Candidacidal activity of myeloperoxidase: Characterization of myeloperoxidase-yeast complex formation

Vol.

154,

No.

July

29,

1988

2, 1988

BIOCHEMICAL

AND

BIOPHYSICAL

COMMUNICATIONS Pages

CANDIDACIDAL

ACTIVITY

Clifford

D. Wright'

Departments

.June 20,

CHARACTERIZATION

OF MYELOPEROXIDASE:

MYKLOPEROXIDASE-YEAST

Received

RESEARCH

809-817

OF

COMPLEX FORMATION D. Nelson I,2

and Robert

of Microbiology1 and Surgery2 University of Minnesota Minneapolis, MN 55455

1988

demonstrated the ability of human FxJmlARY : We have previously -neutrophil myeloperoxidase to bind to cell wall mannan polysaccharide isolated :Erom Candida albicans. This binding capacity provides for association of the enzyme with target yeast which is essential for In this report, we further consider the efficient candidacidal activity. role of the mannan-binding property of myeloperoxidase in the Solubilized mannan antagonizes candidacidal activity of the enzyme. binding o:E the enzyme to yeast, suggesting that mannan may be a primary component of the fungal cell wall which serves as a target for binding of Myeloperoxidase is shown to form complexes with both myeloperoxidase. solubilized mannan and Candida yeast, with Kds of 0.97 x lo-' M and 1.2 x lo-' M, respectively. The interaction between myeloperoxidase and mannan does not allow the enzyme to readily dissociate from the surface of target yeast. As a result, the enzyme may be unable to dissociate from dead yeast to become available for binding to additional fungal targets. 0 1988 ncademlc Press. 1°C.

The microbicidal halide

system

activity

(1)

is

microorganisms.

This

Escherichia -- coli: Candida albicans _ -_

(4).

myelopero'xidase acid

(5)

of this

of the

optimized phenomenon

(2),

Actinobacillus It

enzyme is

has been

at the microbial

thereby

(6,7)

to the for

and its

that for

of

(3),

binding

and

of

generation

of hypochlorous

minimizing

both

interaction

target

killing

actinomycetecomitans allows

surface,

oxidant

peroxide-

bound

demonstrated

has been proposed

to microorganisms

cytotoxic

myeloperoxidase-hydrogen

when the

with

the

dilution

alternate

substrates. We have previously C. albicans interaction

through

was shown

myeloperoxidase 'Current Company,

demonstrated cell

address:

wall

to be ionic,

(p1 = 10.2)

(11)

Pharmacology

Pharmaceutical

that

mannan

Research

myeloperoxidase

polysaccharide

rather and the

Department, Division,

than

binds

to

(4,8,9,10). lectin-like,

phosphate

The between

groups

of the

mannan

Parke-Davis/Warner-Lambert Ann Arbor,

MI

48105 0006-291X/88

809

$1.50

Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol.

154,

outer

No.

2, 1988

chain

(8).

has also

been

The purpose myeloperoxidase of the

BIOCHEMICAL

Binding

shown

BIOPHYSICAL

of myeloperoxidase

to be a mannan-specific

of this

report

to mannan

is

required

RESEARCH

interaction

to further

COMMUNICATIONS

to A. actinomycetecomitans (3).

characterize

to promote

the

candidacidal

the binding

of

activity

enzyme. MATERIALS

Isolation

AND

AND METHODS

of Myeloperoxidase

Human neutrophils were isolated from anti-coagulant treated whole blood according to the procedure of Ferrante and Thong (12). Myeloperoxidase was isolated from human neutrophils according to the method of Andrews and Krinsky (13). The partially purified enzyme had an R. 2. value of 0.62. Myeloperoxidase activity was quantitated in terms of pyrogallol units as described by Baggiolini, et al, (14). The candidacidal activity of the enzyme preparation was characterized in vitro. Candidacidal activity was shown to be both hydrogen peroxideand chloride ion-dependent. Inhibitors of myeloperoxidase activity (0.1 mM azide, 0.1 mM cyanide) and a scavenger of hypochlorous acid (1 mM I-methionine) also effectively inhibited killing of target yeast (4). Isolation

of Mannan

Candida albicans 2252 (ATC 44806) was grown to stationary yeast nitrogen base minimal medium (15). Mannan was isolated Fehling's solution as copper complexes from citrate-extracted described by Peat, et al, (16). Assay

of Myeloperoxidase-Mannan

Complex

phase with yeast

in as

Formation

Myeloperoxidase (3.5 x 10e2 units) and mannan (4.0 mg) were incubated in Dulbecco's phosphate-buffered saline (PBS) (pH 7.4) for 30 minutes at 37%. The sample was then chromatographed on a Sephadex G-150 gel permeation column (18 x 1.5 cm) in distilled water. The elution profile of the myeloperoxidase-mannan reaction mixture was compared with those of isolated myeloperoxidase and mannan. Elution of myeloperoxidase was monitored by its catalytic activity at 435 nm (14). Elution of mannan was monitored by the phenol-sulfuric acid assay for carbohydrates at 490 nm (17). Assay

of Myeloperoxidase-Yeast

Complex

Formation

C. albicans 2252 was incubated overnight in yeast nitrogen base mini&l media at 37%. Following three washes in PBS, the yeast suspension was adjusted to a final optical density of 1.0 at 660 nm (Asso) of 1.0 equals approximately 2 x lo7 yeast per ml. A 0.1 ml aliquot of yeast was incubated with 1.1 x 10e3 units of myeloperoxidase in a final volume of 0.5 ml at 37%. After 30 minutes, the reaction mixture was centrifuged and the amounts of free and yeast-bound myeloperoxidase were determined. Binding of myeloperoxidase to yeast compared in the presence and absence of 2 mg/ml mannan. Dissociation Constants Yeast Interactions

of Myeloperoxidase-Mannan

The dissociation constant of.myeloperoxidase-mannan determined by incubation of varying concentrations 810

and Myeloperoxidasecomplexes was of myeloperoxidase

was

Vol.

154,

No.

2, 1988

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

with 12 mg of C. albicans 2252 mannan in PBS. After a 30 minute incubation at 37OC, the reaction mixture was chromatographed on a G-150 Sephadex gel permeation column (18 x 1.5 cm) in distilled water to separate free myeloperoxidase from mannan-bound enzyme. The peak areas of the chromatographed myeloperoxidase fractions were used to determine the amount of bound and free enzyme for use in Scatchard analysis (18). The dissociation constant of myeloperoxidase-yeast complexes was determined by incubation of varying concentrations of myeloperoxidase with C. albicans 2252. Overnight cultures of C. albicans 2252 were One ml aliquots of the yeast suspension prepared as described above. were centrifuged and resuspended to a final volume of 0.6 ml with a 30-minute incubation at 37OC, the myeloperoxidase in PBS. After reaction mixture was centrifuged and the amount of myeloperoxidase remaining free in the supernate was quantified for use in Scatchard analysis. Molecular

Weight

Determinations

of Candida

mannan

The molecular weight of C. albicans 2252 mannan was determined by meniscus depletion equilibration using a Spinco model E analytical centrifuge with interference optics (19). Samples were prepared at 0.5 mg/ml in 0.1 M KHsPO4, pH 4.7. The sample was centrifuged at 14,000 rpm at 18OC. The mean molecular weight of mannan from two runs was detennined. Assay

of Myeloperoxidase-Mediated

Candidacidal

Activity

2252 were prepared as described Overnight cultures of C. albicans above. A 0.1 ml aliquot ofyeast was incubated with 1.1 x lop3 units of myelopero.xidase at 4nC for 30 minutes. Following preincubation, hydrogen peroxidas,e was added to give a concentration of 0.02 mM in a final All reagents were diluted in PBS to provide a reaction -volume of 1.0 ml. final chl'oride concentration of 0.14 M. The reaction mixture was incubated at 37OC on a rotating table. Myeloperoxidase-mediated killing was stopped by transferring the reaction tube to ice water. Yeast viability was determined by pour plate methodology using yeast extract peptone dextrose agar (15). Control experiments verified that the observed candidacidal activity was generated by the myeloperoxidase-hydrogen peroxide-chloride antimicrobial system (1). RESULTS Myeloperoxidase-Mannan Binding mannan

Complex

was assessed

by gel

of myeloperoxidase of its

catalytic

of the

column.

phenol-sulfuric volume.

myeloperoxidase allow

from

In contrast, acid

association

(435

run),

of the

C. albicans

chromatography G-150

column,

occurred

elution

carbohydrate

and a 4.0

to solubilized

permeation

a Sephadex

activity

The chromatograph

elution did not

Formation

of human myeloperoxidase

monitored

within

of mannan, assay

(490

nm),

of 3.5

mg of mannan

preincubated

showed

the

a shift

volume

by the

occurred

in the

void

of

30 minutes

to

of the peroxidase

peak to the void volume. Binding of myeloperoxidase inhibit the catalytic activity of the enzyme. This 811

Elution

as a function

x 10e2 units for

1).

inclusion

monitored

of a mixture reactants

(Figure

2252

to mannan shift in the

Vol.

154,

No.

2, 1988

BIOCHEMICAL

0.4

-

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

"0 i

0.3

-

:

MPO + mannan 435nm

Iii e

2

MPCJ 435nm

0.2 -

:

II, 0.1

R

-

&!.!L&.

5

10

15

20

25

30

Elution

35

40

Volume

45

50

55

6C

(ml)

FIGIJBE 1. Myeloperoxidase-mannan complex formation. Elution patterns of myeloperoxidase (MPO), -~ C. albicans 2252 mannan, and an MPO-mannan reaction mixture from a Sephadex G-150 gel permeation column are illustrated. Elution of myeloperoxidase was monitored as a function of its catalytic activity at 435 am. Elution of mannan was monitored by the phenol-sulfuric acid carbohydrate assay at 490 nm. Vo, void volume. elution

pattern

for

peroxidase

activity

is

expected

for

an enzyme-mannan

complex. Myeloperoxidase-Yeast Binding

Complex

determining

the

30-minute

incubation

amount

1.1 x 10-e

units

68 percent

of the

corresponding association binding

with that

involves

with enzyme. with

myeloperoxidase

yeast.

in a dose-dependent displace

Analysis

Myeloperoxidase-Candida The dissociation myeloperoxidase-Candida

incubation

Candida

yeast, with

of the

solubilized enzyme with

binding

interaction

of myeloperoxidase

Solubilized

(4).

enzyme.

to solubilized

These

mannan

of the

mannan

was also

results

suggest

and to yeast

of the enzyme.

of Myeloperoxidase-Mannan

and

Interactions constants

(Kds)

interactions

for were

812

myeloperoxidase-mannan determined

the

a common

antagonized

yeast-bound

function

whether

involved

Preincubation

of a

to determine

and yeast

with

manner

a

After

was preincubated

inhibits

of myeloperoxidase

a common binding

In order

by

following

cell-associated

completely

Mannan

2).

2 x lo6

mannan

of yeast. mannan

was assessed

enzyme

(Figure

enzyme became

in free

to addition

to effectively binding

Binding

available

of solubilized

yeast

shown

and yeast-bound

of myeloperoxidase

decrease

to target

to C. albicans

reagents

of myeloperoxidase

mannan prior enzyme

of free of the

function,

2 mg/ml

Formation

of human myeloperoxidase

by Scatchard

and

Vol.

154,

No.

2, 1988

BIOCHEMICAL

120

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-

FIGURE 2. Influence of soluble mannan on myeloperoxidase-Candida comlplex formation. Binding of myeloperoxidase (MPO) to yeast was compared in the presence and absence of soluble mannan (mean 2 S.E.M., n = 3).

analysis

of binding

data

of myeloperoxidase permeation bound

with

enzyme.

of binding

allowed

Scatchard

sites

analysis

available (Figure

myeloperoxidase

with

activity,

allowed

Scatchard

analysis

complexes per results

yeast

cell

3A).

of varying

for

was used of 0.97

for

indicate

to determine

wall-associated

a Kd for

of mannan

interaction

Incubation

of varying

of free 4.12

interactions the

and mannan-

milligram

to determine

that

of free 1.95

of 1.2 x 10e5 M with yeast

concentrations by gel

x 10e5 M with

a determination

was used

followed

determination

2252 stationary -C. albicans and analysis of the supernatant

centrifugation

available

per

Incubation 2252 mannan,

for

complexes

myeloperoxidase

These

3).

albicans

chromatography

myeloperoxidase-mannan

yeast

(Figure C 2

with binding

mannan

for

moles with

concentrations phase

medium

yeast, for

of followed

by

enzyme

and yeast-bound

a Kd for

enzyme.

myeloperoxidase-Candida

x lo-l3

mM of binding

myeloperoxidase affinities

and for

x lo-'

sites

(Figure

3B).

of myeloperoxidase

solubilized

mannan

are

for nearly

identical. In order mannan determined equilibrium

to determine

the

and myeloperoxidase, by analytical method

(19),

stoichiometry

the

molecular

centrifugation. the

Candida

of the weight

of

Using

the

interaction the mannan meniscus

mannan was determined 813

of Candida was

depletion to have

a

Vol. 154,

No.

2, 1988

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

A *O 1

0

10

20

Bound I 15

25

20

Nmoles

30

40

50

60

(micromolar) , 30

Added

Nmoles

Added

FIGURE 3. Binding of myeloperoxidase to Candida mannan (Panel A) and Candida yeast (Panel B). A 30 minute incubation at 37OC allowed for maximal binding of myeloperoxidase to mannan or yeast.

molecular

weight

sites

milligram

per

approximately

of approximately

Capacity

We have yeast.

with

conditions.

viability yeast

units

population

candidacidal under

available

from

for were activity

a lack

peroxide-halide 1.1 x 10-a concentration

is

next

to target performed

resulted

from

of sufficient of myeloperoxidase

of 0.02

yeast

mM to

that

the

percent target

as percent dose

yeast

fungal

shows that

to kill

Thus,

the

Candida optimal

of myeloperoxidase

target

ratio per

These

results

suggest

that

is

limited

by the

amount

for

yeast the of

yeast. to verify a lack

cofactors system

capacity

fungicidal

of available

has a capacity

of myeloperoxidase

of

to the

enzyme was incubated

data

1.4 x lo*.

activity absorbed

optimal

number

7.9 x 1Om8 units

antimicrobial units

of

candidacidal

3, indicate

of the

conditions. binding

is

the

under

of initial

of approximately

Experiments candidacidal

of a plot

logarithm

candidacidal

enzyme

of purified

as the

of myeloperoxidase

activity

ratio

of myeloperoxidase.

characterize

in Figure

increased

experimental

candidacidal

than

the

the

of Candida

illustrated yeast

activity

these

enzyme

to further

Extrapolation versus

1.1 x 10-a

that

concentrations Data

of enzyme binding

to a binding

molecule

when the

1.1 x 10d3 units

of target

decreased.

per

effective

In order

decreasing

killing

equates

of mannan

demonstrated

is most

of myeloperoxidase,

The number

of Myeloperoxidase

previously

myeloperoxidase target

of polysaccharide

two molecules

Candidacidal

280,000.

that

the

of sufficient for

(Table

of

enzyme

rather

the myeloperoxidase-hydrogen 1).

and hydrogen

1 x 10" Candida

limitation

yeast

Simultaneous peroxide

addition

of

to a final

in PBS resulted

in

less

Vol.

154,

No.

2, 1988

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TABLE I Characterization

of Myeloperoxidase-Mediated

Condition

Candidacidal

Supplements Ml'0 a + HzOz b MPO + HzOz + MPO + Hz02 + MPO + HzOz +

1 2 3

4

Yeast Viability

+ Cl-' (-MPO preincubation) Cl (+MPO preincubation) Cl- + (Hz02 at T = 30') Cl- + (MPO at T = 30')

*p
than at

a one percent 37'C

(Fig.

yeast

at

yeast

viability

decrease

4).

4cC for

30 minutes after

(p < 0.01).

Binding

of hydrogen

peroxide

Incubation

(4).

hydrogen reduction treated

provided

a 30-minute

for

of myeloperoxidase is

not

for

of yeast

an additional

viability.

yeast

to kill yeast

with

with

target in

peroxide in

target

the

absence

microorganisms aliquot

resulted

incubation

However,

incubation

reduction

yeast

the

2.5 0.7" 2.0 1.6"

with

hydrogen

a second

30 minutes

an additional

enzyme

percent

with

to Candida

sufficient

a 30 min.

the

a 23.1

f + 2 f

(%)

2 0.02 mM;

after of

incubation

99.0 76.9 80.5 63.5

condition peroxide,

viability

preincubation

of enzyme-treated

peroxide target

in yeast

In contrast,

Activity

of

in no further

of the

1.1 x 10S3 units

previously of

100 90 i

T ~

80-

C. albicans

2252

]

z-l 70.g =.nr 60; 508 40t L 3020-

IO-

4.0

log yeast I

1 1 0.125

0.25

Yeast

5.0

0.5 Concentration

6.0

concentration

1.0 (per

ml x10-9

FIGURE 4. Effect of yeast concentration on myeloperoxidase-mediated candidacidal activity. Percent viability of varying concentrations of C.aLbicans 2252 after incubation with 1.1 mU of myeloperoxidase and -___ 0.012 mM hydrogen peroxide in PBS is illustrated in linear and semilogarithmic plots (mean f S.E.M., n = 3).

815

Vol.

154,

No.

2, 1988

myeloperoxidase after

BIOCHEMICAL

reduced

a 30 minute

myeloperoxidase

activity

Additional

suggest of its

under

from

conditions hour

incubation

availability

the

is

performed

remained

of the

of additional

to desorb

fungal

of

since

a six

incubation

After

a similar

yeast,

the

amount

suggest

may be limited

killed

yeast

not

fungicidal

observations

activity

of this did

hour

optimal

Candida

These

cause

limitation

activity.

from

peroxide

factor the

This

with

candidacidal

enzyme

limiting

under

constant.

percent

of

to determine

enzyme,

catalytic

13.4

conditions.

activity.

on its

COMMUNICATIONS

of hydrogen

the

experimental

peroxide

myeloperoxidase-mediated killing

that

of myeloperoxidase

enzyme

inability

aliquot

hydrogen

had no effect

yeast-bound

by an additional

of the

with

RESEARCH

a second

candidacidal

of myeloperoxidase six

were

autoinactivation

BIOPHYSICAL

substrates

these

experiments

myeloperoxidase-limited result

with

results and not

candidacidal

viability

incubation

These

(p <0.05).

yeast

AND

of

that

by an

to participate

in

targets. DISCUSSION

Myeloperoxidase its

the

cytotoxic

agent

bind

walls

acid. Mannan

of myeloperoxidase that

weight

this

of the

Interaction occurs

without

result,

the

carbohydrate

cell the

provide

for

would

result, yeast.

of a minimum limitation

would

result,

the

may be limited

available

fail

availability by its

to form

is

able

to

cell as a target

as evidenced

for

by the

30 to 50 percent

Candida

through

activity

cell

of the

to this

of the

fungal

of the

both

cell

of the

mannan As a

surface acid

Such localized

dilution

wall

enzyme.

of hypochlorous cells.

candidacidal dose

an inability

activity

of the of the

of myeloperoxidase

also

ion

(20,21).

generation

effective

from

Targeting

mannan

with

in close generation

oxidant

appears

enzyme

of myeloperoxidase-mediated

in part,

chloride

for

of

and its

substrates.

Myeloperoxidase-mediated apparent

essential capable

myeloperoxidase

yeast

with

minimize

alternative

with

may compose

catalytic

components

acid with

readily

of myeloperoxidase

would

of hypochlorous

binding

wall

of myeloperoxidase

to target

interaction

is

is

interaction

to Candida

affecting binding

Second,

polysaccharide

fungal

properties

peroxide

by specific

mannan.

observation

two unique myeloperoxidase

of hydrogen

hypochlorous cell

wall-associated

proximity

First,

reaction

to fungal

binding

to have

activity.

catalyzing

dry

appears

candidacidal

affinity

for

enzyme

between

of myeloperoxidase mannan. 816

to target

candidacidal

to Candida

to discriminate

to require

for

cell

The

activity

to desorb

live

yeast. from

may

killed

wall-associated and dead yeast.

binding

to target

As a yeast

Vol.

154,

No.

2, 1988

We beI.ieve

that

mannan

represents

activity

of the

determine targets

whether for

BIOCHEMICAL

the

ability

additional

interaction

BIOPHYSICAL

RESEARCH

of myeloperoxidase

an essential enzyme.

AND

mechanism

Continued

research

microbial

to

involved must

interact

in the

with

other

with

candidacidal

be performed

polysaccharides

of myeloperoxidase

COMMUNICATIONS

to

may serve

as

microbial

pathogens. ACKNOWLEDGMENT This

work

was supported

in part

by Public

Health

Service

Grant

AI 16785.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Klebanoff, S. J. (1968) J. Bacterial. 95, 2131-2138. Selvaraj, R. J., Zgliczynski, J. M., Paul, B. B., and Sbarra, A. J. (1978) J. Infect. Dis. 137, 481-485. Miyasaki, K. T., Zambon, J. J., Jones, C. A., and Wilson, M. E. (1987) Infect. Immun. 55, 1029-1036. Wright, C. D., Bowie, J. U., Gray, G. R., and Nelson R. D. (1983) Infect. Immun. 42, 76-80. Agner, K. (1972) Structure and function of oxidation reduction enzymes. In A. Akeson and A. Ehrenberg (Eds.), pp. 329-335. Pergamon Press, Oxford. Albrich, J. M., McCarthy, C. A., and Hurst, J. K. (1981) Proc. Natl. Acad. Sci. USA 78, 210-214. Sips, H. J. and Hamers, M. N. (1981) Infect. Immun. 31, 11-16. Wright, C. D., Bowie, J. U., and Nelson, R. D. (1984) Infect. Immun. 43, 467-471. Wright, C. D., Herron, M. J., Gray, G. R., Holmes, B., and Nelson, R. D. (1981) Infect. Immun. 32, 731-738. Wright, C. D. and Nelson, R. D. (1985) Infect. Immun. 47, 363-365. Agner, K. (1941) Acta Physiol. Stand. (Suppl. 8) 2, l-62. Ferrante, A. and Thong, Y.H. (1978) J. Immunol. Methods 24, 389-393. Andrews, P. C. and Krinsky, N. I. (1981) J. Biol. Chem. 258, 4211-4218. Baggiolini, M., Hirsch, J. G., and deDuve, C. (1969) J. Cell Biol. 40, 529-541. Fink, G. (1970) Methods Ezymol. 17A, 59-78. Peat, S., Whelan, W. J., and Edwards, T. E. (1961) 3. Chem. Sot. p. 29-34. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28, 350-356. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. Nakajima, T. and Ballou, C. E. (1974) J. Biol. Chem. 249, 7679-7684. Chattawa, F. W., Holmes, M. R., and Barlow, A. J. E. (1968) J. Gen. Microbial. 51, 367-376. McMurrough, I. and Rose, A. H. (1967) Biochem. J. 105, 189-203.

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