Role of cell surface mobility on bacteriophage infection: Translocation of salmonella phages to membrane adhesions

Vol.

73, No,

BIOCHEMICAL

3,1976

ROLE

OF

CELL

SURFACE

TRANSLOCATION

Toshio The

Institute

Received

OF

MOBILITY

Shintaro

ON

PHAGES

The

RESEARCH

COMMUNICATIONS

BACTERIOPHAGE

IWASHITA,

Science,

October

BIOPHYSICAL

SALMONELLA

TOMITA, of Medical

AND

TO

and

University

INFECTION:

MEMBRANE

Shiro

ADHESIONS

KANEGASAKI

of Tokyo,

P.O.Takanawa,

Tokyo

108

5,1976

SUMWRY: Ph age 615 adsorbed at a low temperature (or by short-time incubation) to the outer surface of Salmonella anatum gathers on further incubation at a high temperature to a certain region where the inner and outer membranes may join. This was demonstrated by separating the inner and outer membranes of the cells in sucrose gradient after addition of 3%-labeled 615 to the cells. Radioactivity adsorbed at 4” was first recovered mainly with the dense outer membrane but disappeared by further incubation at 35” within 5 min. Instead, the radioactivity was recovered with the membrane fraction which had intermediate density. S UC h p h age traslocation was not observed when phage 815 was added to a m mutant of S.anatum to which the phage can adsorb but fail to infect. A host range mutant phage which can infect the a mutant migrated to the intermediate dense region,

The

smooth

antigen, of the

specific

the outer

peripheral surface

of a single

species

polysaccharide the

release

bacterial which

specific

DNA

the

Salmonella and

how

sites,

the

phage

these

limited

randomly

to reach

al I over than

the

to the

the

adhesions

of binding outer

negative acid,

adhesion

reside

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

early

stages

yet

kind

sites.

sites.

The and

the

subsequently

phage

adsorption,

penetrate

into

especially

the those

of DNA

including

adhesion

If the

is that

the

receptors

adsorbed

tothe

adhesion

rhe

are

the

place

be considered over

directly

sites

smooth

between sites

can

is that

the

sites

adhesion

phages

phages

0 antigen

completion

the

to the 807

the

consists

understood.

possibility

and other

and

of possibilities

One

host

After may

to 0

constituent phages

of infection,

the

bacteria. two

the

particles

over

P22 adsorb

of these

acid

clearly phage

preferentially

membrane

migrate

nucleic

located

of Gram

plate

(3,7).

the

various

preferentially

receptors

numbers

(8) that

base

to its receptor not

and

is an important

adsorption

and

are

9341

or deacetylates

phage

of phage

its nucleic

The

in these

however,

membranes

~34, which

cleaves

for

involved

by Bayer

penetrates

which

be triggered

attachment

phages

inner

phage

may

cytoplasm,

as e15,

organisms.

is responsible

Events

demonstrated

the phages

Copyright All rights

protein

between

It was

other

of enzyme

of phage

such

of Iipopalysaccharide, negative

and

into

phages

of Gram

cytoplasm.

transfer

where

portion

(l-6)

occur

outer

Salmonella

the

for

adhesion

recognize are receptor

during

distributed at the incubation.

sites

Vol.

73, No.

3,1976

In this adhesion

BIOCHEMICAL

communication sites.

MATERIALS

we show

The

AND

step

seems

AND

the

BIOPHYSICAL

migration

RESEARCH

of phage

to be essential

for

the

~‘5

from

COMMUNICATIONS

the

attachment

accomplishment

to

of phage

infection.

METHODS:

Most of the methods and materials used in this experiment were the same as those described previously (9). Bacteria and phases: Salmonella anatum A 1, S. gnatum SK59 (a m mutant) (9) and phages oT5vir, eT5hp32 (a host range mutant phage which infects the-bacterium) (9) and ~341 were used. 35S-Labeled phages: 35S-labeled phages eT5 vir, eT5hp32 and ~341 were prepared and purified according to the method described previously (9). Adsorption of radioactive ohaae to bacteria: Bacteria grown in L broth (5 x 108 cells/ml) and the radiactive phage (approximately 100,000 cpm/250 ml) (see figure legends) were mixed at 0’ or 35” and incubated at 0’ or 350. After the incubation, the mixtures were quickly chilled and phagewdsorbed cells were recovered by cenfrifugation. Membrane This was done according to a modified method (10) of the Schnaitman procedure (11). The position of the inner and outer membrane were usually defined by protein profile (12), distribution of cytochrome bT (13), and contents of rhamnose (14). Thedensity of the resultant peaks is comparable to that observed by Smit et al. (IO). The densest peak is outer membrane since it containes the majority of rhamnose. The middle band is unseparated inner and outer membrane as indicated by an intermediate amount of rhamnose and cytochrome bT . The lightest bands are inner membrane as indicated by the lack of rhamnose and high absorbance of cytochrome bl .

RESULTS

:

Salmonella for

anatum

30 min.

cell.

The

The

cells

region

the

membrane

the

radioactive the

region

phage were

decreased of incubation,

indicate

that

are

can

the

to the

further

that

in the

phage

the

receptor

site

intermediate

outer other

than and

previously

reported

bacterial

fail

to lyse

(9).

to its receptor

One

the

gathers

such

The

region

mutants mutant

808

(Fig.

the

outer

the

membrane designated (SK59)

sucrose

fig.

cells

and

When

the

ICI.

(Fig.

and

Id).

The

results

that outer

the

phage

membranes

adhesions. as pep were

to which mixed

cells

After

which

suggest

inner

with

membrane

lipopolysaccharide also

pressure

recovered

lb).

increased

results

ceils

were

when

membrane

where

to the

phage

in the

outer

at 4’

was associated

15 set

region

incubated

a discontinuous

obtained

radioactivity

with

.

onto

of radioactivity were

dense

membrane

migrates

but

the

and

in a French

of the

at 35O for

at 35O,

attaches

applied

rest

results

was found

initially

over

the

incubated

subsequently have

adsorb

and

no radioactivity

randomly

fused, We

incubated

and

mixed

disrupted

of radioactivity

Similar

density.

were

and were

60%

membrane

mixed

e15vir

by centrifugotion

almost

outer

were

while

5 min

adsorbed

la,

of intermediate

phage

distributes

dense

phage

by centrifugation

in Fig.

of the

35 S-labeled

collected

collected

As shown

in the

and

were

membranes

gradient.

and

cells

with

phage the

e15

BIOCHEMICAL

Vol. 73, No. 3,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 10

20 Fraction

Fig.

1.

Translocation

of 35S-labeled

phase

e15 vir

from

the

primary

adsorption

sites

(the

outer membrane) to the membrane with intermediate density. Al cells (5.5 x 108/ml) in 150 ml of chilfed L-broth were mixed with 35S-labeled el%ir (89,000 cpm:MOI+l. 15) in a ice bath. After 30 min for adsorption, the mixture was di vided into three 50 ml portions (a,c and d) and two portions were further incubated at 35” for 1 (c) or 5 min(d). In another experiment, the L-broth culture of Al cells (5.5 x 108cells/ml) in 50 ml was mixed with 3%labeled cT5vir (88,000 cpm:MOI=O. 15) and incubated for 15 set at 35” (b). After incubation, the mixtures were poured into 200 ml (o,c and d) or 150 ml (b) of precooled L-broth containing carrier cells(l, 1 x 10” or 8 x lOTo cells, respectively). The cells washed twice with ice-cold N-2-hydroxyethylpiperazine-N’-2’ethanesulfonic acid (HEPES) buffer, pH 7.4 (10 mM) were suspended in 25 ml of the same buffer, and after addition of 0.5 mg each of deoxyribonuclease and ribonuclease, disrupted in a French pressure cell at 1,200 kg/cm2. Membranes were collected by centrifugation in a Spinco type 60Ti rotor for 3 hrs at 40,000 rpm, suspended into 1 ml of the HEPES buffer and applied to a discontinuous sucrose density gradient (1,5 ml of 2.02 M, 5.5 ml of 1.44 M and 4 ml of 0.77 M sucrose in the buffer). The gradients were centrifuged in a Spinco SW 41 Ti rotor for 15 hrs at 20,500 rpm. The fractions were At iquots of the fractions were spotted on collected by puncturing the bottom of the tubes. 25 mm filter discs (Whatman GF83) and radioactivity was measured (+). The protein profile was also shown (-0 l - - ). 0 and I indicate the positions of the outer and inner membrane, respectively.

radioactive

phage

eT5vir

above

ond

as described to the pels this Lack

m

bacteria

mutant of migration

were lacks

and

incubated

the resultswere recovered

an anchoring

of the

phage-receptor

at 35” shown

with

the

for

5 min.

The

2.

Almost

in Fig. outer

mechanism

membrane.

of phage

complex

809

in the

particles mutant

membranes all The

were

radioactivity results

at the is less

adsorbed

suggest membrane

likely

separated

(see

that adhesions. below).

Vol.

73, No.

3, 1976

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

20

10 Fraction

Fig. 2. Association of 35S-labeled e’5vir with the outer membrane of m mutant celis. SK 59 cells (5.5 x 108/mi) in 250 ml of L-broth were mixed with J% -labeled e15vir (107,000 cpm, MOkO. 04) and incubated for 5 min at 35”. For disruption of the cells and separation of membranes, the similar conditions as described in the legend of Fig. 1 were employed. Radioactivity(+) and protein profile@me0) were shown. 0 and l indicate the positions of the outer and inner membrane, respectively.

20 3 z 0 u

0 ‘0 x

2 1.0 10

-

2 .> .-

1.0

;: ;

0.5

03

0: 3

c .P,

2:

cl cc

1 0

0 20

10 Fraction

Fig.

3.

Miaration

of a host

range

mutant

phage,

c’5h032.

on the

cell

surface

of

--@-e-.(5.5 x 1 /ml) SK 59 cells in 50 ml of L-broth were infected with 35S-labeled c15hp32 (28,000 cpm:MOlrO. 005) and incubated for 15 set (a) or 5 min (b) at 35’. The conditions for disrupting the cells and separating membranes were similar as those described in the legend of Fig. 1. Radioactivity (+) and protein profile (- - l - .) were shown. 0 and I indicate the positions of the outer and inner membrane, respectively.

810

vol.

73, No.

BIOCHEMICAL

3,1976

As previously come

by

reported,

a host

the m

range

bacteria,

o significant

the

with

fraction

with

migration

similar

outer

accomplishment

were

density.

mutants

mutant

phage,

mutant

bocterio, results

in m

As shown

range

pep

to the

such

performed.

These

membrane

Using

host

of the

infection

(9).

of the

membrane

outer

of the

eT5

experiments

the intermediote the

of phage

of phage

of radioactivity

the

from

inhibition

mutation

portion

associated

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

intermediate

dense

was originally

into

suggest

the

that

and

30 and b,

which

migrated

be over-

e 15hp32

in Fig.

phage

strongly

can

membrane

the

phage

mambrane

is essential

for

the

is surrounded

by a rigid

layer

infection.

DISCUSSION: The the

cytoplasmic

membrane

peptideglycan

proteins

and

and

is a limited

(8).

These

saccharide

number

are

translocated

to the

membrane

with

to be

(or at least

penetrates

initially

the

wild

adhesion

sites

wos fast

and

the

of fixation The

mechanism

unclear

bacteria

phage

of the

ot the

has otready

can

(9).

These

phages

not

present.

The

demonstrated

microscope,

the

of migration

of phage-receptor

direction

the by

lateral

diffusion complexes

and

phage

adhesion

( or the

play

diffusion et al.

of the are

at 35O. blocks

by a host

range

more

efficiently

traslocation

infection.

of

In the m although

phage-receptor

on essential

and newly

reverse.

811

The

which

of 2.~

molecules

of the

(17)

membrane.

mutant

sites,

is considered

5 min

the

the

migrated

outer

that

the

the

mutant, mechanism

unknown.

receptor

may

that

region

within

suggest phage

ot the

of the

surface

inner

mutants

accomplishing

MChlradt

of the

pep

which

membrane of this

cells

lipopoly-

through

wos overcome

strongly

is still

and

in a bacterial

the

results

of the

mobility cell

Although

infect

adhesions

of traslocotion

the

but thisdifficulty

for

outer

membrone

observed

to be trapped

ot the

on the

complexes.

mutant

The

of the

we demonstrated

was accomplished

was not stage

dense

between

migration

is essential seems

lipopolysocchoride) of the

early

phage

adhesions

type

in the

the

type

to the

receptor

membranes,

synthesized (16)

paper,

outer

plasmolysis

newly

membrone

In this

and

after

where

of

of lipopolysacchorides,

(inner)

visible

sites

cytoplasm.

include)

ot the

are

to the outer

phage

consists

cytoplasmic

to be the

to the

of the

latter

which

density.

This

wild

the

intermediate

cT5infection phage.

bacteria The

sites

into

adsorbed

translocation

phoges the

acid

of migration

the

of adhesion

molecules

eT5

than

Between

considered

phoge

mutant

(15).

are

nucleic

phage

membrane.

adhesions

phage

The

outer

phospholipids

there

rate

the

of Gram-negative

( in this role

newly

Bayer formed

complex)

for

case,

the

under

the

lateral

synthesized (18)

is also

movement

lipopolysaccharides the

electron

lipopolysaccharides

In addition,

neither

and cteovageof

that

Vol.

73, No.

0 antigen plate

polysaccharide

of the

smooth since

phws, or phage

Felix

saccharide gathered

The

enzymatic

especially

specific

0,

the

and

action

phages

the

the mechanism

intermediate

dense

region

formed

treatment

can

release

( unpublished of the

phage

of the

traslocation

of the

membrane

number

observation

). Further

ACKNOWLEDGEMENTS:

acid

into

We

to have

of phage

the

thank

cytoplasm

Dr.

We also thank Dr. Masanosuke Yoshikawa work was supported in part by a grant-in-aid of Education, Science and Culture, Japan.

Yoshito for

most

with

takes

the

of the

polysaccharide

smooth

phages

specific

(l-6)

allow

(5)

of the

lipopolyphages,

(unpublished

which

base

movement

portion

incubation

of the

its DNA,

are

of the

observation). may

correct

be required orientation

DNA. is,

particles

lateral

R-core

from

phages,

ejected

action

0 antigen

specific

their

experiments

the

cleave

after

smooth

fraction

enzymatic

to be the

of the

to eject

seemed

not

different

of the

surface

a significant

nucleic

plate

the explain

region

movement

cell

even

is known

dense

perpendicular

above

does

is quite

intermediate

the

after

of which

base

nor

sufficiently

which

morphology

of the

e15

may

(c341),

receptor

whose

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by phage

phages

9341

to the

for

Whatever

shown

phage

(19)

also

of the

BIOCHEMICAL

3, 1976

radioactive

since

EDTA-Triton associated

needed place

Kaziro

his support for scientific

with

few

plaques

X100.

This

the

to clarify through

for

phage

outer

whether the

in the were,

treatment membrane the

penetration

region.

his interest

and encouragement. research from

and the

criticism. This Ministry

REFERENCES: S. (1973) Biachem. Biophys. Res. Comn., a, 4031. Iwashita, S., and Kanegasaki, 409. 2. Kanegasaki, S., and Wright, A. (1973) Virology, 2, 160-173. 3. Iwashita, S., and Kanegasaki, S. (1975) Virology, &, 27-34. 4. Iwashita, S., and Kanegasaki, S. (1976) Eur. J. Biochem., 45, 87-94. 5. Iwashita, S., and Kanegasaki, S. (1976) J. Biol. Chem. , in press. S. (1975) Abstr., 3rd Int. Cong. Viol., 6. Takeda, K., Uetake, H., and Toyama, hAcldrid, p.213. 7. Israel, J. V., Anderson, T.F., and Levine, M, (1967) Proc. Natl. Acad. Sci. U.S., z, 284-291. 8. Bayer, M. E. (1974) Ann. N.Y. Acad. Sci., 235, 6-28. 9. Kanegasaki, S., and Tomita, T. (1976) J. Bacterial., m, 7-13. 10. Smit, J., Kamio, Y., and Nikaido, H. (1975) J. Bacterial., 124, 942-958. 11. Schnaitman, C. A. (1970) J. Bacterial., 104, 890-901. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J . (1951) J. Biol . Chem ., 193, 265-275. 13. Osborn, M. J., Gander, J. E., Parisi, E., and Carson, J. (1972) J. Biol. Chem., 247, 3962-3972. 14. Bray, D., and Robbins, P.W. (1967) J Mol. Biol., 30, 457-475.

812

Vol. 73, No. 3, 1976

15. 16. 17. 18. 19.

Wright, Mijhlradt,

A.,

and P. F.,

BIOCHEMICAL

Kanegosaki, Menzel,

S. J.,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(1971) Golecki,

J.

physiol. Rev., 51, 748-784. R., and Speth, V. (1973)

Biochem., 35, 471-481. Miihlradt, P. F., Menzel, J., Golecki, J. R., and Speth, Biochem., &3, 533-539. Bayer, M. E. (1975) In Proceedings of the 1st Intersectional Vol. 2, pp. 43-49, Univ. of Tokyo Press. Lindberg, A. A. (1967) J. Gen. Microbial., 48, 225-233.

813

V.

(1974)

Congress

Eur.

J.

Eur.

J.

of the

I.A.M.S.