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. ([email protected])
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