Science - USA (2021-07-16)

sufficiently to capture lipid extraction and
blebbing by live confocal imaging of giant
unilamellar vesicles (GUVs) and negative-
stain EM of liposomes (fig. S13, A and B, and
movies S10 and S11). These effects were
completely lost with rAPOL3DAHthat could
not undergo lipid-triggereda-helical conver-
sion for insertional disruption of the bilayer,
as measured by circular dichroism (fig. S13,
C and D). This solubilizing activity was re-
quired for bacterial restriction inside human
cells, as shown by mutating four Phe resi-
dues to Ser (4F-S) on the hydrophobic face

of AH-2 and -3 that structural homology

modeling predicted were critical to positive

curvature induction and membranolytic ac-

tivity (Fig. 5F and fig. S14, A and B). The APOL3

4F-S mutant exhibited decreased liposome

solvation and bacterial killing in vitro and

could not restore IFN-g–induced immunity

when reintroduced intoDAPOL3human cells,

despite continuing to traffic to bacteria (Fig. 5,

G and H).

Finally, we used high-energy native mass

spectrometry (nativeMS) ( 27 , 28 ) to study

APOL3 structural dynamics during membrane

solubilization. In aqueous solution, rAPOL3

existed as a partly disordered“open”mono-

meric species with exposed surface area ac-

cumulating many positive charges (+16 to

+10) during ionization. Upon engaging lip-

osomes, rAPOL3 underwent a marked shift,

adopting a tightly folded“closed”conformer

of lower charge state (+7 to +5) associated with

multiple lipid adducts, confirming lipoprotein

particle assembly (Fig. 6A). This same confor-

mational change occurred with live bacteria.

Here, open rAPOL3 monomers converted to

closed monomers and dimers (44% of soluble

Gaudetet al.,Science 373 , eabf8113 (2021) 16 July 2021 6 of 14

ΔAPOL3

ΔAPOL3 /GBP1

ΔSTAT1

0.25 0.5 1 2 4 8 16

Log 2 rAPOL3 (μM)

Stm

viability (% dialsate)

100

50

Extracted Stm from

IFN-γ-activated cells

P<0.0001

++++

++

+ +

rGBP1

GTP

rAPOL3

+

+

−−

−−−

− − −

100

50

Stm

viability (% input)

***

rGBP1RFP + GTP

AB

E

F

P<0.0001

mock + mock

rGBP1 + mock

mock + rAPOL3

rGBP1 + rAPOL3

0.46

2.67

15.42

104 105 106 107

IM permeability

(Sytox fluoresence)

104

105

106

107

OM permeability(NPN fluoresence)

Bacterial

ATP (nM)

rGBP1RFP − GTP^

APOL3 GBP1 APOL3

P2A

Empty GBP1

(−) Dox

(+) Dox

Stm

replication 6h (fold)

TRE TRE TRE TRE

***

CD

G

0

10

20

30

40

ns

ns ns

Sytox

Stm

GFP

WT ΔGBP1 ΔAPOL3 ΔAPOL3/GBP1

6 hour

0246

0

2

4

6

8

10

0246

0

2

4

6

8

10

0246

0

5

10

15

20

0246

0

5

10

15

20

+IFN-γ

H

R

Stm

foci (% total)

Sytox

+ cells (% total)

−IFN-γ

Time (h)

WT

ΔAPOL3

ΔGBP1

ΔAPOL3/GBP1

*

***

ns

ns

***

*ns

ns

Time (h)

Wildtype ΔAPOL3ΔGBP1ΔAPOL3/GBP1

Stm

IFN-γ

++++

+ + + + +

− −

−

GBP1

APOL3

β−actin

(Pro)

p32

(Pro)

(IL-18)

CASP4

IL-18

φ

75

37

50

37

20

15

37

H

Unprimed HeLa cells + IFN-γ

Stm

0

-2

0

2

4

6

HeLa + IFN-γ + Stm (2h)

APOL3FLAG O-antigen GBP1 merged

WTΔGBP1/2

*

Penetrating foci per

Stm

merged

WT

ΔGBP1/2

APOL3 OM

infiltration

Stm

Fig. 4. Human GBP1 potentiates APOL3 bactericidal activity.(A) Viability
of cytosolicStmextracted from the indicated HeLa cell genotype (+IFN-g)
and exposed to rAPOL3. (BtoD)Stmtreated with 5mM recombinant human
GBP1RFP(rGBP1) (1 hour) with or without GTP and imaged by confocal
microscopy (B), washed, treated with 5mM rAPOL3 for 1 hour, and then analyzed
by colony counting (C) or ATP (bubble size) in conjunction with both OM
and IM permeability by NPN or Sytox uptake, respectively (D). Bubbles represent
five independent experiments in technical duplicate. (E) Fold replication of
Stmin unprimed HeLa cells expressing doxycycline-inducible (TRE, Tet
response element)APOL3,GBP1, or both in tandem separated by the self-
cleavable P2A peptide. (F) IFN-g–activated HeLa cells expressing APOL3FLAG
infected withStmfor 2 hours were analyzed by SIM (mid-2D single-plane

imaging) after immunostaining for FLAG, GBP1, andStmLPS (O-antigen).

Arrows indicate penetrating APOL3 foci and quantification (n= 50). (Gand

H) IFN-g–activated HeLa cells infected withStmGFPwere analyzed by live

microscopy for HRStm(foci 10 to 35mm) and cell death (Sytox+)(G)or

whole-cell lysates probed by immunoblot after 3 hours (H). Representative

images and immunoblots from one of three independent experiments and

quantification of total events per well (% total cells) are shown. Data are means ±

SEM from three to five independent experiments. Micrographs in [(B) and (F)]

are representative of three independent experiments. Statistics indicate

significance by one-way ANOVA [(C) and (G)], two-way ANOVA (E), unpaired

ttest (F), or nonlinear regression (A). *P< 0.05, ***P< 0.001. Scale bars, 10mm

(B), 1mm (F), 80mm (G).

RESEARCH | RESEARCH ARTICLE