ULTRASTRUCTURAL LOCALIZATION OF S M 1 5 A N D S M 2 5 , T W O MAJOR TEGUMENTAL ADULT W O R M ANTIGENS
OF SCHISTOSOMA MANSONI
ABATH F.G.C.*, HIRST E.M.A.", HAGAN P.*** & SIMPSON A.J.G.****
Summary :
S m l 5 and Sm25 are two of the principal tegumental antigens recognized by antibodies from mice protectively vaccinated with adult worm tegumental membranes and may therefore be potential vaccine candidate antigens. Using antibodies affinity purified from anti-tegumental membrane anti-sera, and antibodies raised against the recombinant antigens, S m l 5 and Sm25 were shown to be located specifically in the tegument of adult worms being distributed throughout the syncitium but not associated with the outer membrane.
K E Y W O R D S : Schistosoma mansoni, tegumental antigens, immunolocalization, Sm25, Sm 1 5.
Résumé : LOCALISATION EN ULTRASTRUCTURE D E DEUX DES PRINCIPAUX ANTIGÈNES TÊGUMENTAIRES D E SCHISTOSOMA MANSONI, S M 1 5 E T S M 2 5 Sm I5 et Sm25 sont deux parmi les principaux antigènes têgumentaires reconnus par des anticorps produits par des souris qui ont été vaccinées avec des membranes têgumentaires de Schistosoma mansoni adultes et qui sont, en conséquence, des candidats potentiels à un vaccin spécifique contre ce parasite.
Des anticorps purifiés par affinité à partir de sérum anti-membrane tégumentaire, ainsi que des anticorps obtenus contre des
antigènes recombinants, ont été employés pour
l'immunolocalisation de Sml5 et de Sm25. Les résultats ont montré que ces antigènes se trouvent spécifiquement dans le tégument des vers adultes, distribués dans tout le syncitium mais n'étant pas cependant associés à la membrane externe.
MOTS CLES : Schistosoma mansoni, antigènes têgumentaires, immu- nolocalisation, Sm25, Sml 5.
T
h e principal m e m b r a n e associated antigens o f the tegument o f adult Schistosoma mansoni d o not cross react with the parasite egg that is the focus o f immunopathological reactions (Simpson et al., 1 9 9 0 ) . Since immunization o f m i c e with isolated tegu- mental m e m b r a n e s has b e e n found to stimulate a level o f protective immunity that is c o m p a r a b l e with that sti- mulated in the s a m e animal m o d e l by vaccination with highly irradiated cercariae, an anti-schistosome v a c c i n e b a s e d o n tegumental antigens would avoid p r o b l e m s o f e x a c e r b a t i n g p a t h o l o g i c a l r e s p o n s e s induced b y egg antigens (Smithers et al, 1 9 8 9 ) . T h e principal tegumental antigens recognized by antibodies from protectively v a c c i n a t e d m i c e in immunoblotting are integral m e m b r a n e proteins o f 2 5 , 15 and 13 k D a (Smithers et al, 1 9 9 0 ) . T h e full length g e n e for the* Instituto A g g e u M a g a l h ä e s , F u n d a c á o O s w a l d o Cruz, Av. M o n i e s R e g ó s / n , C i d a d e U n i v e r s i t a r i a , 5 0 6 7 0 - 4 2 0 , R e c i f e , B r a z i l .
** N a t i o n a l Institute for M e d i c a l R e s e a r c h , Mill Hill. L o n d o n , N W 7 1AA, U K .
*** D e p a r t m e n t o f I n f e c t i o n a n d I m m u n i t y , I B L S , U n i v e r s i t y o f G l a s g o w , G l a s g o w , G 1 2 8 Q Q , S c o t l a n d , U K .
**** I n s t i t u t o L u d w i g d e P e s q u i s a s s o b r e o C á n c e r , R. Prof. A n t o n i o P r u d e n t e 1 0 9 , 0 1 5 0 9 - 0 1 0 , S A O P a u l o - S P , B r a z i l .
C o r r e s p o n d e n c e : F r e d e r i c o G . C . A b a t h . Instituto A g g e u M a g a l h a e s ( F I O C R U Z ) , Av. M o r a e s R e g ó s/n. C i d a d e U n i v e r s i t a r i a , C E P : 5 0 6 7 0 - 4 2 0 , R e c i f e , P E , B r a z i l .
Fax: ( 0 8 1 ) 4 5 3 1 9 1 1 - E-mail: f a b a t h @ g e n e . d b b m . f i o c r u z . b r
25 kDa antigen ( S m 2 5 ) ( O m e r Ali et al, 1991; El-Sher- beini et al, 1 9 9 1 ) and the 15 k D a antigen ( S m l 5 ) (Abath et al, 1 9 9 3 ) have b e e n c l o n e d and s e q u e n c e d and the mature g e n e product analyzed in s o m e detail (Karcz et al, 1 9 8 8 ; P e a r c e et al, 1 9 9 1 ; Abath et al, 1 9 9 4 ) .
W e have investigated the distribution o f S m l 5 and S m 2 5 within adult w o r m s by immuno-gold electron microscopy. Both S m l 5 and Sm25 are distributed throu- g h o u t t h e adult t e g u m e n t but are a p p a r e n t l y not e x p o s e d o n adult w o r m surfaces.
MATERIALS AND METHODS
PARASITES
W
orms from a Puerto Rican strain o f Schisto- soma mansoni maintained in the laboratory w e r e o b t a i n e d from s i x - w e e k infected ham- sters by perfusion from the hepatic portal system (Smi- thers & Terry, 1 9 6 5 ) .ANTISERA
Rabbit anti-tegumental m e m b r a n e a n t i b o d i e s w e r e raised by immunization with tegumental m e m b r a n e proteins solubilized in SDS (sodium dodecyl sulphate), as described by Simpson et al. ( 1 9 9 0 ) . T h e experiments of immunolocalization w e r e undertaken with sera from Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1999063243
ABATH F.G.C., HIRST E.M.A., HAGAN P. & SIMPSON AJ.G.
rabbits immunized with purified r e c o m b i n a n t S m 2 5 ( O m e r Ali et al, 1 9 9 D and affinity purified antibodies, specific for S m l 5 (Abath et al, 1 9 9 4 ) .
IMMIINO-GOLD LABELLING OF ADULT WORM SECTIONS Adult w o r m s o f S. mansoni w e r e fixed either in 0.1 % high vacuum distilled glutaraldehyde for 3 0 min at 4 ° C , and then q u e n c h e d in 1 % a m m o n i u m chloride, or 2 % paraformaldehyde/1 % acrolein for 3 0 min at r o o m temperature. Samples were then washed in PBS pH 7.2, dehydrated at progressively l o w e r e d temperatures and e m b e d d e d in Lowicryl K4M at - 3 5 ° C (Roth et al, 1 9 8 1 ) . Sections 6 0 nm thick w e r e cut o n a Reichert Ultracut M i c r o t o m e and placed o n t o formvar/carbon coated 2 0 0 mesh gold grids. Sections w e r e pre-blocked with 10 % n o n fat dry milk in T B S (tris buffered saline) pH 8.2 for 3 0 min at r o o m temperature. Grids w e r e then transferred to antibodies specific for S m l 5 diluted 1:100 in T B S containing 1 % BSA and 0.25 % T w e e n 2 0 ( T B S / B S A / T w e e n ) , and incubated overnight at 4 ° C . Control sections w e r e treated with the s a m e dilution o f normal rabbit serum. T h e grids w e r e washed for o n e hour at r o o m temperature with T B S / B S A / T w e e n , then incubated at r o o m temperature with goat anti-rabbit IgG, conjugated to 10 nm gold particles (diluted 1:50 in T B S / B S A / T w e e n ) . After two hours o f incubation, the grids w e r e rinsed in P B S - B S A - T w e e n , followed by dis
tilled water. T h e sections w e r e dried, stained with uranyl acetate and v i e w e d in a Phillips 3 0 0 electron m i c r o s c o p e . Studies involving the localization o f S m 2 5 used rabbit antibodies raised against a S m 2 5 / g l u t a - thione-S-transferase ( G S T ) fusion protein p r o d u c e d u s i n g t h e p G E X - l N e x p r e s s i o n v e c t o r . N e g a t i v e controls utilized rabbit anti-GST antibodies. Evaluation o f titers o f t h e t w o antisera b y ELISA, using G S T ( e n z y m e linked i m m u n o s o r b e n t assay) indicated that the negative control serum had a fourfold higher titer o f antibody against G S T than the anti-fusion protein serum. T h e sections used for the localization o f S m 2 5 w e r e processed exactly as for studies with S m l 5 except that only 0.1 % glutaraldehyde was used as a fixative.
In the c a s e o f S m 2 5 an estimation o f the distribution o f gold particles w a s performed b y counting the total gold particles in two distinct areas o f the tegument and calculating the proportion for those associated to the discoid bodies.
RESULTS
G
old immunolabelling o f adult w o r m sections using affinity purified antibodies specific for S m l 5 and antibodies raised against r e c o m b i nant Sm25: w e have previously demonstrated by immun o f l u o r e s c e n c e that affinity purified antibodies, w h i c h
r e c o g n i z e S m l 5 , s h o w e d a positive and specific reac
tion with the tegument o f adult w o r m s (Abath et al, 1 9 9 4 ) . T h e immunolabelling for electron m i c r o s c o p y with antibodies specific for S m l 5 was performed with different fixative regimens. In figure 1, samples A and B w e r e fixed with glutaraldehyde, w h e r e a s samples C and D w e r e fixed with paraformaldehyde/acrolein.
Although the labelling was not intense (Fig. I B ) , it was associated with the tegument, possibly with s o m e pre
ference for the discoid ( e l o n g a t e ) b o d i e s . Figure I D shows tegumental localization o f the labelling, although structural preservation is poor. T h e gold particles w e r e not associated with the tegumental outer m e m b r a n e or the muscle.
I m m u n o f l u o r e s c e n c e using antibodies raised against a Sm25-p-galactosidase protein h a v e b e e n previously u s e d to d e m o n s t r a t e that S m 2 5 is e x p r e s s e d o n l y within the tegument o f adult w o r m s (Knight et al, 1 9 8 9 ) . Here antibodies against a S m 2 5 - G S T r e c o m b i nant w e r e used to study the distribution o f the antigen at the ultrastructural level (Fig. 2 ) . T h e antibody exhi
bited a generalized binding throughout the tegument often, but not exclusively, associated with the discoid bodies. Analysis o f two distinct fields demonstrated that 39 % o f the gold particles w e r e associated with these structures. T h e r e w a s n o e v i d e n c e for the association o f the antigen with the spines, muscle layer or the tegu
mental outer m e m b r a n e . T h e control serum e x h i b i t e d an extremely low level o f binding within the tegument, less than 10 % o f that e x h i b i t e d b y the positive serum despite its m u c h higher titer against G S T demonstra
ting the specificity o f the binding exhibited b y the anti- r e c o m b i n a n t S m 2 5 antibodies.
DISCUSSION
S
m l 5 and S m 2 5 are major antigens r e c o g n i z e d by antibodies from m i c e v a c c i n a t e d with isolated t e g u m e n t a l m e m b r a n e s (Smithers et al, 1 9 8 9 ; Smithers et al, 1 9 9 0 ) . In addition, e v i d e n c e from other experimental systems is consistent with S m 2 5 b e i n g a key target antigen o f host protective humoral i m m u n e r e s p o n s e s (Wright et al, 1 9 8 8 ; El-Sherbeini et al, 1 9 9 0 ) .T h e m e m b r a n e fraction o f adult w o r m s u s e d for vac
cination studies was isolated by the induced shedding o f tegumental c o m p o n e n t s b y incubation o f live adult w o r m s in p h o s p h a t e buffered saline ( S i m p s o n et al, 1 9 8 1 ) . This preparation contains m e m b r a n o u s c o m p o n e n t s o f the tegumental syncitium, including mito
chondria, discoid and m e m b r a n o u s bodies, in addition to the tegumental outer m e m b r a n e itself. B o t h S m l 5 and S m 2 5 have b e e n s h o w n to b e h a v e as integral m e m b r a n e proteins as judged by their quantitative
Fig. 1. - Immunoelectron micrographs demonstrating Sml5 in the tegument of adult S. mansoni. Gold labelling (arrows) can be observed on the matrix of the syncitial tegumental cytoplasm. In A and B the specimens were fixed with glutaraldehyde while in C and D they were fixed with paraformaldehyde/acrolein. Micrographs A and C show the reactivity of normal rabbit serum. Micrographs B and D show the reactivity of affinity purified antibodies specific for Sml5. The bars represent 1 |im. S, spine; M, muscle bundle.
partitioning into the detergent p h a s e u p o n T X - 1 1 4 fractionation and are d e t e c t e d in purified tegumental m e m b r a n e s o f adult m a l e and female w o r m s ( O m e r - Ali et al, 1 9 9 1 ; Abath et al, 1 9 9 4 ) . H o w e v e r , the pre
dicted a m i n o acid s e q u e n c e o f S m l 5 is highly hydro- philic (in contrast to S m 2 5 ) with n o e v i d e n c e o f h y d r o p h o b i c domains characteristic o f either a trans
m e m b r a n e a n c h o r or the binding o f a GPI anchor.
I n d e e d the s e q u e n c e is uniformly highly acidic w h i c h is consistent with the electrophoretic mobility o f the antigen in t w o dimensional gels (Smithers et al, 1 9 9 0 ) . T h e basis o f its tight m e m b r a n e association remains to b e established, although it is possible that it interacts with m e m b r a n e m o l e c u l e s ( A b a t h & W e r k h a u s e r , 1 9 9 6 ) . As highly purified tegumental surface m e m b r a n e s isolated by adsorption to poly-lysine b e a d s contain S m 2 5 as their dominant c o m p o n e n t (Payares
& Evans, 1 9 8 7 ) , the possibility exists for the interac
tion o f S m l 5 with S m 2 5 . In addition, e x p e r i m e n t s
involving trypsin digestion s h o w e d that during a o n e hour incubation o f live adult w o r m s a substantial por
tion o f the extractable Sm25 was available to the exter
nally applied e n z y m e . From this it w a s c o n c l u d e d that the antigen is e x p o s e d o n the adult parasite surface ( P e a r c e etal, 1 9 9 1 ) .
T h e data derived from ultrastructural localization stu
dies failed to demonstrate the p r e s e n c e o f either S m l 5 or S m 2 5 at the parasite surface o r i n d e e d associated with the outer m e m b r a n e o f the tegument. B o t h anti
g e n s had a similar distribution being specifically asso
ciated with and distributed throughout the tegument, with a tendency for both o f them to b e associated with the discoid b o d i e s . Although the majority o f the par
ticles in both cases are not clearly associated with iden
tifiable m e m b r a n e structures within the tegument, their quantitative isolation in m e m b r a n e pellets and distri
bution in T X - 1 1 4 p h a s e partitioning indicates that the proteins d o not exist in a soluble form. T h e discoid
LOCALISATION OF SM1.5 AND SM25
A BATH F.G.C., HIRST E.M.A., HAGAN P. & SIMPSON A.J.G.
Fig. 2. - Immune-electron micrographs demonstrating Sm25 in the tegument of adult 5. mansoni. Gold labelling (arrows) can be observed on the matrix of the syncitial tegumental cytoplasm, and in association with discoid bodies (some are indicated by lines). The specimens were fixed with glutaraldehyde. Micrograph A show the low reactivity of anti-GST anti-serum. Micrograph B shows the reac
tivity of rabbit antibodies raised against a Sm25-GST fusion protein.
The bars represent 1 Urn. S, spine; M, muscle bundle; DB, discoid bodies.
b o d i e s h a v e b e e n s u g g e s t e d to b e involved in b i o g e nesis o f the surface bilayers and m a i n t e n a n c e o f the integrity o f the t e g u m e n t (McLaren, 1 9 8 0 ; Zhou et al, 1 9 9 0 ) . Interestingly, s c h i s t o s o m e paramyosin, a pro
tective cytoskeletal protein, has a predominant locali
zation in discoid b o d i e s (Matsumoto et al, 1 9 8 8 ) , what c o u l d suggest that the discoid b o d i e s m a y also b e rele
vant for the b i o g e n e s i s o f the tegumental cytoskeleton.
O n the o t h e r hand, o t h e r tegumental proteins are typi
cally present in discoid b o d i e s and b o t h lipid bilayers that c o v e r the tegumental surface (Jianget <^-> 1 9 9 6 ; Zhou et al, 1 9 9 0 ) . T h e d i v e r g e n c e o f the b i o c h e m i c a l
and ultrastructural data c o n c e r n i n g the localization o f S m l 5 and S m 2 5 necessitates further investigation. It is possible that surface a s s o c i a t e d m o l e c u l e s are present but t h e y are not c o n s e r v e d b y the fixation p r o c e s s e s used. It s h o u l d also b e n o t e d that the adult t e g u m e n t is a d y n a m i c structure highly sensitive to its environ
ment a n d r e s p o n d s to n o n ideal c o n d i t i o n s b y the shedding o f m e m b r a n o u s fragments a n d b l e b s . Cer
tainly the results here indicate a c o m p l e x and dynamic interaction b e t w e e n the t e g u m e n t a l m o l e c u l e s a n d d e m a n d further investigation.
ACKNOWLEDGEMENTS
The w o r k w a s partially funded by FACEPE (Fun- d a c a o de amparao a ciencia e tecnologia de Per- n a m b u c o ) a n d CNPq ( C o n s e l h o Nacional de P e s q u i s a ) .
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Recu le 8 décembre 1998 Accepté le 16 avril 1999
