Expression of A β-lactamase activity in Mycoplasma capricolum transfected with the liposome-encapsulated E. coli pBR 322 plasmid

Vol. 108, No. 3, 1982 October

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

15, 1982

Pages 982-986

EXPRESSION OF A B-LACTAMASE

IN MYCOPLASMA CAPRICOLUM TRANSFECTED

ACTIVITY

E.COLI pBR 322 PLASMID

WITH THE LIPOSOME-ENCAPSULATED Claude

Nicolau'

and Shlomo

Rottem 2

1 Centre de Biophysique Moleculaire, C.N.R.S., lA, avenue de la Recherche Scientifique 45045 Orleans cedex, France 2

Received

Department of Membrane and Ultrastructure The Hebrew University, Hadassah Medical Jerusalem, Israel

August

Research, School,

2, 1982

pBR 322 plasmid of E.co%i was used to transfect in culture. 12 hours after transfection the M. the enzyme B-lactamase as shown by the hydrolysis of cephalosporin by extracts of the transfected cells. Extracts of cells incubated with empty-liposomes or with free DNA failed to hydrolyse cephalosporin. The expression of this bacterial enzyme and of acquired tetracycline resistance by the M.caprico7,m cells was evidenced also by plating the cells in the presence of tetracycline, indicating a transformation rate of 'L 10m6. This represents direct evidence of fusion of mycoplasma with liposomes as well as the capacity of the former to express an exogenous, bacterial enzyme. Liposome-encapsulated

Mycoplastna capricoh cells capricobn cells expressed

Mycoplasmas The small

size

model

genetic

for

of these

although

with

studies

these (2)

lipid

smallest

(1).

very

engineering

there

is

and simplest

genome would Unfortunately,

few mutants experiments

organisms

are were

incorporate

no evidence

encapsulated

cells in

were

liposomes

by the assays

of eucaryotic

culture

a restriction

fragment

appear

to render

because for

genetic

performed

with

these

concerning

the

procaryotes. it

an appealing

of the fastidious

available

exogenous

successfully

phospholipids

studies cells. from

mechanisms

(3)

but

the

of the acquired cells

with

of this

transfected

resistance the

plasmid

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

with

transformation

of cell

nature and very Moreover,

the

growth

interaction

encoding

982

the pBR 322 plasmid

rate

was very

to tetracycline.

liposome-entrapped

0006-291X/82/190982-05$01.00/0 Copyright All rights

self-replicating

vesicles. E.co%i

cated

the

of the mycoplasma

organisms,

few genetic

medium

are

the

low as indi-

Transfection

pBR 322 plasmid enzyme

B-lactamase

or with led

to

Vol. 108, No. 3, 1982 the

appearance

BIOCHEMICAL

of this

The purpose

followed

with

liposome-encapsulated

tive

result

cally

would

transformed

the capacity Materials

enzymatic

activity this

in the

paper

DNA and assay

not

only

but

could

of fusion

in

AND BIOPHYSICAL

have

of these

cells

for

also

these

(2,3).

mycoplasma

organisms

to answering

cells

activity.

A posi-

may be geneti-

the question

about

liposomes.

and Methods

MycopZasma capricohn cells were grown containing 0.5 % bovine serum albumin (Sigma), ml of each) and 20 rig/ml of cholesterol. with

cells

the B-lactamase

that

with

transfected

was to transfect

indicated

contribute

RESEARCH COMMUNICATIONS

pBR 322 plasmid was prepared 32P by nick-translation.

according

in a modified Edward medium (6) oleic and palmitic acids (10 ug/ to (7).

The plasmid

was labeled

For the preparation of DNA-labeled liposomes, several phospholipids were used : egg yolk lecithin (PC) (Sigma, purified according to (9), ox brain phosphatidylserin (PS) (Sigma, used without further purification), phosphatidylglycerol (PG) (Sigma). Cholesterol (Ch) was also from Sigma. The following molar ratios were used : PC:PS:PG:Ch (8:1.7:0.3:10), PC:PS:Ch (8:2:10), or PC:PG:Ch (8:2:10). In a typical experiment 10 uM of PC or PG at the molar ratios indicated were dissolved in 10 ml of chloroform in the presence of cholesterol (molar ratio PL:Ch = 1.0). The lipids were then evaporated to dryness under a nitrogen stream and redissolved in 10 ml of freshly distilled ether. 10 ng of the E.coZi pBR plasmid with traces of 32P-nick translated plasmid were dissolved in 5 ml of Tris-histidine-NaCl buffer (25 mM Tris-HCl, 2 mM histidine and 145 mM NaCl, pH 7.4) and heated to 60°C in a water-bath. 10 ml of the ether solution of the lipid-cholesterol mixture were slowly injected into the warm DNA solution. Under these conditions liposomes were formed, entraping DNA molecules. After injection, nitrogen was bubbled until the ether was completely removed. The liposome suspension was incubated for 30 min with 50 ug ml-l of DNAse I (Sigma) in PBS supplemented with 10 mM of MgC12,in order to hydrolyze the non-entrapped DNA. After incubation the suspension was filtered through a Sepharose 4B column and the 32P-radioactivity of DNA was counted in the fractions. The presence of the liposomes was monitored in the fractions by following the optical density at 620 nm. The liposome-DNA suspension was eluted from the column with a Hepes buffer (5 mM Hepes, 0.1 M NaCl/KCl pH 7.4). The fractions containing the liposomes and the associated DNA were used for the transformation experiments. Typically, 1.5 ng of the pBR 322 plasmid were encapsulated in the liposomes. Before starting the transfection experiments, the cytotoxicity of the 1 ml of each liposome suspension was different types of liposomes was assayed. added to 10 ml of an early exponential phase culture (A640 = 0.10 - 1.12) in a modified Edward medium (6). Every 4 hours the absorbance of the cell suspension at 640 nm was measured using a Unicam SP-700 spectrophotometer at 37°C. As the PC:PS:Ch liposome preparation did not exert any growth inhibitory effects, these liposomes were selected to perform the transfection experiments. 4 ml of the DNA-loaded liposome suspension were added to 16 ml containing lo8 colony forming units (CFU) per ml and incubated at 37°C hours. Control cultures were incubated under the same conditions, with of free pBR 322 plasmid, or with a DNA-free liposome suspension. After of incubation the cells were harvested by centrifugation at 12,000 xg

culture for 12 5 rig/ml 12 hours for 20

Vol. 108, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

min, washed twice in PBS and incubated with 10 pg/ml of DNAse I for 30 min at 37"C, in order to remove possibly adsorbed DNA. The cells were then collected by centrifugation, resuspended in 1 ml of PBS (pH 7.0) containing 0.1 % Triton X-100, and briefly sonicatecl. The soluble fraction was then separated from non soluble residues by centrifugation at 34,000 xg for 30 min and B-lactamase activity in the soluble fraction was determined spectrometrically using 10B4 M cephalosporin (10). The optical spectra of the reaction mixture were recorded with a Unicam SP-7000 recording spectrophotometer at 37°C. Cephalosporin shows ring is cleaved and a strong absorption at 398 nm at pH 7.0. When the 8-lactam the conjugation of the dinitrostyrene moiety with the 8-lactam is destroyed, a new, strong absorption band appears at 510 nm and the 398 nm band decreases rapidly.

determined containing

The resistance of transfected M.caprico%wn by plating the cell suspension on solid 2 ug/ml of tetracycline.

Results

and Discussion Liposomes

containing

M.caprico%m and were Figure

I shows

Fifteen

percent

liposomes loaded

the

PC:PS:Ch

therefore

elution

profile

of the plasmid

and thus liposomes

not

were

used

sensitive used for

(8:2:10 to entrap

of

added

ratios)

were

DNA and transfect

the DNAse treated

not

associated

The fractions

was (11)

toxic

to

mycoplasmas.

DNA-liposome

(10 pg of DNA) were

to DNAse I. the

molar

cells to tetracycline Edward medium plates

containing

suspension with

the

the DNA-

transfection.

=pm

t5750

.p:

OD

T

,,p

5650

3000 +-+‘I3 0....o

620nm cpm

32P

2000

1000

012345

Fr.ct,on

N’

Figure I - Efficiency of the DNA entrapment by the PC:PS:Ch liposomes. 5 ml of a DNAse I treated DNA-liposome suspension was filtered through a Sepharose 48 column. The column was eluted with Hepes (5 mM Hepes, 0.1 M NaCl/KCl, pH 7.4). To the cold pBR 322 plasmid traces of 32-P-nick-translated plasmid was added 1.2~106 cpm/ml). The liposome presence was monitored by following the O.D. at 620 nm in the samples.

984

Vol. 108, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

00 2.0

a)

00; ,’ 08 06 OL 02 00 b)

300

100

500

600

700

Figure 2 - Spectroscopic assay of B-lactamase. I ml of cell extract pH 7.0 was reacted, at 37'C with 10-b M cephalosporin. The spectra ded over on hour at 37°C. a) Cells incubated with the free pBR 322 plasmid. b) Cells incubated with the liposome-entrapped pBR 322 plasmid.

The expression cell

is

shown

of

in Fig.

B-lactamase

2. Cells

mes show

no spectral

change

transfected

with

liposome

mainly appears absorption kinetic

a rapid

the decrease

incubated

of the

free

(Fig.

(Fig.

levels

after

40 min.

B-lactamase

The rate

Comparison

when cephalosporin

reacts

indicates

985

Za).

show dramatic

2b).

M. capricolton

DNA and with

at 398 nm and a new absorption fast

mycoplasma-expressed

with

encapsulated-DNA

quite

modification

by the transfected

cephalosporin

and increases off

activity

in PBS, were recor-

"empty"

liposo-

M. caprico%um cells spectral

changes,

band at 510 nm that of increase

in the

of the spectra

and of their

with

B-lactamase

practical

bacterial identity

in

the

510 nm

and two

cases

Vol. 108, No. 3, 1982 (4,5).

These

fected

with

fusion

mechanism

results the

The extent

BIOCHEMICAL strongly

liposome

may be very

it

phospholipid

was claimed

rather

than

sicles

that

reported but

reaction

of the with

the evidence

product

cells

with

with

low but

grown

were

lipid

for

with

the

lipid

(12).

8-lactamase

cription

and translation

machinery

of the mycoplasma

argument viable

for cells)

so that with point

this

conclusion.

is well

beyond

the expression

this out

eucaryotic

frequency, that cells,

Acknowledgements:

the rate

fusion, a rare

though

place

Fusion

(II), mechanisms

was,

however,

features

appearance

when

indicate

that

trans-

the

the pBR 322 is an additional

of Q 10 -6 (out mutation

could low

of the

of its

to tetracycline

of spontaneous

The relatively taking

of

the cells

recognize

of transformation

of the new genetic by accident.

react

of resistance

The rate

quantities

The identity

and of the kinetics

and mycoplasmal

The expression

Although,

or exchange

vesicles.

circumstantial

cephalosporin

by a

AcholepZama laid2awi-i and PC ve-

mycoplasma,

was rather

trans-

liposomes.

into

is due to transfer

cells

being

apparently

large

to be incorporated

B-lactamase

sequences.

are

transfection.

dispersions,

bacterial

plasmid

cells

the DNA containing

sufficient

with

found

another

M. caprico%wn

that

such incorporation

to fusion

previously

are

RESEARCH COMMUNICATIONS

E.coZi pBR 322 plasmid,

of the mycoplasma

of fusion

exogenous

suggest

encapsulated

when M. capricolwn cells the

AND BIOPHYSICAL

in these

not have

in this

case

of Martine

Dubois

is

cells

rate

as in the

might case of

event.

The excellent

help

acknowledged.

References 1. Stanbridge, E.J. and Reff, M.E. (1979) in "The Mycoplasmas" Vol. I, p. 157186, M.F. Barile and S. Razin eds., Academic Press, N.Y. 2. Razin, S., Kutner, S., Efrati, H. and Rottem, S. (1980) Biochim. Biophys. Acta 598, 628-640. 3. Fraley, R.T., Fornari, C.S. and Kaplan, S. (1979) Proc. Nat. Acad. Sci. USA 76, 3348-3352. 4. Wong, T.K., Nicolau, C. and Hofschneider, P.H. (1980) Gene 10, 87-94. 5. Sen6, C. and Nicolau, C. (1982) Biochim. Biophys. Acta (in the press). 6. Razin, S. and Rottem, S. (1976) in "Biochemical Analyses of Membranes", p. 3-26, A.E. Madday ed., Chapman and Hall, London. 7. Ish-Horowitz, D. and Burke, J.F. (1981) Nucleic Acids Res. 9, 2989-2998. 8. Singleton, W.S., Gray, M.S., Brown, M.L. and White, J.L. (1965) J. Amer. Chem. Sot. 42, 53-61. 9. Deamer, D. and Bangham, A.C. (1976) Biochim. Biophys. Acta 443, 629-634. 10. O'Callaghan, C.H., Morris, A., Kirby, S.M. and Shinger, A.H. (1972) Antimicrob. Agents Chemotherap. 1, 283-288. II. Gross, Z., Rottem, S. and Bittman, R. (1982) Eur. J. Biochem. 122, 169-174. 12. Grant, C.W.M. and McConnell, H.M. (1973) Proc. Acad. Sci. USA 70, 1238-1240. 13. Stanbridge, E. (1971) Bacterial. Rev. 35, 206-227. 986

9

(13),

appeared,

transformation

is,

of 'L 10