Inhibition of bacterial wall lysins by lipoteichoic acids and related compounds

Vol.67,

No. 3, 1975

BIOCHEMICAL

INHIBITION

OF BACTERIAL WALL LYSINS BY LIPOTEICHOIC ACIDS AND RELATED COMPOUNDS

J.-V. H ltje2, A.J. Wicken', Cleveland' L. DaAeo-Moore ? and G.D. Shockmanl

R.F.

Department of Microbiology Philadelphia, Pa.1

Received

AND BIOPHYSICAL RESEARCH COMMllNlCATlONS

October

and Innnunology, and the Rockefeller

A. Tomasz',

Temple University School of Medicine, University, New York, N.Y.2

16,1975

SUMMARY - Lipoteichoic acid (LTA) from several Gram-positive microorganisms, mn antigen (Fag) from Diplococcus pneumoniae R36A, and an acidic lipopolysaccharide (ALP) from Micrococcus luteus were examined for effects on four wall lysis systems. The LTAs inhibite=N-acetylmuramidases of Streptococcus faecalis and Lactobacillus acidophilus, and the amidase of Bacillus subtilis 168.acylated LTA failed to inhibit. LTAs failed to inhibit amidase of pneumococcus. Fag inhibited the pneumococcal amidase, but had no effect on the other three systems. Bacterial division wall

autolytic

(1,2)

enzymes

and in the

inhibitory

bacteriolytic

antibiotics

of these

enzymes

faecalis

(2).

lead More

to the detection recently,

Also,

Fag added

against

lysis

erable It

amidase

antigen

concerning

has been suggested

the (5)

autolysin

We now report

the results

validity

to that

in the of the

Abbreviations: lipopolysaccharide;

plasma

choline

bacterium

was found

of action

(5).

to protect

observations

may play

cells

have consid-

of bacteriolytic

acids

autolysin

as the choline-

of this

These

lipoteichoic

in 2.

antibiotics. a physiological

activity. of experiments

of the pneumococcal

Fag is an amphipathic localized

that

control

factor

was identified

pneumococci,

mechanism

and other

of cellular

inhibitory

acid")

(5).

in cell

of the pneumococcal

E.C.3.5.1.28)

of growing

both

of penicillins

the mechanism

inhibitor

and vancomycin

in regulating

general

for

"lipoteichoic

(Fag:

by penicillin

action

of an autolysin

a powerful

to the medium

importance

also

role

Forssman

(killing) A search

(3).

(an N-acetylmuramyl-L-alanine containing

have been shown to be important

substance membrane, and ribitol

LTA = lipoteichoic SDS = sodium

findings which

designed for contains

to test

other

bacterial

structure

acid; dodecyl

wall

teichoic

Fag = Forssman sulphate 1128

systems. bound

and seems to have a primary containing

the possible

lipids,

structure acid

antigen;

is analogous

of this ALP - acidic

VoL67,No.

3, 1975

BIOCHEMICAL

There

species

(6,7).

between

Fag and other

positive

bacteria, (ALP)

chain

glycerol

and are widely (8,9).

possess

membrane

succinylated from

each of the compounds

as potential autolytic

systems and are

and a type

RESULTS AND DISCUSSION - Lysis hydrolases (12,13)

of &. faecalis was inhibited

trations

of five

terial

species

2. faecalis

wall

(Fig.

dry weight indicate

casei

residues

of,

of the LTA (8),

Estimates

(9),

and of cell 2. faecalis

the LTA concentrations found

of cellular wall

1).

about

used to inhibit

glycan

Low concen-

4 different

bac-

2. lactis

system

of glycosyl

(Fig.

ATCC 9936, 1).

substitution

on the basis

of nmoles

3 streptococcal of LTA/mg

of LTA at 1 to 2% of bacterial

at 25 to 40% of bacterial

contains

3 to 5 nmoles muramidase

dry weight of LTA/mg

activity

(14)

wall.

are well

Thus within

in cells.

LTAs of two lactobacillus (Fig.

from

1).

to all

content

of such

63AM Gasser

dose responses

when compared

1).

(Table

NCTC 6991,

nature

of the

system.

strain

the 2. faecalis

or chemical

not all

N-acetylmuramide

and purified

inhibited

show that

occurrence

each

of LTAs

isolated

compounds, homologous

reported

general

by the

NIRD R094, -L. fermentum

indistinguishable

that

amounts

LTAs,

of the degree

glycerol

LTAs were

by low concentrations

39 and NCIB 8191)

Irrespective of the

(L. -

the for

walls

as a

amphipathic

by one or more but

both

shown to

of 4 different,

The results

of specificity of cell

of these

ATCC 9790 and -L. acidophilus

different

has been characterized

inhibitors

with

a linear

in Gram-positive

(lysodeikticus)

can be inhibited

lipo-

to a glycolipid,

components

systems.

consistent

LTAs contain linked

a variety

of Gram-

and the acidic

has been recently

We examined

location

species

8,9)

covalently

associated

ALP, which

bacterial

activities

(LTA:

of an LTA, -M. luteus

sources,

in other

Conventional

residues

as membrane

(10,ll).

autolytic

acids

RESEARCH COMMUNICATIONS

and cellular

found

(10,ll).

phosphate

several

tested,

inhibitory

of K. luteus

associated

and heterologous,

compounds

lipoteichoic

Instead

mannan

in chemistry

amphipathic

distributed

bacteria

obtained

similarities

notably

polysaccharide of 25-30

are

AND BIOPHYSICAL

For these

2 LTAs only

species

also

inhibited

concentrations

1129

higher

lysis than

of 5. faecalis 0.2

nmoles/mg

walls in-

Vol.67,

No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 Effect

A. On Lysis

of Walls

LTA of 2. faecalis

Deacylated3

of LTAs, Fag, and ALP on the Wall Lytic Systems of S. faecalis and L. acidophilus nmoles LTA 96 of control w/ml per mg wall2 lysis rate of S. faecalis

ATCC 97901

39 (Exp.

1)

0.7

0.1

(Exp.

2)

P4 9:o

it; 1:3

;;

24.3

5.2

100

LTA of 2. faecalis

39

Fag of pneumococcus4 ALP of M -- luteus

B. On Lysis

of Walls

LTA of C. fermentum

86 54

25.3

1395

3.3

106 101

166.: 34:2

1;:

of L. acidophilus7 NCTC 6991

4.6

0.9 ;:

2::; LTA of 2. faecalis

39

Deacylated3

LTA of S_. faecalis

Fag antigen

of pneumococcus4

1The assay system as previously weight of wall-enzyme complex The sodium phosphate, pH 6.8. to 0.52 hrs -1 determined from

39

E

9

i-6" 712

i.5" 1:o

:i 13

24.3

3.6

82

13.3

98

described (12) consisted of 1.3 to 2.0 mg dry from exponential phase cells in 3.0 ml of 0.01 fi rate of wall lysis in control cultures was 0.40 the slope of the first order reaction.

2Concentrations of LTA in nmoles were determined on the basis of their phosphorus content and the assumption of an average glycerol phosphate chain length of 27.5 (8). All LTAs were prepared by phenol extractions and gel filtration (18). 3Deacylation was carried out in 10 volumes of methanolic 0.2 M KOH at 37°C for min. followed by passage through a Dowex 50 column as descrii?ed (18). Dry weight was estimated from phosphorus content as acylated LTA equivalents. 4Fag was prepared

as described

previously

15

(7).

5Stimulation of the rate of wall autolysis in the S. faecalis system was attributed to the probable presence of a small residue of trypsin used in the preparation of Fag (7). Low concentrations of proteinases are known to activate a latent form of the S. faecalis enzyme (12). A similar increase in wall lysis rate was not observea in the C. acidophilus system which is unaffected by trypsin (13).

1130

BIOCHEMICAL

Vol.67,No.3,1975

AND BIOPHYSICAL

s. faecah

l

39

A s. faecalis l

01

1

1

0

’

Inhibition species.

hibited,

and the slope

tococcal

LTAs.

of the

crepancy

remains

LTAs have substituents, of several

’

0.8

’

1

’

1.2

(nmoleslmg

concentration

1.6

wall)

response

LTA was also

muramidase

system

less (Table

walls by purified LTAs of details as described in

paralleled

that

effective

than

of the strep-

the

1B).

The reason

phosphate,

but

for

streptococcal this

dis-

to be determined. a common backbone

and in the LTAs reduced

muramidases,

8191 9936

of lysis of 5. faecalis Assay system and other

The C. fermentum

LTA on the C. acidophilus

lacris

’

0.4 LTA

Figure 1. several bacterial Table 1.

S.

RESEARCH COMMUNICATIONS

nature

of polyglycerol of their

or completely

even at high

concentrations

to the effect

of LTAs,

6Preparations 6.7 and 16.7 (11)).

of ALP were kind gifts pg/ml, (10)) and from

7The assay system from exponential

pneumococcal

glycolipids abolished (over

(8). inhibitor

'24 pg/ml;

differ

Chemical activity Table either

in sugar deacylation

for 1).

both

In contrast

Fag failed

to inhibit

of the muramidases,

from Drs. Drs. Pless

Owen and Salton (assays at 3.3, and Lennarz (assay at 34.2 ug/ml,

(13) consisted of 1.3 to 2.0 mg dry weight of wall-enzyme phase cells in 3.0 ml of 0.05 E sodium citrate, pH 5.0.

1131

complex

Vol.67,No.3,

1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2 Effect of LTA on the Action of Soluble N-acetylmuramidase on SDS-inactivatea

NJ/ml LTA of 2. faecalis

39 (Exp.

1)

4.3 8.5 21.3

LI;A of 2. faecalis 39 (Exp. 2) I, II ' Autolysin pre-bound to wall (10 min.,

6.8 6.8

to substantially

concentration

inhibit

LTA also tivated

high

the 2.

inhibited

walls

of inhibition

2).

of autolysin

to its

These

wall

of lysis

activity crude

strong

Figure).

g. subtilis

Fag nor

purified

amidase

faecalis

39 LTA (Table

of only

68% of activity

of 2. faecalis, the wall

deacylated

to increase

substrate.

32 29

in O.OOSpl sodium

that

also

failed

1A).

5. faecalis

suggest

phosphate

ALP of E. luteus

(Table

or I-. fermentum wall-enzyme

amidase

of the crude

of g.

1’:;

muramidase

of enzyme to walls

did

on SDS-inac-

not affect

LTA does not

(purified)

complex

on SDS-walls

and purified

Preincubation

failed

system

the degree

affect

binding

3).

(15) In these

inhibition,

enzyme alone

not affect

ZA).

However,

although alone"

LTA does not seem to affect effects

Inhibition

the LTAs (see

produced

no loss this

inhibited

experiments

with resulted

As with

binding

of B. subtilis

of LTA either

directly

1132

legend

to

of activity. A highly

by low concentrations

incubation

control.

by both was parti-

system.

preincubation

the

the

2B) was inhibited

inhibited

was also

of an "enzyme

At present,

(Fig.

with

LTA of C. fermentum

subtilis

(Fig.

did

LTA of C. fermentum.

when the enzyme was preincubated

Neither

min.

(crude)

LTA of g. subtilis

cularly

41 24 11

1).

of soluble

results

of the B. subtilis

of crude

0.7 1.5 3.7

substrate.

LTAs of EJ. subtilis rate

(Table

Prebinding

observed.

% of control Lysis rate

determined

faecalis

the action

(Table

nmoles LTA per mg wall

O°C)

lCrude autolysin was obtained and activity pH 6.7 as previously described (12).

even at a relatively

S. faecalis Wm

LTA for

of 2 10

in recovery

the muramidase amidase on enzyme

to

Vol.67,No.3,

BIOCHEMICAL

1975

AND BIOPHYSICAL

. No add,t;on l L fermen turn L TA 0 L?. subfih L TA A Deacylared L. fermenturn

t\

RESEARCH COMMUNICATIONS

\

I

L TA \‘!\

0 SDS walls . No addibon l L. fermen tom L TA

030

Figure 2. The effects of a B. subtilis wall-enzyme amidace on SDS-inactivated

90

1500

30 MINUTES

90

150

of several LTA preparations and Fag on (A) lysis complex and (B) on the action of crude B. subtilis B. subtilis walls.

(A). B subtilis wall-enzyme complex was isolated from cells grown in Spizizen's medium to 5 x 103 cells/ml, by mechanical disintegration and washing three times with cold 0.15M NaCl. Walls were suspended at an A560 of 0.75, in 1.0 ml (0.05M pH 3) Tris containing the indicated compounds at 100 pg/ml, and the turbidity was measured during incubation at 37°C. (E). The supernate (15,000 x g, 30 min) of an autolysate of the wallenzyme complex described above was used as a source of crude amidase. The Fag at 190 pg/ml) were added to amidase various preparations (LTAs at 100 ug/ml, (100 ~1) in 1.0 ml of 0.05M Tris, pH 3. After 20 min. at 37"C, SDS-inactivated (30°C, 30 min.) walls (200--11) were added and turbidity measured as in (A). ), the SDS-walls were added at time zero. B. subtilis LTA In one tube ( -awas prepared by hot aqueous phenol extraction (IB). The sources ofher preparations are given in Table 1.

activity

or on wall The various

stimulated

the

trations

antibody

amidase

assay

at least

amidase

assays.

amidase (3)

seem equally

LTA preparations

used were

5. subtilis coccal

substrate

at 5 and 12.5 to the

possible.

failed system

100 fold

to inhibit,

of D. pneumoniae those

previously,

pg/ml

Addition

(5). system

(Table

used either

As reported

pneumococcal

and in some instances

did

1133

4).

The concen-

in the muramidase Fag inhibited

or

the diplo-

of antipolyglycerolphosphate

not affect

Fag inhibition,

suggesting

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 3 Effect

of LTA on the Release of [3H]Glucosamine label Walls by the Action of a Purified k. subtilis

w/ml LTA of 2.

faecalis

LTA of S. faecalis with amidase, Deacylated

39

39 preincubated (10 min., 37°C)

LTA of 2. faecalis

39

from B. subtilis Amidare

nmoles LTA/pmole amino sugar

% of control lysis rate

E

3.8

64

18:0

30.5 7.6

z:,

2.3

3.8

72

19.5

33.0

100

1The assay system as previously described (15) consisted of 50 ul [3H]glucosamine labeled walls (150 nmoles of amino sugar, 1.7 x 102 dpm) 2 ~1 of amidase, in 0.05 M Tris, pH 8.0, containing 0.05 ELiCl, 0.01 MMgC12 and lrntj EDTA in a total vaume of 200 ~1. The reaction was stopped after 60 min. at 37°C. Purified amidase and labeled wall substrate were kindly provided by Dr. L. Glaser. For LTA preparations, see Table 1.

TABLE 4 Effect

of LTAs,

Fag,

and ALP on the Pneumococcal

Amidase

% of control

m/ml LTA LTA LTA LTA ALP Fag

of of of of of of

S. faecalis 5. E -936

39

500 500 500 350 1000

i. casei7094

B. subt'il. is 168 w. lur teus Fneumococc 3s

Fag of pneumococcus phosphate antibody

plus (6)

System1

121 158 135 131 100

125.5

z;

5 12.5

47 29

antipolyglycerol-

TThe assay system as previously described (5,19), consisted of [methyl 3Hlcholine labeled walls (2.0 ug, 104 cpm) plus crude amidase (15 pg protein from about 1.5 x 106 cell equivalent units of cell lysate) in 0.05 MTris maleate, pH 6.9 in a total volume of 220 ~1. The reaction was stopped after 10 min. at 37°C. For materials used see Table 1.

that

inhibition The data

phosphate

type

is

not due to contaminating

presented

indicate

LTAs can inhibit

that, three

LTA. at low concentrations,

autolytic

1134

enzyme systems.

polyglyceroThe unique

Vol.67,No.3,

BIOCHEMICAL

1975

specificity

of Fag for

the pneumococcal

polyglycerophosphate

type

phosphate

polymers

containing

Deacylation addition which

is

work

will

in

both

in --in vivo

not

be required

especially

since

not precisely

rence

in pneumococci

the

known. acylated

regulation

in loss

degree

The membrane and deacylated of autolytic

(8)

in

compounds with

membranes

of the two muramidases. of the observed

of each compound

association forms

activities

association

of a

investigated.

amphipathic

specificities

of acylation

absence

of polyglycero

been carefully

Other

inhibitors the

nature

biological

including

to unravel

be due to the

has not

of several

LTA,

effective

could

RESEARCH COMMUNICATIONS

and chemical

activity.

common with

were

amidase

The presence

inhibitory

in

ALP and Fag),

Further fects,

of lytic

have properties

(i.e.,

of LTA.

of LTA results

to loss

AND BIOPHYSICAL

(16,17),

of LTA as well are appropriate

ef-

or preparation as its

occur-

for

a role

activity.

ACKNOWLEDGEMENTS This work supported Institutes of Allergy and from the National Science the American Association Cancer Institute Training

by grants AI 05044 and AI Infectious Diseases and Foundation. A.J.W. was of Dental Research. R.C. grant CA 05280.

06888 from the National grants GB 31121 and GB 20813 a senior foreign Fellow of was supported by a National

REFERENCES 1. 2. i: 5. 6. 2 10": 11. 12. ii: 2 17. ii:

Rogers, H.J. (1970). Bacterial. Rev. 34, 194-214. L. and Higgins, M.L. (1974). Ann. N.Y. Shockman, G.D., Daneo-Moore, Acad. Sci. 235, 161-197. Tomasz, A. (1974). Ann. N.Y. Acad. Sci. 235, 439-447. Goebel, W.F., Shedlovsky, T., Lavin, G.I. and Adams, M.H. (1943). J. Biol. Chem. 148, 1-15. Holtje, J.-V. and Tomasz, A. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 1690-7694. Fujiwara, M. (1967). Jap. J. Exp. Med. 37, 581-597. Briles, E.B. and Tomasz, A. (1973). J. Biol. Chem. 248, 6394-6397. Wicken, A.J. and Knox, K.W. (1975). Science 187, 1161-1167. Knox, K.W. and Wicken, A.J. (1973). Bacterial. Rev. 37, 215-257. Owen, P. and Salton, M.R.J. (1975). Biochem. Biophys. Res. Comm. 63, 875-880. Pless, D.D., Schmit, A.S. and Lennarz, W.J. (1975). J. Biol. Chem. 250, 1319-1327. Shockman, G.D., Thompson, J.S. and Conover, M.J. (1967). Biochemistry 6, 1054-1065. Coyette, J. and Shockman, G.D. (1973). J. Bacterial. 114, 34-41. Shockman, G.D. (1965). Bacterial. Rev. 29, 345-358. Herbold, D.R. and Glaser, L. (1975). J. Biol. Chem. 250, 1676-1682. Joseph, R. and Shockman, G.D. (1975). Infect. and Immun., 12, 333-338. Markham, J.L., Knox, K.W., Wicken, A.J. and Hewett, M.J. (1975). Infect. and Immun., 12, 378-386. Wicken, A.J. and Knox, K.W. (1970). J. Gen. Microbial. 60, 293-301. Mosser, J .L. and Tomasz, A. (1970). J. Biol. Chem. 245, 287-298.

1135