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unpublished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.
112
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.
113
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
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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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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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