Science - USA (2021-11-05)

Huet al.,Science 374 , eabe6723 (2021) 5 November 2021 4 of 13

A C

DE

FGH

Listeria monocytogenes

Buffer BSA

SPRR2A SPRR2A

I SP

Buffer

I SP

PC

I SP

PC:PS

I SP

PC:CL

250

150

100

75

50

37

25

20

15

10

kDa

Buffer

SPRR2A

PC:PS liposome

Triglyceride

Cardiolipin

Diacylglycerol

Phosphatidic acid

Phosphatidylserine

Phosphatidylethanolamine

Phosphatidylcholine

Phosphatidylglycerol

Phosphatidylinositol

PtdIns(4)P

PtdIns(4,5)P2

PtdIns(3,4,5)P3

Cholesterol

Sphingomyelin

Sulfatide

Blank

0246810

0

20

40

60

80

100

120

SPRR2

% CFU remaining

Bacteroides thetaiotaomicron

Escherichia coli K12

Citrobacter rodentium

Lactobacillus reuteri

Enterococcus faecalis

Listeria monocytogenes

Gram-negative:

Gram-positive:

B

0

20

40

60

80

100

120

Listeria monocytogenes

%

C

FU

remaining

11

0.5 0.5

0 0

anti-SPRR2A 0 0

SPRR2A

****

**

**

0 200 400 600 800 1000

120

100

80

60

40

20

0

Dye release (%)

SPRR2A

OG

80% PC

20% PS

CF-loaded

0 200 400 600 800 1000

Time (s)

80% PC

20% CL

CF-loaded

SPRR2A

OG

0 200 400 600 800 1000

100% PC

CF-loaded

SPRR2A

Buffer

SPRR2A

OG

Liposome

composition:

Buffer

SA

SPRR2A

SPRR2A

SPRR2A

SPRR2A

SPRR2A

0 10 20 30 40 50 60

Time (min)

0 10 20 30 40 50 60

Listeria monocytogenes Lactobacillus reuteri

0

20

40

60

80

Propidium iodide uptake

(% of max)

100

I

1

10

100

% CFU remaining

05

0.0

01

0

0 5

0 0.1

SPRR2A

LPS (mg/mL):

0.010.1

55

Listeria monocytogenes

***

**

*

0 200 400 600 800 1000

0

20

40

60

80

100

120

Time (s)

SPRR2A

SPRR2A + LPS

LPS

Buffer

Dye release (%)

Treatment

OG

Liposome composition:

80% PC

20% PS

CF-loaded

J

0

20

40

60

80

100 ****

SPRR2A

SPRR2A + LPS

Dye release (%)

K Liposome composition:

80% PC

20% PS

CF-loaded

Fig. 2. SPRR2A is a bactericidal protein that targets Gram-positive
bacteria by membrane permeabilization.(A) Recombinant SPRR2A was
expressed in a baculovirus expression system and purified by size exclusion
chromatography. SPRR2A was added to ~10^4 CFU of log-phase bacteria for
2 hours, and surviving bacteria were enumerated by dilution plating. (B) SPRR2A
was added to log-phaseL. monocytogenesin the presence of anti-SPRR2A
antibody, and surviving bacteria were enumerated by dilution plating.
(C) Transmission electron microscopy ofL. monocytogenesafter incubation

with SPRR2A. Bovine serum albumin (BSA) was used as a negative control.

Examples of cell surface damage and cytoplasmic leakage are indicated with red

arrowheads. Scale bars, 200 nm. (D)L. monocytogenes(left) andL. reuteri

(right) were treated with SPRR2A or BSA as control, and propidium iodide (PI)

uptake was measured over 1 hour. (E) Membranes displaying various lipids were

incubated with 1mg/ml of SPRR2A and detected with anti-SPRR2A antibody.

(F) SPRR2A was incubated with liposomes having the indicated lipid

compositions. After ultracentrifugation, the liposome-free supernatant (S)

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