Science - USA (2022-01-14)

four-gene operon fromLysobacter enzymogenes
exhibited robust defense against coliphages T4,
T5, and T6 (Fig. 2, C and D, and fig. S6, B and
C). Deletion of the bGSDM gene from the
Lysobacteroperon abolished protection (Fig. 2,
CandD,andfig.S6C).Thus,thebGSDMis
essential for defense.
Expression of some of the bGSDM-protease
systems inE. coliinduced potent cellular tox-
icity in the absence of phage infection (Fig. 2,

E and F; fig. S7, B and C; and table S5). Particu-

larly strong toxicity was observed for aRunella

system, which required bGSDM palmitoylation

(Fig. 2E and fig. S7, B and C). Time-lapse mi-

croscopy in the presence of propidium iodide

(PI) showed that cells expressing theRunella

system ceased dividing and lost membrane in-

tegrity, which suggests that bGSDM activation

induces membrane disruption (Fig. 2, E and F;

fig. S7D; and movies S1 and S2). Mutation of

the predictedRunellacaspase-like protease

active site residues H796 and C804 ablated all

cellular toxicity (Fig. 3A). TheRunellabGSDM

and protease only induced cellular toxicity

when expressed together, which suggests that

the protease targets bGSDM during system

activation (Fig. 3A and fig. S8A). In fact, a muta-

tion that disrupted the active site of the second

trypsin-like protease in theLysobacterbGSDM

system abolished antiphage defense (fig. S8B).

222 14 JANUARY 2022¥VOL 375 ISSUE 6577 science.orgSCIENCE

A

Vitiosangium

bGSDM

B

mouse

GSDMA3

400 aa

100 aa

Prokaryotes

Fungi

Invertebrates

Vertebrates

NTD

CTD

N-term cys

E

Bradyrhizobium

VitiosangiumRunella

(WT)

Runella

(C3A)Gillisia

Rhodoplanes

30

40

50

60

70

Tm

(°C)

hydroxylamine +hydroxylamine

N-term cys

no N-term cys

C BradyrhizobiumbGSDM

D

Bradyrhizobium FO−FC omit map

C3

C4

C

N

C

N

N

C

CTD

N

F211

L24

F253

M12

C3

L22

V90

I163

L115

V86

P26

L78

C

B

DA E P

L91

α 1

β 2 β 1

β 3

β 4

β 5

α 2

α 3

β 6 β 9

β 7 β 8

α 4

β 10 β^11

N N

C C

autoinhibition?

Fig. 1. Structures of bGSDMs reveal homology with mammalian cell
death effectors.(A) Gasdermin phylogenetic tree. The sizes of the gasdermin
NTDs and CTDs are depicted. Vertebrate gasdermins are labeled with
single letters (“A”to“E”) indicating human gasdermins GSDMA to GSDME,
and“P”depicts pejvakin. The black teardrop indicates a conserved N-terminal
cysteine (N-term cys). A representative set of 20 fungal gasdermins are
included in the tree. aa, amino acid. (B) Crystal structures of bGSDMs
from species of the generaBradyrhizobiumandVitiosangium. bGSDM
structures reveal homology to the NTD of mammalian gasdermins in an
inactive conformation, including mouse GSDMA3 (Protein Data Bank ID
5B5R). (C) Gasdermin topology diagrams indicate a conserved central core
of the bacterial and mammalian NTD. bGSDMs notably lack the CTD required

for autoinhibition of mammalian gasdermins and instead encode a short

C-terminal peptide. (D) Simulated annealing FO−FComit map (contoured at

3.0s)fromtheBradyrhizobiumbGSDM fit with a palmitoyl modification

at C3. Omit map is shown as green mesh, and select residues forming

a hydrophobic pocket around the palmitoyl group are indicated. (E) Melting

temperatures (Tm) of bGSDMs with and without N-terminal cysteines, as

determined with thermofluor assays. Data are the means and standard

deviations of three technical replicates and are representative of three

independent experiments. WT, wild-type. Single-letter abbreviations for the

aminoacidresiduesareasfollows:A,Ala;C,Cys;D,Asp;E,Glu;F,Phe;

G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg;

S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

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