Inhibition of adsorption and replication of the RNA-phage MS-2 in escherichia coli C 3000 by levallorphan

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Vol. 41, No. 6, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INHIBITION OF ADSORPTIONAND REPLICATION OF THE RNA-PHAGE MS-2 IN ESCHERICHIA COLI C 3000 BY LEVALLORPHAN C. Raab and R. Roschenthaler Institut

Received

fiir Hygiene und Medizinische Mikrobiologie, Technische Universitat Miinchen

November

5, 1970

SUMMARY - Multiplication

of the RNA-phage MS-2 in its host Escherichia coli C 3000 is strongly inhibited by levallorphan. This inhibition takes place at concentrations of levallorphan at which growth of the host is not inhibited. It could be demonstrated that levallorphan effects a desorption of the phage from its host. Most of the inhibition is due to a failure to infect the host. Inhibition of phage replication by levallorphan was found to be less affected than adsorption and infection. INTRODUCTION- It

has been shown that

morphinans selectively and that

inhibit

levallorphan

RNA synthesis

they induce a loss of polyamines (4).

protein synthesis decreased.

is affected

that

cells

At higher

(3) and the cellular

Brdar and Fromageot (6) reported

and other

in E.coli

(l,2,3)

concentrations

ATP level

in mouse fibroblasts

is the

synthesis of mengo virus RNA was strongly inhibited. The synthesis of mengo virus RNA was at least as sensitive to levallorphan as that of the host cell. As the loss of polyamines and other observations probable that levallorphan permeability of the cells

(7,8)

make it

might also induce some changes in we became interested in the question

whether

the adsorption of the phage MS-2 to the sex pili or the RNA synthesis of this bacterial virus might be affected by levallorphan. It could be shown that at concentrations which do not affect growth of the bacterial host levallorphan inhibits the adsorption of the phage to the host cell. At the same concentration the RNA synthesis of the phage is also inhibited but to a lesser degree. MATERIALS & METHODS- Materials: Levallorphan-tartrate was generously provided by Hoffmann-La Roche, Basel. Actinomycin D was purchased from Serva, Heidelberg and Freund's adjuvant from Hyland Laboratories, Los Angeles. Bacterial strains and Media: The RNA phage MS-2 and its host were a gift of Dr. B. Weisblum, Madison, Wise. USA. The cells were grown in a medium described

by E.J.

Simon and D. Van Praag (9) at pH 1429

8.1.

Vol. 41, No. 6, 1970

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Sodium succinate

(0.5

7:) served as carbon source and the medium was

supplemented with casamino acids (0.2 $, Difco). Assays for plaque forming units were performed by the agar layer method (IO). For mass propagation of MS-2 phage the defined medium described by Shimura, Moses and Nathans (11) was used. 10 Liter cultures were grown in a New Brunswick Microferm 4 x lo*. Cells

destined

for

fermentor actinomycin

and infected D treatment

at a cell

density

of

were grown in TPG-medium

supplemented with 0.2 $ of casamino acids (Difco). (13), Phage preparation: MS-2 phage was prepared and purified entirely according to Shimura, Moses and Nathans (11). The final phage suspension was purified Preparation

by CsCl-gradient

of antiphage

centrifugation.

serum: Antiphage

serum was produced by

immunization of rabbits (10,12). The rabbits were injected subcutaneously with a phage suspension at a concentration of IO "PFU/ml twice a week. The phage suspension was mixed 1 : adjuvant prior ear vein after

1

with Freund's

to injection. The animals were bled from the marginal four weeks. The blood was allowed to clot at 37'C,

the red cells were removed by low speed centrifugation, the serum was filtered and preserved frozen. The K-value of the antiserum was determined according to Adams (10) and was found to be 255. Measurement of burst size: The burst size of MS-2 phage was measured according determine used.

to the method described

plaque forming units

by Adams (10).

(PFU) the agar layer

To

technique

was

Actinomycin treatment of the host cells: The aotinomycin D treatment of E.coli cells was performed according to the method of Oeschger and Nathans (13). The actinomycin-treated cells were infected with phages at a concentration of 10 l2 PFU/ml and labelled with 14C-phenylalanine and 1 kc/ml 3 H-Uraoil. One vial contained 0.15 PC/ml -4 100 ~1 samples were taken at intervals 6.9 x 10 M levallorphane. of 10 minutes, precipitated with 5 $ trichloracetic acid, filtered on Sartorius membrane filters and the dried filters were counted in a Packard liquid scintillation counter. 14C-phenylalanine was SO mC/mM, that of The specific activity of the 'H-uraoil was 20 C/mM. These substances were products Saclay, France. HESULTS a) Inhibition

of adsorption

and infection

1430

of C.E.N.

- WThenlevallorphan

Was

Vol. 41, No. 6, 1970

added to a culture

BIOCHEMICAL

of E.coli

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C 3000 during

of the phage MS-2 (up to 8 min after

the time of adsorption

addition

of the phage) the time

at which the burst occured was retarded. This retardation depended on the concentration of levallorphan. The concentration ranged from 0.69 mM, at which no inhibition of the growth of the host cells was observed, to 1.1 mM, which caused slight inhibition (20 - 30 $). The levallorphan was diluted out after the adsorption time. The burst size of the phage multiplication remained constant but the background of PFU during

the latent

decreased sharply.

period

The latter

- representing infected bacteria observation suggests that either

adsorption of the phages or their levallorphane. FIG. 1

infection

0 10 20304050607080

FIG.

is inhibited

by

MIN.

1 Burst of MS-2 phage after treatment with levallorphan at different concentrations during adsorption time. Cells at a density of 2 x 10 cells/ml were infected with phages at a multiplicity of 40. At the same time levallorphan was added at a concentration of I) 0.69 mu +-+, 2) 0.92 a The closed circles represent the x-x, 3) 1.1 mM o-o. control with no addition (0). Levallorphan was present only during the adsorption time i.e. the time interval from addition of phage until 8 minutes thereafter. After this time levallorphan was diluted out by treating the suspension with phage antiserum (1:50) for 5 minutes. Then the serum was diluted out I:400 before plating. At 1) the burst size = 36, at 2) burst size = 30, 3) burst size = 32, and at 0 it is 33

In order to distinguish between adsorption and infection the metabolism of the cells was inhibited with KCN 2 minutes prior to infection and during the 8 minutes adsorption time, after which it was diluted out. If the phages were added to a culture with a 1431

Vol. 41, No. 6, 1970

multiplicity

BIOCHEMICAL

of 40 and it

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was assumed that

all

phages were adsorbed,

then about 40 phages should give rise to only one plaque. If the phages are detached from the bacteria by rising concentrations of levallorphan the plaque forming units should also rise form plaques with the indicator bacteria. and is shown in FIG. levallorphan. phage after

If

28. The PFTJrise

as more free phages can This experiment was done

with

the concentration

the test was performed with

8 minutes adsorption

were observed indicating inhibition with KCN. On the other hand, if

that

time with

no bacteria

the bacteria

inactivation

antiphage

of free

serum no plaques

were infected

were infected

of

during

during

the 8 minutes

and then KCN added during 2 minutes thereafter the infected could be counted by the plaques they formed. With different

bacteria

concentrations

time the

of levallorphan

number of infectious after this treatment

present

during

the infection

centers also increased. If antiserum was added the infectious centers decreased indicating that 0

A

500. 400.

0 0____1__1

2 300. Y -:-:::.: u 200 aI

$ ‘“j :‘--------._-mm7---t I

1

I

I

.23 .46 .69 .92 mM

FIG.

130

0

LEVALLORPHAN

0.69

092

110

mM LEVALLORPHAN

28 Detachment from cyanide-treated host cells of MS-2 phage by levallorphan. Cells at a density of 2 x IO8 cells/ml were treated with 0.01 M KCN. After 2 minutes phages were added at a multiplicity of 40, the culture divided into three tubes and the levallorphan added in the appropriate concentrations. The phages were allowed to adsorb during 8 minutes. Then the samples were diluted I:100 and plated according to the agar layer method (9) B Liberation of free ES-2 phage from the host cells by levallorphan. were infected with phage at a E.coli C 3000 cells (lO'/ml) multiplicity of 40 and different concentrations of levallorphan IL6KCN was were added. After the adsorption time (8 min) 0.01 present during 2 minutes. Then the samples were divided in two series, one treated with phage antiserum o---o to inactivate free phage, the other one was plated without The calculated difference between the serum treatment o--o. two represents free (non adsorbed) phage o-e 1432

Vol. 41, No. 6, 1970

BIOCHEMICAL

fewer bacteria difference

were infected

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in the presence of levallorphan.

The

between both curves as shown in FIG. 2B is mainly

a

reflection of the number of phages detached from the host bacteria. From these data it appears that the inhibition of phage infection by levallorphan b) Inhibition burst

is a consequence of decreased phage adsorption.

with increasing

not only adsorption levallorphan an inhibition less strong

- The increasing

of phage production concentrations is inhibited

retardation

of levallorphan

suggests that

but also phage synthesis.

When

is added after the infection with phage (after 8 min) of phage synthesis is also found. This inhibition is than in the case when the levallorphan

is present prior

to infection. After 39 minutes levallorphan had the least effect since most of the phages are already completed: FIG. 4. The fact the plaque forming units later. To investigate

decrease after

further

the burst will

the inhibition

DNA dependent synthesis

of RNA and protein

was inhibited

0

10 20304050607080

that

be discussed

of the phage synthesis

according to the technique of Oeschger and Nathans (13). levallorphan concentration was 0.69 mM, a concentration culture. FIG.3 to inhibit growth of a bacterial

FIG.

of the

the

by actinomycin The not sufficient

MINUTES

5 Measurement of burst size at different times of levallorphan addition. Levallorphan was added to phage infected cultures at a concentration of 0.69 m&I. 0 = at 39 minutes after phage 1 = after 8 minutes (adsorption time) I,, addition 70, 2 = at the same time as the phage addition X, and 3 = addition of levallorphan 10 minutes before phage addition

1433

Vol. 4 1, No. 6, 1970

BIOCHEMICAL

The synthesis (20 $) than that At this

of phage RNA was found to be somewhat more inhibited of phage protein (9 7;).

concentration

60 to 05 p, if

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the burst

levallorphan

the time of infection 30 minutes. The reason for FIG. 4 is not yet

size of the phage is reduced about

remains in contact

until

plating

the decrease in burst completely

understood.

size with

double labelling to indicate that levallorphan specific

with

biological

different

4

from about

time as shown in

Preliminary

We therefore suspect that when remaining in the results

obtained

experiments and CsCl-gradient centrifugation some phage is produced in the presence of buoyant density

with seem

and about 50 $ less

activity.

0 10 20 3040

FIG.

occurs.

is retarded

The PFU/ml decrease and

after a certain time reach a constant value. phage is produced which loses its infectivity medium in which the lysis

with the cells

and the burst

506070

0 10 20304050607080 MINUTES

Phage development in actinomycin treated cells in presence of levallorphan. Cells at a concentration of 8 x lo8 cells/ml were treated according to Oeschger and Nathans (1212 The actinomycin inhibited cells wgre infected with IO phages/# and labelled C-phenylalanine with 10 PC/ml of II-uracil (A) and 0.15 PC/ml (B). One sample obtained levallorphan in a concentration of 0.69 mh! (2). As controls an uninfected sample (3) and an infected sample (1) were used.

DISCUSSION - Brdar and Fromageot (6) reported strong inhibition of mengo virus in mouse fibroblasts by levallorphan. Simon et al. (4) reported leakage of polyamines from bacterial cells, and Green and Magasanik (5) have described a leakage and destruction of ATP in presence of high together with observations made doses of levallorphan. These facts, by Dr. P.L. Boquet (7) and by Gale (8) make it probable that levallorphan has an effect on the cell membranes. The attachment of the phage to the sex pili could therefore also be affected. Cur 1434

BIOCHEMICAL

Vol. 41, No. 6, 1970

results

indicate

that

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infection

liberated

from uninfected

The burst

size with different

is inhibited

bacteria

and that

phage is

in the presence of levallorphan.

concentrations

of levallorphan

is not

changed when the drug is present only during the adsorption period but the base line during the latent period is drastically decreased, indicating present

that

c

during

antiphage

this

time.

fewer infected

cells

in the untreated the culture

develop normally cells.

when levallorphan

Free phages were inactivated

serum. Once the levallorphan

the infected

burst

s are far

If,

is diluted giving

in contrast,

is

by the

out the phage in

the same burst levallorphan

size as

remains in

medium to the end of the phage development the normal

size is reduced by 83 $. However when levallorphan

after the adsorption time the phage synthesis The concentration of levallorphan during this

is added

is decreased only 28 $. treatment is 0.69 m&I

and is not sufficient to show a visible inhibition of growth of the host during the time period in question. The maximal retardation of the burst

of 30 minutes at this

concentration

is obtained

levallorphan is present from the time of addition the end of phage development. These facts

show clearly

that

it

is possible

effects of levallorphan on phage multiplication: infection and ii) inhibition of the synthesis

when

of the phages until to dissociate

two

i) inhibition of of phage. The inhibition

of infection may result from a decreased ability of the bacteria to adsorb phages. This is suggested by the increasing number of free phages found when the levallorphan

concentration

is increased

under

conditions under which infection is both permitted and prevented. That phage synthesis is also inhibited is supported by the experiments with actinomycin D. However the synthesis of phage is also inhibited by this drug. The comparison of incorporation of 14C-phenylalanine with

that

of 'H-uracil

incorporation

of uracil

shows that

there is a certain

being more inhibited

difference,

than that

the

of the amino

acid, which is in agreement with results obtained when the overall incorporation of these precursors into the cell in presence of levallorphan is compared (3). We therefore conclude that of the 83 $ inhibition obtained when levallorphan is present throughout the whole cycle of phage development less than 30 $ can be ascribed to inhibition of phage replication, while about 30 $ is due to inhibition of adsorption and/ or penetration. As this manuscript was prepared for publication a report on the action of levorphanol on replication and synthesis of RNA phages appeared (14). 1435

Vol. 41, No. 6, 1970

Our results

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are in agreement concerning

retardation

of burst

and

inhibition of phage specific RNA and protein synthesis. With low concentrations of levallorphan however we found that most of the inhibition was due to decreased infection of the phage.

ACKNOWLEDGEMENTS - We wish to thank Dr. P. Fromageot for generous gifts of radioactive material, the Deutsche Forschungsgemeinschaft for providing the scintillation counter, and Prof. K. Liebermeister for providing laboratory facilities. We are also grateful to Dr. B. Weisblum who gave us the MS-2 phage and its host, and to Dr. D. Jarvis for valuable discussion and reviewing the manuscript. REFERENCES

1. :: 4.

2: 7. ;: 10. 11.

12. 13. 14.

Simon, E.J., Science, 144, 543 (1964) Simon, E.J. and D.Van Praag, Proc.Nat1.Acad.Sci.U.S. 2, 1151 (1969) Roschenthaler, R., M.A. Devynck, P. Fromageot and E.J. Simon, Biochim.Biophys.Acta 182, 481 (1969) Simon, E.J., S.S. Cohen and A. Raina, Biochem.Biophys.Res.Comun. 3, 482 (1966) Greene, R. and B. Magasanik, Mol.Pharmacol. 2, 453 (1967) Brdar, B. and P. Fromageot, FEBS Letters 6, 190 (1970) Boquet, P.L., C.E.N. Saclay in preparation Gale, E.F., J.Gen.Microb. 2, part 3 (1969) Simon, E.J. and D. Van Praag, Proc.Nat1.Acad.Sci.U.S. 1, 877 (1964 Adams, M.H., wBacteriophages", Interscience, New York f- 1959) pe 461 Shimura, Y., R.E. Moses and D. Nathans, J.Mol.Biol. 12, 266 (1965) Clowes, R.C. and W. Hayes, "Experiments in Microbial Genetics", Blackwell, Oxford (1968), p. 232 Oeschger, M.P. and D. Nathans, J.Mol.Biol. 22, 235 (1966) Simon, E.J., D. Grawes and J. Rand, Biochem.Biophys.Res.Comun. &, 1143 (1970)

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