Science - USA (2021-07-16)

Stmand reversed inDAPOL3cells treated
with IFN-g(Fig. 2D and movies S3 and S4).
Stmlacking the Cpx-driven IM repair path-
way (StmDcpxR:FRT) had exacerbated damage
and was more susceptible to APOL3-driven

restriction (fig. S5, A to C), underscoring the

importance of bacterial membrane repair for

resisting APOL3-dependent immunity. In

addition, a dual-reporterStmstrain respon-

sive to de novo arabinose-induced GFP ex-

pression became completely unresponsive to

this stimulus when targeted by APOL3 (Fig.

2E), indicating that these bacteria were com-

promised in their ability to respond transcrip-

tionally to external cues. These fitness defects

Gaudetet al.,Science 373 , eabf8113 (2021) 16 July 2021 3 of 14

A

P = 0.0817

P < 0.0001

B

Time (h)

036

0

3

6

9

Stm

replication (fold)

WT

ΔAPOL3

ΔAPOL3 + APOL3

ΔAPOL3 +

LAP137QSS

Stm:mCherry Stm:araB-GFP APOL3FLAG Merged

mCherry

araB-

GFP

infectarabinose

30 min 2.5 h

Stm de novo mRNA reporter

Functional IM IM dysfunction

Stm:minD-GFP IM reporter

IM

OM

3D-SIM stack

APOL3

LPS

APOL3GFP

IFN-γ150'

0

1

2

3

APOL3−(n= 466,450)

APOL3+(n= 104,123)

Bacterial coating

ΔAPOL3

(n = 638)

IFN-γ−−++

ns

***

***

GFP/mCherry ratio

−IFN-γ+IFN-γ

45 min

150 min

036

0

50

100

150

Time (h)

Stm

ΔinvA

::pR1203

replication (fold)

03 6

Time (h)

−IFN-γ+IFN-γ

WT + LLOMe

ΔAPOL3

+ LLOMe

WT

ΔAPOL3

***

***

C

APOL3−

APOL3+

IFN-γ−−++

45min 150min

0

25

50

75

100

Stm

(%)

Exterior Interior

D F

E

+IFN-γ+−IFN-γ IFN-γ

0

5

10

15

20

Stm

with minD

aggregates (%)

APOL

1

APOL1bAPO

L2

AP

OL

3

APOL4APOL5APOL6

0

10

20

30

Stm

targeted (%)

150 min + IFN-

γ

+IFN-γ

APOL3 position:

APOL3StmRFP

APOL3 WT LAP137QSS StmΔinvA::pR1203^

−LLOMe +LLOMe

−IFN-γ

+IFN-γ

2D SIM

46:00 56:00 67:00 84:00 93:00

APOL3

StmRFP

3D-mid z

−IFN-γ

+IFN-γ

568 ApoL3

−IFN-γ

+IFN-γ

Stm:minD-GFP APOL3RFP

WT (n = 604)

Bacterial fitness

APOL3mnGFPStmRFP

Fig. 2. Human APOL3 targets and inflicts damage to cytosolic bacteria.
(A) APOL3mnGFPtargetingStmRFPby live imaging in HeLa cells (movie S1).
Percentage of totalStmtargeted by HA-tagged APOL family members (2 hours)
is shown at right. (B)StmRFPtargeting and replication in IFN-g–primed
DAPOL3cells complemented with the indicated APOL3 variant. (C) Deconvolved
wide-field images of APOL3mnGFPtargeting vacuole-confinedStmRFP
(StmDinvA::pR1203) with or without vacuole release with LLOMe; fold replication is
shown at right. (D) Inner membrane (IM) integrity as measured by minDmnGFP
aggregation withinStmin HeLa cells expressing APOL3RFPat 2 hours with
or without IFN-g. Quantification reflects aggregation in APOL3-coated versus
uncoated bacteria or total bacteria in WT versusDAPOL3cells via Fisher’s

exact test. (E) Arabinose-induced GFP inStmtargeted by APOL3FLAGin

HeLa cells with or without IFN-g. Maximal-intensity GFP/mCherry ratios are

shown (mean ± SD,n=50).(F) Immunofluorescence and SIM of APOL3HAand

LPS onStmwith or without IFN-gat selected times. Mid-2Dz-planes are

shown. Quantification of LPS penetrance (25 bacilli, mean ± SEM,n= 3)

and 3D surface rendering are shown below. Blue arrows indicate cryo-

immunogold EM staining of APOL3GFPinStm-infected HeLa cells; OM, outer

membrane. Micrographs are representative of at least three independent

experiments. Data are means ± SEM [(A), (B), (C)] with significance by one-

way ANOVA at 6 hours. ***P< 0.001. Scale bars, 5mm [(A), (B), (C), and (E)],

2 mm (D), 1mm (F).

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