) flies, PGRP-LE112 flies, and PGRP-LC7454

flies were dissected in PBS at the times indicated, the body debris were discarded, and

the resultant humoral and cellular fractions were separated by centrifugation. L.

monocytogenes growth in each fraction was quantified by determining colony-forming

units by plate assay. (a) L. monocytogenes growth in the humoral (hemolymph)

fraction. (b) L. monocytogenes growth in the cellular fraction. Results are

representative of two independent experiments.

5

Figure S3

WT ∆hly WT ∆hly

a b c d

6

Supplementary Figure 3

Cytoplasmic invasion of wild-type L. monocytogenes into drosophila cells. (a, b) Ex

vivo-cultured hemocytes from PGRP-LE112 larvae infected with (a) wild-type or (b)

∆hly strain L. monocytogenes for 2.5 h (0.5 h incubation with bacteria and additional 2 h

incubation in gentamicin-containing medium). (c, d) S2 cells were incubated with

wild-type (c) or ∆hly strain (d) L. monocytogenes for 0.5 h, and additionally incubated

in gentamicin-containing medium for 1.5 h. (a-d) Fixed cells were stained with

rhodamine-labeled phalloidin (red) and DAPI (cyan). Arrowheads indicate some of

the actin tails at the poles of bacteria. Scale bars, 5 µm.

7

RNAi:

Cell line:

Co

py

num

ber

(PG

RP

-LE

/rp

49)

0.06

0.03

0

S2 S2-LE

–

Atg5

Dif/

dlRel

Dif/

dlRel

Atg5

Co

py

num

ber

(dl/

rp49

)

Co

py

num

ber

(Dif

/rp

49) 0.001

0.0005

0

0.001

0.0005

0

S2 S2-LES2 S2-LE

–

Atg5

Dif/

dl Rel

Dif/

dl Rel

Atg5

Atg5

Dif/

dl Rel

Dif/

dl Rel

Atg5

Co

py

num

ber

(Atg

5/rp

49)

0.004

0.002

0

S2 S2-LE

–

Atg5

Dif/

dl Rel

Dif/

dl Rel

Atg5

Co

py

num

ber

(Rel

/rp

49)

0.08

0.04

0

S2 S2-LE

Atg5

Dif/

dl Rel

Dif/

dl Rel

Atg5

RNAi:

Cell line:

– – –

– – ––

Figure S4

a b c

d e

8

Supplementary Figure 4

Quantification of the expression of PGRP-LE, Atg5, Relish, Dif, and dorsal in S2 cells

and S2 cells expressing PGRP-LE by real-time reverse transcription-polymerase chain

reaction. (a) PGRP-LE (b) Atg5 (c) Relish (Rel) (d) Dif (e) dorsal (dl). rp49 was

used as an internal control. Bars indicate the variance of duplicate measurements.

9

Co

py

num

ber

(Dp

t/rp

49) 0.08

0.04

00 10 20

Time (h)

Cell line L. monocytogenesS2-LE WT

∆hlyS2-LES2-LE No infection

a

b

Figure S5

S2 WTS2 ∆hlyS2 No infection

∆hly ∆hly ∆hly

Co

py

num

ber

(Drs

/rp

49)

0.04

0.02

00 10 20

Time (h)

Co

py

num

ber

(Att

/rp

49) 0.4

0.2

00 10 20

Time (h)

Co

py

num

ber

(LE

/rp

49) 0.06

0.03

00 10 20

Time (h)

0.03

0.02

0.01

0

Co

py

num

ber

(Dp

t/rp

49)

RNAi :

0.03

0.02

0.01

0

Co

py

num

ber

(Drs

/rp

49)

0.4

0.2

0

Co

py

num

ber

(Att

/rp

49)

LC imd

WT No infection

Atg5 LC imd

Atg5 LC imd

Atg5 LC imd

WT No infection

Atg5 LC imd

Atg5 LC imd

Atg5 LC imd

WT No infection

Atg5 LC imd

Atg5 LC imd

Atg5‒ ‒ ‒‒‒‒‒‒‒

10

Supplementary Figure 5

AMP induction in response to L. monocytogenes infection in S2 cells expressing

PGRP-LE is dependent on imd, but not on Atg5. (a) Real-time reverse

transcription-polymerase chain reaction of Diptericin (Dpt), Drosomycin (Drs), Attacin

(Att), PGRP-LE (LE), and rp49 (internal control). S2 cells and S2 cell lines stably

transfected with a metallothionein-PGRP-LE construct (S2-LE). After 1.5 h infection

with wild-type or ∆hly L. monocytogenes, cells were cultured in CuSO4- and

gentamicin-containing medium, and the RNA was extracted for analysis. (b) S2-LE

cells were transfected with double-stranded RNA specific for the indicated genes (RNAi,

below graph) and then infected with wild-type or ∆hly L. monocytogenes for 1.5 h.

After 9 h incubation in CuSO4-and gentamicin-containing medium, the RNA was

extracted for real-time reverse transcription-polymerase chain reaction analysis. Bars

indicate the variance of duplicate measurements.

11

Figure S6

Bac

teri

a/ce

ll

60

40

20

00 2 4 6

Time (h)

S2 WTS2-LE WTS2 ∆hlyS2-LE ∆hly

Cell line L. monocytogenes

12

Supplementary Figure 6

PGRP-LE is crucial for the clearance of intracellular L. monocytogenes. S2 cells or S2

cells expressing PGRP-LE (S2-LE) were infected with wild-type (WT) or ∆hly L.

monocytogenes (approximately 250 bacteria per cell) for 0.5 h, followed by incubation

in CuSO4- and gentamicin-containing medium for the indicated time. L. monocytogenes

growth was quantified by determining colony-forming units by plate assay.

13

WTWT Lm

WTRapamycin

WTNo infection

WT∆hly Lm

LE112WT Lm

LE112Rapamycin

DAPI

Merged

EGFP-LC3

a b c ed f

Figure S7

14

Supplementary Figure 7

PGRP-LE is required for the induction of autophagy in response to L. monocytogenes

infection in the cytoplasm. Ex vivo-cultured hemocytes from EGFP-LC3 expressing

wild–type (WT) or PGRP-LE112 (LE112) mutant-background third-instar larvae infected

with wild-type (WT Lm) or ∆hly strain (∆hly Lm) L. monocytogenes. EGFP-LC3 (green

or white), rhodamine-labeled phalloidin staining of the actin cytoskeleton (red), and

DAPI (blue or white). All images were obtained using a confocal microscope. Filled

arrowheads indicate some of the dot-shaped EGFP-LC3 signals. The open arrowhead

indicates Lm DNA. Bars represent 10 µm.

15

WTTCT

LE112

DAP-PGNWT

Lys-PGNLE112

TCTWT

DAP-PGNLE112

Lys-PGN

EGFP-LC3

Figure S8

c d e gf h

a b

16

Supplementary Figure 8

PGRP-LE is required for the induction of autophagy in response to TCT or DAP-type

peptidoglycans. (a) A fluorescence microscopy image of S2 cells expressing PGRP-LE

and GFP-LC3 (green) transfected with TCT. (b) An electron microscopy image of the

fluorescence-positive field in (a). Arrows indicate double-membrane structure. Scale

bars represent 5 µm in (a) and 500 nm in (b). (c-h) Ex vivo-cultured hemocytes from

GFP-LC3 expressing wild-type (c, e, g) or PGRP-LE112 (d, f, h) mutant-background

third-instar larvae treated with 100 nM TCT (c, d), 100 µg/ml highly purified DAP-type

peptidoglycans from L. plantarum (DAP-PGN) (e, f), or lysine-type peptidoglycans

from S. epidermidis (Lys-PGN) (g, h). All images were obtained using a confocal

microscope. Some of the dot-shaped GFP-LC3 signals are indicated by filled

arrowheads. Bars represent 10 µm.

17