Vol. 70, No. 1,1976

BIOCHEMICAL

GRADIENT,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

POLYACRYLAMIDE GEL RLECTROPRORRSIS OF PROTEINS FROM CYTOTOXIC MYCOPLASMA MEMBRANES

M. G. Gabridge, Department

S. E. Singer,

of Microbiology, Urbana,

Received

February

and R. A. Esposito University

Illinois

of Illinois

61801

23,1976

SUMMARY : Membranes of Mycoplasma pneumoniae were prepared following several different cell lysis procedures (freeze-thaw, french press, digitonin, sonication, osmotic shock, pH shock). All possessed some degree of toxicity for ciliated epithelial cells, but the french press and freeze-thaw processes were optimal in terms of lysis efficiency, membrane yield, and cytotoxicity. Membrane proteins from virulent and attenuated mycoplasmas were electrophoresed with a new, high resolution electrophoresis technique (gradient polyacrylamide gels), and strain differences were detectable among the 50+ discernible bands.

INTRODUCTION: Mycoplasma respiratory not

pneumoniae

epithelium,

been elucidated

formation

(1,Z).

enough

proposed

(4,5)

mediation

to those is

that

must

with

enzyme systems

pose of this

(7).

(8)

account

the

fact

that

membrane

as defined

by high-resolution

the

preparations,

Copyright 0 19768 by Academic Press, Inc. All rights of reproduction in any form reserved.

of pathogenesis

cytotoxic

that

in detail as well

cells.

polyacrylamide 271

nor has been or

of J$ pneumoniae organ

This

cultures

biological contains

indicate

that

cytolytic

the relative

gel

peroxide

injury

membrane

as their

has

effect.

in trachea

have

It

cellular

membranes

reports

also

between

relationship.

the mycoplasma

(9)

to ciliated

substantiated

direct

viable

recent

was to examine

been well

(e.g.,

(6)

with

In addition,

mycoplasma

mechanism

causal

alterations

infection

attachment

of an association

for

reported

and erythrocytes

study

reports

mechanism

and histopathologic

consistent

the precise

a definite

recently

for

(3) have not

some other

seen following

lymphocytes

Early

production

by toxin(s))

gross

as yet,

to establish

Our laboratory induce

but

and disease

consistent

has a predeliction

activity

membranes

cytotoxicity

electrophoresis.

similar

numerous

potential.

relative

can

protein

from

The purof various composition

Vol. 70, No. 1, 1976

MATERIALS

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND METHODS:

M. pneumoniae, strain FH, was provided by J. Tully (National Institutes of Health) and strains Mac and Pl 104166 were furnished by W. Clyde (University of North Carolina). Strain 1428 was obtained from R. Chanock (National Institutes of Health) and 3. fermentans, strain KlO, was provided by Wm. Murphy (University of Michigan). Acholeplasma laidlawii was purchased from the American Type Culture Collection (i/14192). Organisms were in the 6 to 11th passage since receipt, and were grown in G-199 medium (10). Trachea organ cultures were prepared from adult Golden hamsters as described previously (6). Relative ciliary activity measurements, i.e., percentage of a trachea ring remaining intact (0 to 100) multiplied by the vigor of ciliary motion (0 to 3), were made daily during observations with inverted phase optics (x 225). Organ cultures were maintained in 90% minimal essential medium (Eagle's with Hanks' salts) and 10% fetal calf serum, supplemented with L-glutamine (40 mM), penicillin G (200 U/ml), and N-2-hydroxyethylpiperazine-N'-2'-ethane-sulfonic acid (25 mM, HEPES, Sigma Chemical Co., St. Louis, MO.). The lysis procedures, including 20 cycles of freeze-thaw (6), 2 cycles in a French pressure cell at 25,000 psi (ll), digitonin (12), osmotic shock (13), carbonate/bicarbonate buffer at pH 11 (14), and 5 one-minute cycles of sonication at 20 kHx (13), have been described previously in conjunction with the preparation of mycoplasma membranes. The methods, along with the criteria used to ascertain membrane "purity," have been reviewed (7,ll). Suspensions were cleared of unbroken cell aggregates by centrifugation at 4,000 x g for 5 min at 4OC. Membranes were collected by ultracentrifugation of supernatants at 41,000 x g for 45 min at 4oC, and were washed 4x with sterile PBS1 prior to resuspending them in 10 ml of PBS. Where indicated, membranes were subjected to an additional purification step by layering them on a sucrose step gradient (35% and 50%) according to the method of Engleman et al. (15) prior to the 4 successive PBS washes. Protein content was determined with the Lowry method, and membranes were resuspended in organ culture medium at 40 ug protein/ml. A modification of the discontinuous SDS2 systems of Laemmle (16) and O'Farrell et al. (17) was used for electrophoresis. The apparatus, described in detail by Studier (18), contained a running gel (14.5 cm x 8.5 cm x 0.08 cm) with a linear, 7.5 to 20% concentration of acrylamide. The stacking gel (3% acrylamide) was 1.6 cm from the bottom of the well formers to the top of the running gel. Both gels contained 0.1% SDS, and the stacking gel also had 0.5% agarose for stability. The gel was pre-run at 12.5 milliamps for 30 min at room temperature. Prior to application, samples were lyophilized and rehydrated in sample solvent (49.6 mM Tris, pH 6.8; 32% glycerol; 1% SDS; 1% 2-mercaptoethanol; O,l% bromphenol blue) to yield a final concentration of 4 mg protein/ml. The sample was heated at 100°C for 10 min, and a 10 ~1 aliquot was placed in one of the 9 preformed wells (1.2 cm x 0.6 cm x 0.08 cm). Running buffer contained 0.1% SDS in 49.5 mM Tris and 384 mM glycine, adjusted to pH 8.3 with 0.1 N HCl. Electrophoresis was at 12.5 milliamps until the marker dye migrated 0.5 cm into the stacking gel (approx. 40 min at 22OC). The apparatus was then held at 4oC and the current was increased to 20 milliamps. Electrophoresis 1phosphate buffered saline, 2 sodium dodecyl sulfate

pH 7.4

272

Vol. 70, No. 1,1976

BIOCHEMICAL

TABLE 1. Comparisons of yields, of membranes from -M. pneumoniae,

Method

of lysis

Membrane

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

efficiency PI 1428,

of lysis and relative cytotoxic prepared by various methods.

yield

Viable

(mg protein/L)

cellsa

CFU

potential

Cytotoxicityb

% initial

RA

% control

Freeze-thaw

6.6

0

0

111

53

French

6.2

0

0

70

33

Digitonin

6.4

2 x lo3

6 x 1O-5

76

36

Sonication

8.0

1 x lo3

3 x lo-5

147

70

2.7

2 x lo3

6 x lO-5

153

73

0.8

0

0

98

47

press

Osmotic

shock

Carbonate,

pH 11

aViable cells remaining after lysis procedure and membrane washing, expressed as total CFU remaining from 1 L of cells, or as percent of the initial, total CFU count/L. The highest residual level of viable cells was still 10 to loo-fold less than that required to cause significant cytopathogenicity within 5 days. b Effect on relative ciliary activity, EA (percent of trachea ring remaining intact x relative Mean data

vigor of ciliary from 12 trachea

beating), rings.

after

5 days of exposure

to 40 ug protein/ml.

continued until the marker was within 1 cm of the bottom of the running gel (3-4 hrs:). The gel slab was stained in 0.2% Coomassie Blue in 50% methanol with 7% acetic acid for 1 hr at 37OC. The background was destained overnight in 25% methanol - 7% acetic acid at 4oC. Gels were scanned at 550 nm with a Gilford densitometer with a Zeiss chromometer. RESULTS: Influence

of preparation

method

representative

methods

osmotic

and pH shock)

in

shock,

Table

1 show that

Membranes toxic,

from

whereas

the relative osmotic in eliciting

the

(freeze-thaw,

all

the other

and sonic

activity shock

an effect.

applied

preparations

methods

press,

tested

shock

than

yielded

The digitonin

digitonin,

PI 1428.

procedures

were

the least

which

control

could

values.

membranes

which

were

and french

press

methods

273

The data

of toxicity.

preparations

53% of the

six

sonication,

had some degree

gave membrane

to less

of membranes.

to g. Pneumoniae,

and osmotic

methods

potential

french

were

sonication

ciliary

on cytotoxic

consistently

reduce The slow

gave membranes

Vol. 70, No. 1, 1976

Fig.

Electrophoresis and membranes M. pneumoniae 2. pneumoniae -%. fermentans

1

which

caused

ture,

the

values

BIOCHEMICAL

an obvious

trachea

rings

of about

and french

yielded

press

cytotoxic

methods

potential

drop

proved

contained

intact

cells,

relative

complexity

PI 1428,

yielded

5 days in culciliary

The freeze-thaw

activity

and carbonate

toxicity.

when yields,

The freeze-thaw

efficiency

of lysis,

and

overall. In order

protein

techniques

cytoplasm,

After

had relative

of intermediate

some unique

of each.

membranes

membranes.

electrophoresis

48 hrs.

value.

optimal

compared

toxic

-M. pneumoniae,

to these

membranes

were

compared

within

of the control

of mycoplasma

polyacrylamide

in activity

exposed

Electrophoresis membranes

RESEARCH COMMUNICATIONS

banding patterns of proteins from mycoplasma cells (freeze-thaw). Left to right: M. fermentans membranes; cells; I. pneumoniae cytoplasm; E. pneumoniae membranes; membranes purified on a sucrose step gradient; membranes.

one-third

(pH 11) methods

AND BIOPHYSICAL

were

and membranes

As shown approx.

in figure

to establish

component(s), employed.

high Initial

in an attempt 1, whole

50 individual

274

whether resolution studies to ascertain

cells

bands,

or not

while

of virulent cytoplasm

the

Vol. 70, No. 1, 1976

(lyophilized were

osmotic

extremely

components pattern hood

that

in staining

in terms

of quantity.

since:

c) additional

a) intact

gradient

during

potential

are

distribution

those

associated

with

When cells compared

clearly

PI 1428, organ

lent

related.

in

strains,

a deeply

could

which

was the

both

the

strains.

near

the

intact

cells;

and

pattern

possess

cytotoxic

similar

in number

cells

and

than

essentially

tracings

or membranes in some of the species gels

strain

were

molecular

apparently

decreased

to the virustrains

of the

gel,

difference

present

in

differorganisms proteins

in

obtained

that

also

significant

weight

had

whereas

reaction

and attenuated

of the PI 1428 membranes

275

similar

This

The only

strains

in trachea markedly

discernible

of virulent large

length

2

strains

of the virulent

bands. 4).

for

with

pattern

barely

doublet

(Fig.

Acholeplasma

when tested

(15% down the

(Fig.

pattern

identical

All

weaker,

of mycoplasmas

of the -M. pneumoniae

had a banding

of the

protein

In addition,

and all

are virulent

top

species

the non-pathogen,

exception.

the

first

or absence

'Jhe other

viable

same basic

Mac (an attenuated

cultures)

in densitometer

decrease

the latter.

were

a consistent

doublet

represents

cells

but more complex

several

the

seen between

Mac had a noticeably

be seen

ence between

organ

showed

of which

Strain

but with

staining

which

cells,

from

g. pneumoniae,

- all

(6,lO).

the attenuated area

were

The patterns

trachea

intact

electrophoresis

species,

by intact

all

differences

FH, and 104166

virulence

protein

The likeli-

them on a sucrose-step

the

membranes

the banding

by differential

to detect

alter

(freeze-thaw)

electrophoretically,

cultures

removed

minor

the cytoplasm.

and the pathogen,

laidlawii, were

presented

and membranes

Clbvious

and 3).

with

by layering

not

of numerous

to those

contaminated

failed

Mycoplasma

composed

cells.

techniques

did

reports,

intact

were

of the membranes

preparation. thus

seen with

The latter

represented

earlier

aggregates

ultracentrifugation

of the membrane

as many bands.

with

was simply

plating

RESEARCH COMMUNICATIONS

and presumably

to that

cell

purification

half

Consistent

preparation

b) direct

about

intensity,

was similar

the membrane

AND BIOPHYSICAL

had only

faint

centrifugation;

were

lysate)

of membranes

was remote

size

BIOCHEMICAL

Samples

of Of

from various

Vol. 70, No. 1, 1976

Fig,

2

BIOCHEMICAL

Polyacrylamide gel cell preparations. strains PI 104166, minimal staining at

lysis

procedures

showed

toxic

potential)

also

The nature

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

electrophoresis of proteins from various mycoplasma Left to right: A. laidlawii; M. pneumoniae, Mac, PI 1428; FH;A.- laidlawii.Arrow indicates doublet in the attenuated strain, Mac.

sonicated

membranes

had the weakest

and potential

protein

cytotoxicity

(those

reaction

of these

with in

specific

the least

cyto-

doublet

area.

this

proteins

is

now under

investigation.

DISCUSSION: The characterization gel

electrophoresis

of their

has proved

extremely

of protein

composition.

sheet,

as opposed

requiring have

minimal

recently

been

of mycoplasma

valuable

to the amounts

proteins, for

cells

and membrane

as developed

identification

cylinders, of sample

used in conjunction

markedly (e.g., with

276

by Razin and for

The use of such gels

by polyacrylamide et al.

comparative

in the configuration increases

30-80

ug).

mycoplasmas

(7,19),

resolution Though (20,21),

studies of a thin while

such flat the

gels technique

Vol. 70, No. 1, 1976

Fig.

3

BIOCHEMICAL

AND BIOPHYSICAL

Polyacrylamide gel electrophoresis various mycoplasmas. Order is

RESEARCH COMMUNICATIONS

of proteins from membranes identical to that of Fig. 2.

of

Membranes

-

Fig.

4

MAC

Densitometer tracings of stained protein bands of cells and membranes of -M. pneumoniae, PI 104166 (virulent) and Mac (attenuated).

277

Vol. 70, No. 1, 1976

developed

for

discernible

BIOCHEMICAL

this

study

bands).

dodecyl

sulfate

This

(7.5%

banding

uniqueness illustrated

through

technique

strains

approx.

the combined gel,

and a linear

has shown

differences

and for in high

50

use of sodium gradient

the homology

of J-i. pneumoniae,

and J-i. fermentans, protein

(i.e.,

a stacking

This

in several

laidlawii

potential

resolution

the proteins,

to 20%).

patterns

of 4.

improved

was achieved

to denature

of polyacrylamide protein

provides

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

the relative

the first

time,

and low virulence

has isolates

of -M. pneumoniae. The possibility on other

cells

is

associated

with

numerous

enzymes

tase

(23),

has also Given

that not this

remote

this

(7),

associated

(24),

of biological

or

(3)

membrane

integrity.

currently

under

active

Proteolytic

of &

pneumoniae

component;

(2)

orale it

of these

is

is

alternatives

contain phospha-

enzyme

activity

and E. salivarium

(25).

conceivable

that of:

or specific

such as cell

activity

RNase (22),

a manifestation

generalized

processes,

Which

(22).

effect

enzymatic

membranes

contain

activities,

of &

biophysical

the extensive

membranes

and AlPase

a biological

exert

Acholeplasma

in membranes

cytotoxicity

could

one considers

and g. pneumoniae

of a membrane

dation;

membranes

For example,

demonstrated

catalog

if

site.

phospholipase been

toxicity

mycoplasma

fusion

membrane-

(1)

direct

enzymatic

followed

degra-

by loss

has the most validity

of

is

investigation. ACENOWLEDGFMBNTS

This Institutes Drs.

study

was

supported

of Health.

S. Razin

in part

The critical

and S. Kaplan

by grant reviews

are greatly

#AI 12559 of this

from

manuscript

the

National

provided

by

appreciated.

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W. A.

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