Science - USA (2021-07-16)

coating occurred in unprimed cells, indicating
that cytokine activation was needed for APOL3
expression but not its relocation. Even so, once
APOL3 relocated to bacteria within IFN-gÐ
activated cells, it conferred antibacterial activity;
identification and mutation of a hydrophobic
patch (LAP137QSS) (fig. S3D) that prevented
bacterial targeting also abolished restriction
(Fig. 2B). Targeting was specific to cytosol-
exposed bacterium, because vacuole-confined
StmDinvA::pR1203failed to recruit APOL3 unless
released into the cytosol with the lysosomotropic
agentL-leucyl-L-leucine methyl ester (LLOMe),

which restored both bacterial targeting and sub-

sequent restriction (Fig. 2C). Moreover, APOL3

targeted cytosolicS.flexneri,B. thailandensis,

andListeria monocytogenesbut not compart-

mentalizedS. Typhi (fig. S4A), which is generally

in keeping with the susceptibility profiles of

these pathogens to APOL3 restriction. Notably,

sterile damage triggered by LLOMe was suffi-

cienttomobilizeAPOL3tothelumenofrup-

tured LAMP1+endolysosomes independent of

bacteria (fig. S4B and movie S2). It is therefore

likely that initial APOL3 targeting signals are

damage-associated molecular patterns (DAMPs)

induced by pathogenic bacteria when they

rupture their LAMP1+vacuole to enter the cy-

tosol, akin to the defense protein Galectin-8 ( 13 ).

To assess the fate of APOL3-coated bacte-

ria, we engineered reporter strains to diag-

noseStmfitness inside human cells.Stm

inner membrane (IM) integrity was tracked

via minD, a bacterial cell division protein that

loses its lateral membrane oscillatory behavior

when IM potential is perturbed ( 14 ). Loss of

both IM localization and oscillation (demar-

cated by minD aggregates) was significantly

elevated in APOL3-coated versus uncoated

Gaudetet al.,Science 373 , eabf8113 (2021) 16 July 2021 2 of 14

9.4

65.3

6h

68.2

1h

101

102

103

104

90.6

6h + IFN-γ

102103104 105 102103 104 105 102103104 105

Uninfected

0.4

69.9

99.6

5.5

A

WT

ΔAPOL3

ΔAPOL3

+ APOL3

Untreated + IFN-γ

9.9% 0.9%

11.2% 6.0%

9.7% 1.9%

101

102

103

104

101 102 103 104 101 102 103 104

101

102

103

104

101

102

103

104

StmGFP

FL-3 (auto)

C Single-cell Stm burden Population Stm burden

SRHR

Infected

31.8

Uninfected Uninfected

0 24 6

Time (h)

Stm

replication (fold)

HR

Infected

31.1

Infected

34.7

1

10

100

1000

Bacteria per cell

245

HR

lentiviral

CRISPR

library

StmGFP

Enrichment of sgRNAs in

HR versus SR populations

SR HR

• IFN-γ + IFN-γ

Human epithelia

0

10

20

30

0

10

20

30

0

10

20

(^30) −IFN-γ
+IFN-γ
11.8
2.2
14.3
HR
B
SR
StmGFP
FL-3 (auto)
SR HR SR HR
0
1
2
3
0
1
2
3
0
25
50
75
0
1
2
3
4
Cytosol-invasive + IFN-γ Vacuole-confined + IFN-γ
StmΔsifA B. thailandensisS. flexneri StmΔinvA::pR1203^ S. Typhi
8h 18h 6h 8h 8h
** ns
ns
Bacterial load (rel: WT)
D
0
5
10
15
20
StmmScarlet
siCtrl
siAPOL3

• IFN-
  γ
  − IFN-
  γ
  Brightfield
  siCtrl
  Time (h)
  H
  R foci per well
  E
  Primary human intestinal fibroblasts (10h)
  0510
  0
  1000
  2000
  3000
  P = 0.0002
  − IFN-γ siCtrl


• IFN-γ siAPOL3


• IFN-γ siCtrl
  HR Stm foci
  10-35 μm
  ns
  Inset
  FACS
  Overlay
  WT
  ΔSTAT1
  ΔAPOL3
  Lamp1StmGFP Slow replicating (SR)Hyper-replicating (HR)
  Population (6h)
  SR HR
  IFN-γ-induced gene expression (Log 2 )

  ---

  
  

  Enrichment (-Log
  10
  P
  vlaue) APOL3
  STAT1
  HR vs. SR Enrichment (19,050 genes)
  -IFN-γ +IFN-γ^
  -5 0 5 10 -5 0 5 10
  5 4 3 2 1 0
  siCtrlsiA3
  IFN-γ−++
  37 kD−
  φ
  APOL3
  β−actin
  SR HR i
  10 μm
  Fig. 1. Genome-wide identification of humanAPOL3as an antibacterial
  ISG.(A) FACS of HeLa cells infected with GFP-expressingSalmonellaenterica
  Typhimurium (StmGFP). HRand SRgates are percentages of infected cells.
  Below, 3D confocal microscopy and calculated bacterial load per cell from each
  population (mean ± SEM). (B) Genome-wide CRISPR-Cas9 screen schema
  and gene-level enrichment scores in HRversus SRpopulations in the presence or
  absence of IFN-g. Each gene is plotted against relative (fold) induction of its
  mRNA in IFN-g–activated cells determined by RNA-seq. (C)StmGFPgrowth by
  FACS at 6 hours (left) and gentamicin protection assays (right) in APOL3-
  deficient HeLa cells (DAPOL3) genetically complemented with APOL3
  (bottom row) or empty retroviral control (top two rows). Fold is given as
  relative to 1-hour starting time point (input). (D) Increase in bacterial load
  [relative to wild-type (WT) cells] recovered from APOL3- or STAT1-deficient
  IFN-g–activated HeLa cells after the indicated time. **P<0.01, ***P<0.001
  (one-way ANOVA); ns, not significant. (E) Human primary intestinal myofibro-
  blasts treated withAPOL3siRNA or nontargeting scrambled control (siCtrl)
  (immunoblot, bottom right) and infected withStmmScarlet. Shown are represen-
  tative final micrographs (10 hours) and quantification of HRStm(foci 10 to
  35 mm) every hour (mean ± SD,n= 3, representative of two independent
  experiments. In (C) and (D), data are means ± SEM from four independent
  experiments and FACS plots representative of at least four independent
  experiments.fdenotes a nonspecific band. Scale bar, 75mm.
  RESEARCH | RESEARCH ARTICLE