Trypanosoma cruzi: Inhibition of metacyclogenesis by mannose

Vol. 187, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 108-113

August 31, 1992

Trypanosoma cruzi:

M.A.

Barbieri*,

E.M.

Inhibition

La~nel,**

of Metacyclogenesis

, E.L.D.

Isola**,

by Mannose

and F. Bertini*, 1

*Instituto de H i s t o l o g i a y Embriologia, F a c u l t a d de Ciencias M 6 d i c a s U n i v e r s i d a d Nacional de Cuyo, M e n d o z a and ** D e p a r t a m e n t o de Microbiologia, Facultad de Medicina, Universidad de Buenos Aires, A r g e n t i n a Received June 2, 1992

M e t a c y c l o g e n e s i s of T r v D a n o s o m a cruzi e p i m a s t i g o t e s was e v a l u a t e d in a medium supplemented with Triatoma J.nfestan~ i n t e s t i n a l homogenate in the presence of sugars and derivates as are mannose, galactose, fucose, N - a c e t y l g l u c o s a m i n e , m a n n o s e 6-P, and fructose 1,6-P at a concentration of 25 n~d. 0nly mannose significatively inhibited metacyclogenesis. Sodium metaperiodate and t r y p s i n t r e a t m e n t of the intestinal h o m o g e n a t e also i n h i b i t e d differentiation. In our o p i n i o n there e x i s t s a p r o t e i n i c factor in the i n t e s t i n of the v e c t o r that p r o m o t e s m e t a c y c l o g e n e s i s and is i n c o r p o r a t e d b y the parasite. T r e a t m e n t of the intestinal h o m o g e n a t e w i t h a l k a l i n e p h o s p h a t a s e had no effect. Instead, h i g h ionic s t r e n g t h in the medium (0.4 M NaCI) s t r o n g l y inhibited metacyclogenesis indicating that, in these conditions, the possible b i n d i n g of the d i f f e r e n t i a t i o n factor to the p a r a s i t e surface w a s inhibited, ©1992AcademicPress,Inc.

It

has b e e n

stimulate to

demonstrated

differentiation

metacyclic

addition

forms

of

intestinal

a

homogenate. revealed

treatment

factor

Triatoma

analyzed

major 16

with

in the

1

homogenate

the

KD.

0006-291X/92 $4.00 108

a whole

bands with

a

Preliminary showed

that

morphogenesis

(or more)

proteinic

is involved

To whom correspondence should be addressed at Inst. Histologla y Embriologfa, Casilla de Correo 56, Mendoza 5500, Argentina.

Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

from

electrophoresis,

abolished one

the

has

of

observations)

that

that

obtained

protein 75

vector

epimastigotes

(3) showed

gel

to

trypsin

intestinal

cr~zi

that

by

from

it is p o s s i b l e

insect

infestans

to

five

unpublished

of

fraction

of

fraction

Thus,

al.

varying

(Isola,

present

et

equivalent

fraction,

presence

of the

stimulation.

of

activity

weight

experiments

Isola

chromatographic

This

the

molecular

extracts

of T r v D a n o s o m a

(1,2).

homogenate

metacyclogenic

that

in the

Vol. 187, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

differentiation in

other

process

cells

receptors

located

in

only c a r r i e d

from

external

located

in

derivative In

mannose of

by

6-phosphate

of

that

to

secreted

by the p a r a s i t e

lumen

of the

Consequently,

hexoses

and

its

cruzi,

described

by Isola ~

MATERIALS

AND METHODS

Effect

of sugars

derivates

using al.

by

enzymes

recaptured

due to

figure the

and r e c o g n i z e d

some

Lysosomal

recognize

we

vector

TrvDanosoma

that

receptor~

the

hexose

(5).

epimastigotes

organ.

known

recognized

but also

the cell,

findings,

by some g l y c o p r o t e i n

are

(4).

cytoplasm,

plasmale~na

these

differentiation triggered

in the

It is well

factors

the plasmalenmaa

environment

the

view

parasite.

differentiation

are not the

of the

a model

on the

that

metacyclic

by the

during

we

out

the

form

is

intestin

of the

its stage

in the

studied

the

effect

metacyclogenesis

of d i f f e r e n t i a t i o n

of of

previously

(6).

on m o r m h o ~ e n e s i s

The d i f f e r e n t i a t i o n model d e s c r i b e d by Isola et al. was used (6). Breafly, e p i m a s t i g o t e s (4 x i0 ~ / ml) from biphasic medium (BP) were stimulated in Grace medium (GM) plus intestinal h o m o g e n a t e (IH) for 15 min at 28°C (stimulated e p i m a s t i g o t e s ) , then t r a n s f e r r e d to GM for 15 days at 28°C. A b a t c h of n o n stimulated parasites was transferred to GM as a control. Morphogenesis was evaluated with the light microscope according to parasite motility, shape, and relative k i n e t o p l a s t - n u c l e u s p o s i t i o n by c o u n t i n g at least 200 cells in wet p r e p a r a t i o n s (6). It w a s e x p r e s s e d as the p e r c e n t a g e of m e t a c y c l i c forms, and it w a s e s t i m a t e d by q u i n t u p l i c a t e every 48 hours. E p i m a s t i g o t e s from b i p h a s i c m e d i u m were w a s h e d w i t h i0 n~ p h o s p h a t e b u f f e r (pH 7.2) c o n t a i n i n g 0.15 M NaCI. The pellet was s u s p e n d e d in i0 mM T r i s - H C l buffer (pH 7.2) c o n t a i n i n g 0.25 M sucrose. A v o l u m e of 150 ~i of h e x o s e in Tris-HCl buffer was added to 1.5 ml of parasite suspension (3x107 cells). The final c o n c e n t r a t i o n of each sugar w a s 25 mM. The samples were incubated at 28°C for 20 min, and c e n t r i f u g e d at 3,000 rpm for i0 min. E a c h p e l l e t was r e s u s p e n d e d in GM + IH or in GM + IH plus each sugar at a c o n c e n t r a t i o n of 25 n~ and incubated at 28°C for 15 min. A f t e r this s t i m u l a t i o n in the p r e s e n c e of IH, the samples w e r e c e n t r i f u g e d and transferred to 0.5 ml of GM, b e i n g i n c u b a t e d at 2 8 ° C for i0 days. The p e r c e n t a g e of m e t a c y c l i c forms w a s then e v a l u a t e d as e x p l a i n e d above. The e f f e c t of high ionic strength on morphogenesis was t e s t e d in c u l t u r e s c o n t a i n i n g 0.4 M NaCI. In order to detect if a sugar stimulates the m e t a c y c l o g e n e s i s in the a b s e n c e of IH, the e p i m a s t i g o t e s were t r a n s f e r r e d to GM w i t h each sugar, c u l t u r e d at 28°C for I0 days, and m o r p h o g e n e s i s was m e a s u r e d as e x p l a i n e d above.

109

VoI. 187, No. 1, 1992

Chemical

and

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

enzymatic

treatment

of the m a r a s i t e s

Epimastigotes from biphasic cultures were washed with PBS and treated with agents that may modify cell membrane composition. For this p u r p o s e , i0 ~ cells w e r e i n c u b a t e d in 1 ml of the buffer containing 0.08 units of neuraminidase (Sigma) at 3 7 ° C for 20 min. (7). A f t e r the i n c u b a t i o n , the sample w a s d i l u t e d to 2.0 m l w i t h c o l d PBS, a n d cent.rifuged for i0 m i n at 3 , 0 0 0 rpm. T h e c e l l 3 were washed once w i t h the b u f f e r , a n d p r o c e s s e d for the e v a l u a t i o n of m o r p h o g e n e s i ~ . The effect of sodium periodate was studied by incubating l0 s c e l l s w i t h t h e salt in a c o n c e n t r a t i o n 1 ng-~ at 4 ° C for 15 m i n in t h e darkness. The reaction was s t o p p e d b y d o u b l i n g the v o l u m e of t h e s a m p l e w i t h 2% g l y c e r o l in P B S b u f f e r (8). The samples were centrifuged, washed once with PBS, and the parasites were processed for the evaluation of the morphogenesis with suitable controls. Chemical

a n d eczvmati~,

treatment

o f the

intestinal

h~oeenat~

The IH w a s p r e p a r e d from the i n t e s t i n of ~ infestans a c c o r d i n g to I s o l a et al. (5). A v o l u m e o 1 m l of f i l t r a t e d IH (I0 m g o f p r o t e i n s in PBS) w a s incubated with I0 r~,1 s o d i u m m e t a p e r i o d a t e at 4 ° C for 6 h in the d a r k n e s s . T h e r e a c t i o n w a s stopped with the a d d i t i o n of g l y c e r o l to a f i n a l c o n c e n t r a t i o n of 0 . 2 M (9). T h e m i x t u r e w a s t h e n d i a l y s e d a g a i n s t one l i t e r of P B S at 4 ° C f o r 24 h, t h e n d i l u t e d 1:9 (v/v) w i t h t h e GH. The morphogenesis of the dialysed s. a. m. p. ~ was evaluated t o g e t h e r w i t h s u i t a b l e c o n t r o l s c o n t a i n i n g u n t r e a t e d IH. To study the effect of t r y p s i n , 150 u g o f e n z y m e (Sigma) w e r e a d d e d to o n e ml o f IH (I0 m g of p r o t e i n ) , a n d the m i x t u r e was i n c u b a t e d at 3 7 ° C for 20 min. A f t e r the i n c u b a t i o n , 150 u g of trypsin soybean i n h i b i t o r (Sigma) were added, and the m o r p h o g e n e s i s w a s t r i e d o u t as e x p l a i n e d e l s e w h e r e . The treatment with alkaline phosphatase was carried out according to U l l r i c h et al. (I0). A volume of 1 ml of IH (i0 m g of p r o t e i n ) w a s d i a l y s e d o v e r n i g h t a g a i n s t 50 m M T r i s - C i H b u f f e r (pH 7.5) containing 1 n-~i M g C I 2 . A l k a l i n e phosphatase (Sigma type III) w a s a d d e d to a final c o n c e n t r a t i o n of 2.3 u n i t s p e r ml. T h e m i x t u r e w a s d i a l y s e d for 4 h o u r s at 4°C, a n d for 6 hours at 37°C against the Tris-CiH buffer. The morphogenesis was then assessed together with controls containing alkaline phosphatase. RESULTS Table

I shows

differentiation. of p a r a s i t e s

equal

in

sugar

tested, caused

in

free only

absence

without

(column

with A).

N-acetylgalactosamine

a moderate

and with

its IH

inhibition. llO

of

IH.

derivates (GM + IH)

form, The

IH

(column

only

to

a

of

level

B).

a

moderate

this

sugar,

Regarding

while

on 66%

presence

metacyclogenesis

presented

pre-washed

medium

sugars

to t h e m e t a c y c l i c

inhibited

of c o n t r o l s

were

of

supplemented

the

metacyclogenesis

the p a r a s i t e s a

GM

the medium

to t h a t

The

effect

differentiated

10Z d i f f e r e n t i a t e d mannose

the

In the

increase and

the

and galactose

~en

cultured other

in

sugars

6-phosphate

VoI. 187, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Tablel: E f f e c t of sugars, d e r i v a t e s (0.4 M NaCI) on the Trvmanosoma

Compound

and high ionic s t r e n g t h cruai metacyclogenesis

Percentage

of m e t a c y c l i c s

A

Sugars

B

(25 mM):

Sucrose

66 ± 16

66 ±

7

Mannose

73 ± 12

i0 ±

6

Galactose

55 ± 15

59 ±

6

Fucose

64 ± 14

58 ±

6

N-Acetylglucosamine

55 ± 15

60 ± Ii

N-acetylgalactosamine

58 ± 17

45 ± ii

Mannose

56 ±

6

58 ±

60 ±

8

47 ± i0

6-P

Galactose

6-P

Fructose

Salt

1,6-P

65 ± I0

(0.4 NaCI)

7

68 ±

15 ±

3

lO ±

3

8

Controls: Not s t i m u l a t e d Stimulated

with

with

IH

IH

66 ± I0

E p i m a s t i g o t e s were washed with the sugar b e f o r e s t i m u l a t i o n in Grace m e d i u m s u p p l e m e n t e d with the i n t e s t i n h o m o g e n a t e (IH) of Triatom~ infestanm. They were then c u l t u r e d in the Grace m e d i u m without (A) or in the presence of the sugar (B). In the e x p e r i m e n t s with 0.4 M NaCI, stimulated e p i m a s t i g o t e s were used. The data represent mean and standard deviation of three e x p e r i m e n t s run in q u i n t u p l i c a t e (sugars) or of one experiment run in q u i n t i p u l i c a t e (sugar derivates and salt).

The

addition

epimastigotes while

of strongly

neither

the

differentiation

epimastigotes of +

were

IH

to

24 of

percentage

not

show

to

I%.

5

±

NaCI

to

sugars

stimulated

with

signifieative

forms

86

10% no

any

71

±

results

(58

111

(15

17%).

on

the

When

the

percentage

controls (68

a decrease 10%

were

alkaline ±

the for

± 3%)

effect

effect

caused

while

effect

had

±

had

from

Similar 8%),

stimulated

epimastigotes.

metaperiodate

1%.

of

metaperiodate, from

Neuraminidase

(5 ±

cultures

metacyelogenesis

the

decreased

metaeyolio

treatment a

treated

IH with of

controls) trypsin

±

nor

non

forms

H

inhibited

salt of

metacyelic

Treatment

0.4

(IH

in G H ± in

I0%). the

stimulated

obtained

phosphatase,

with did

Vol. 187, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION The

results

inhibition

show

of

or

glycoprotein

the

the

of

with

indispensable In

a

during

recognized the

previous of

first

and the hours

becomes

irreversible

factor

does not

by a s p e c i f i c

that

binding

of

the

(or

to

contains

located the

results

would

(2)

on

not

be

factor.

showed occur

if

that the

from the m e d i u m After that

~n~n~dlate,y incorporated

this

period,

the

after into

exposure,

the p a r a s i t e

process.

to o b s e r v e

factor

that

as to

high

it may

ionic

be

plasmalelrm]a

strength

expected occurs

also

if

the

before

the

process.

Though

not

increase

of

mannose.

This

elimination competing We

of

epimastigote

site

not

indicating

act

metacyclogenesis,

endocytic

al.

exposure.

it may be

pinocytic

It is i n t e r e s t i n g inhibited

et does

of

stimulating

us

of

acid

IH r e m o v e d

stimulation

and s u g g e s t s

According

sialic

Iso!a

sodium

one

glycoprotein

of the d i f f e r e n t i a t i o n

report,

four

IH c o n t a i n s

the

metacyclogenesis

are washed,

the

the

stimulation

parasite.

IH w i t h

stimulation

by an a f f i n i t y

neuraminidase,

for r e c o g n i t i o n

stimulation parasites

in the

our opinion,

ligand

surface

obtained

involved In

that

a significative

of

inhibited

suggesting

differentiation.

causes

Treatment

trypsin

metacyclogenesis,

mannose,

only m a n n o s e

metacyclogenesis.

periodate

more)

that

statistically metacyclic c o u l d be

from

for the

believe

strategies

the site our

forms

interpreted surface

with

of b i n d i n g

parasites

as the result

of the may be

was

washed

an with

of an of

compounds

factor. useful

the m e t a c y c l o g e n e s i s ,

and m e c h a n i s m s

there

of the e p i m a s t i g o t e

experiments

to u n d e r s t a n d

of virulence,

significative,

of h o s t - p a r a s i t e

to

open new

the a c q u i r e m e n t relationship.

Acknowledgments; This w o r k was s u p p o r t e d by a grant from the Government of the P r o v i n c i a de Mendoza, Argentina, and from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET) of Argentina. We thank Mrs Nilda Pochettino for c o r r e c t i n g the m a n u s c r i p t .

REFERENCES i- Wood, D.E., and Pipkin, A.C. (1969) Exp. Parasit. 24,176183. 2- Isola, E,L.D., Lan~nel, E.M., Katzin, V.J., and G o n z a l e z C a p p a S.M. (1981) J. Parasit. 67,53-58.

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Vol. 187, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3- Isola, E.L.D., La~nel, E.M., Giovaniello, O., Katzin, A.M., and Gonzales Cappa, S.M. (1986) J. Parasit. 72, 467-469. 4- Von Figura, K. and Hasilik, A. (1986) Ann. Rev. Biochem. 55, 167-193. 5- Goldstein, J.L., Brown, M.S., Anderson, R.G.W., Russel, D.W. and Schneider, W.J. (1985) Ann. Rev. Cell Biol. 1,1-39. 6- Isola, E.L.D., Lanmael, E.M., and Gonzales Cappa, S.M. (1986) Exp. Parasit. 62, 329-335. 7- Pereira, M.E.A., Luores M.A., Villata F., and Andrade A.F.B. (1980) J.Exp. Med. 152,1375-1392. 8- Toowicharanont, P. and Chulavatnatol, M. (1983) J. Reprod. Fert. 67, 275-280. 9- Sosa, M.A., Mayorga, L.S., and Bertini, F. (1987) Biochemo Biophys. Res. Colmnun. 143,799-807. i0- Ullrich, K., Mersmann, G., Weber, E., and Von Figura, K. (1978) Biochem. J. 170,643-650.

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