C
e l l s u r f a c e a n a l y s i s o f t r y p a n o s o m e s o f t h e s u b g e n u sSCHIZOTRYPAN UM ISOLATED FROM BATS
P IN T O A .S .*, T E IX E IR A L .F.M .*, S O U T O -P A D R Ó N T .** & A N D R A D E A .F .B .***
Summary :
Two stocks (M 5, M 2 9 ) of trypanosomes of the subgenus Schizotrypanum were isolated from the bat Phylloslomus hastatus and analyzed for cell electrophoretic mobility (EPM) and lectin binding surface sites. Epimastigotes from the M 5 and M 2 9 stocks presented a mean EPM of around - 0 .5 7 and - 0 . 5 6 μm, s- 1.V-1.cm, respectively. Differences in the agglutination profiles were detected between epimastigotes or trypomastigotes from the two parasite populations using lectins with specificity for D -GlcNAc, D-GalNAc, D-Gal and D-Man as probe. M ajor variation w as observed between epimastigote forms. Additionally, the D -GlcN Ac binding lectins W G A and BS II strongly interacted with the trypomastigote from both M 5 and M 2 9 stocks; this fact is evidence that these trypanosomes are distinct from Trypanosoma (Schizotrypanum) cruzi.
KEY WORDS : Schizotrypanum from bats, electrophoretic mobility, lectin agglutination.
R ésu m é : Analysedelasurfacecellulaired estrypanosom es APPARTENANT AU SOUS-GENRE SCHIZOTRYPANUM ISOLÉS DE LA CHAUVE- SOURIS PHYLLOSTOMUS HASTATUS
Deux stocks d e trypanosomes appartenant au sous-genre Schizotrypanum, isolés d e la chauve-souris Phyllostomus hastatus, ont été analysés pour leur mobilité électrophorétique (M EP) et leurs profils de liaison d e sites d e superficie lectiniques. tes
épimastigotes des stocks M 5 et M 2 9 ont présenté des moyennes M EP approximatives d e - 0 .5 7 et - 0 .5 6 μm s-1. V-1.cm respectivement. Des différences des profils d'agglutination ont été détectés entre les épimastigotes et les trypomastigotes dans les deux populations d e parasites, en utilisant des lectines de spécificités D-GlcNAc, D-GalAc, D-Gal et D-Man. Les variations les plus fortes ont été observées dans les formes épimastigotes. De plus, les lectines W G A et BS II, d e spécificité D G Ic N A c , ont présenté une forte interaction avec les trypomastigotes des deux stocks. C e résultat montre que ces trypanosomes ne se rattachent pas à l'espèce Trypanosoma (Schizotrypanum) cruzi.
MOTS CLÉS : Schizotrypanum de la chauve-souris, mobilité électrophorétique, interaction avec lectines.
IN T R O D U C T IO N
T
rypanosom es o f the sugenus S ch iz o try p a n u m are m orphologically alm ost indistinguishable from each other and those from bats have co smopolitan distribution (Hoare, 1972). In Latin Ame
rica, T ry p a n o so m a (S ch iz o try p a n u m ) cru zi, the cau
sative agent o f Chagas’ disease, can be found in several mammalian orders, including bats (Pinto and Costa Bento, 1986). Thus, in this region, it is o f considerable importance to public health to determine whether iso
lates from bats are distinct from T. (S.) cru zi.
Cell electrophoretic mobility (EPM) and lectin aggluti
nation patterns have successfully been applied to cha
racterize trypanosom es o f the subgenus S ch iz o try p a n u m , as w ell as to d iscrim in a te T .(S .) c r u z i
* Departamento de Microbiologia, Instituto de Ciencias Biológicas, CP 486, Universidade Federal de Minas Gerais 31270-010, Belo Horizonte, MG, Brasil.
** Instituto de Biofísica Carlos Chagas Filho,
*** Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, 21949-900, Rio de Janeiro, RJ, Brasil.
developmental stages (Pereira et a l , 1980; Shottelius et al., 1983; Souto-Padron et al., 1984; Souto-Padron et al., 1990).
To determine w hether bats isolates are distinct from T. cru zi, w e analyzed the cell surface charge and the surface lectins binding sites o f two stocks o f parasites, o f the subgenus S chizotrypan u m , isolated from the bat P. h asta tu s in Minas Gerais, Brazil.
MATERIALS A N D M ETH O D S
Pa r a s it e s
T
he M5 and M29 stocks o f the subgenus S ch i
z o try p a n u m w ere isolated from P. h a sta tu s bats collected in Pedro Leopoldo and Serrania, Minas Gerais, Brazil, respectively. Isolation was per
formed by hemoculture in Brain-Heart-Infusion (BH I) medium supplem ented with 10 % (v/v) heat-inacti
vated fetal calf serum (FCS) and 2.5 (v/v) o f 10 % rabbit hem oglobin solution, 100 IU penicillin/ml, and 50 g streptomycin/ml. Flagellates w ere maintained by serial passages every 10 days. They w ere cryopre- Parasite, 1996, 3, 143-146
Mémoire 143
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1996032143
PINTO A.S., TEIXEIRA L.F.M., SOUTO-PADRÔN T. & ANDRADE A.F.B.
s e r v e d in liq u id n i t r o g e n a f t e r a d d in g 1 0 % g l y c e r o l t o t h e c u lt u r e s .
Pa r a s i t e m a s s
P a r a s it e c e l l s w e r e o b t a i n e d in 1 8 X 1 8 0 m m s c r e w - c a p p e d t u b e s c o n t a i n i n g 5 .0 m l o f B H I m e d iu m s u p p l e m e n t e d w it h F C S a n d h e m o g l o b i n a s d e s c r i b e d a b o v e b u t w it h o u t a n t ib io t i c s . I n o c u la a lw a y s c o n s i s t e d o f a b o u t 4 % (v / v ) o f a m id - lo g p h a s e g r o w t h c u l t u r e k e p t a t 2 8 °C . E p im a s t ig o t e a n d t r y p o m a s t ig o t e s fo r m s w e r e h a r v e s t e d f r o m 8 - d a y s o r 2 7 - d a y s - o l d c u l t u r e s , r e s p e c t iv e ly .
El e c t r o p h o r e t i c m o b i l i t y a s s a y
E p im a s t ig o t e s w e r e c o l l e c t e d b y c e n t r if u g a t io n ( 1 .5 0 0 g, 1 0 m in , 4 ° C ) , t w i c e w a s h e d w it h 0 .1 M p h o s p h a t e b u f f e r , p H 7 .2 a n d f i x e d f o r 1 h r in 2 .5 % g lu t a r a ld e - h y d e in 0 .1 in p h o s p h a t e b u f f e r . A f te r f i x a t i o n c e l l s w e r e w a s h e d t w i c e in a 0 .8 5 % s o d iu m c h l o r i d e s o l u t i o n w it h a n io n i c s t r e n g t h o f 0 .1 4 5 m o l. d m , p H 7 .2 . T h e e l e c t r o p h o r e t i c m o b ilit y ( E P M ) o f t h e c e l l s w a s d e t e r m i n e d in a Z e is s c y t o p h e r o m e t e r w it h a c u r r e n t o f 4 - 6 m A a n d a f in a l v o l t a g e o f 1 0 0 V . T h e c e l l s u s p e n s i o n w a s p l a c e d i n t o a c h a m b e r a n d t h e n a llo w e d t o e q u i l i b r a t e f o r 1 0 m in u t e s . M e a s u r e m e n t s w e r e m a d e a t 2 5 °C . W h e n t h e c u r r e n t w a s s w i t c h e d o n , t h e t im e n e c e s s a r y f o r o n e c e l l t o t r a v e l a c r o s s t w o v e r t ic a l lin e s , s e p a r a t e d b y a d is t a n c e o f 1 6 m m w a s m e a s u r e d . T h e n t h e p o la r i t y w a s r e v e r s e d a n d t im e w a s m e a s u r e d a g a i n f o r t h e c e l l t r a v e lin g in t h e o p p o s i t e d i r e c t i o n . A b o u t 3 5 c e l l s w e r e m e a s u r e d f o r e a c h s a m p l e a n a ly z e d . C a lib r a t io n o f t h e e q u i p m e n t w a s m a d e b y m e a s u r i n g t h e e l e c t r o p h o r e t i c m o b ilit y o f f r e s h h u m a n e r y t h r o c y t e s . S t a t is t ic a l a n a ly s is w a s p e r f o r m e d u s in g t h e T - t e s t .
Le c t i n a g g l u t i n a t i o n a s s a y
E p i m a s t i g o t e s o r t r y p o m a s t ig o t e s w e r e c o l l e c t e d a n d w a s h e d a s d e s c r i b e d a b o v e . A g g lu t in a t io n a s s a y s w e r e p e r f o r m e d w it h a T a k a s k y m ic r o t r it a t o r p la t e ( C o o k e E n g i n e e r i n g , V A ). E q u a l v o lu m e ( 2 5 m l) o f t h e p a r a s it e s u s p e n s i o n c o n t a i n i n g 1 -5 x 1 0 8 c e lls / m l a n d t h e le c t i n d ilu t io n w e r e m i x e d a n d p l a c e d a t r o o m t e m p e r a t u r e f o r 1 h . T h e a g g lu t in a t io n o f c e l l s w a s a lw a y s s c o r e d v is u a lly w it h a h a n d l e n s a f t e r g e n t ly r e s u s - p e n d i n g t h e s e t t l e d c e l l s a n d b y m i c r o s c o p i c o b s e r v a t io n s . A g g lu t in a t io n i n h ib it io n a s s a y s w e r e p e r f o r m e d w i t h f o u r a g g l u t i n a t i n g u n it s o f l e c t i n a n d 0 .1 M c o n c e n t r a t i o n o f e a c h s p e c i f i c c a r b o h y d r a t e . T h e l e c t in s u s e d a n d t h e ir c o n c e n t r a t i o n s a r e id e n t if i e d in T a b l e I. A ll l e c t i n s w e r e p u r c h a s e d f r o m S ig m a C h e m ic a l C o . ( S t .- L o u is ) .
RESULTS
El e c t r o p h o r e t i c m o b i l i t y
E
p im a s t ig o t e f o r m s o f t h e t w o s t o c k s o f t r y p a - n o s o m e s s t u d i e d u n d e r s t a n d a r d c o n d i t i o n s h a d n e g a t i v e s u r f a c e c h a r g e . P o p u l a t i o n a n a ly s is in d i c a t e d t h a t t h e c e l l s o f b o t h M 5 a n d M 2 9 s t o c k s w e r e h o m o g e n e o u s in t h e i r E P M ( F ig . 1 ). T h e o r i e n t a t i o n o f t h e p a r a s it e s w a s r a n d o m d u r in g t h e m ig r a t io n t o w a r d s t h e p o s it iv e e l e c t r o d e .Fig. 1. — Electrophoretic mobility (EPM) distribution among epi- mastigote forms o f trypanosomes of the subgenus Schizotrypanum isolated from the bat Phyllostomus hastatus in Minas Gerais, Brazil.
A, M5 Stock; B, M29 stock.
144 M ém oire Parasite, 1996, 3, 143-146
NUMBEROF CELLS
C e ll s u rfa c e o f Scwzotrÿ&àkum trypaxosomes^
Le c t in a g g l u t in a t io n
The agglutination patterns o f epimastigote and trypo- m astigote stages o f the tw o stocks are show n in Table I. Results are presented as minimum concentra
tion required to agglutinate the flagellates. The binding reaction is considered to be most specific with cells that are agglutinated at the lowest lectin concentration.
W hen epimastigotes and trypomastigotes w ere agglu
tinated, mixed types o f clumps w ere observed, i.e., body-body, body-flagellum and flagellum-flagellum.
Quantitative and qualitative differences in the aggluti
nation patterns w ere observed. Major variations w ere observed betw een epimastigotes, w here WGA, SBA, HP, dB, NP and RCA I lectins clearly discriminated the two parasite populations. On the other hand, WGA, WFH, SBA and Con A strongly interacted with epi
m astigote and trypom astigote stages from the two stocks o f parasites. Evidence for the interaction of WGA with sialic residues on carbohydrate determinants was observed. The digestion o f epimastigotes o f the M5 or M29 stocks with C lostridiu m p e r frin g e n s siali- dase completely abolished the ability o f WGA to agglu
tinate the cells. Such treatment exposed receptor sites for the interaction with PNA lectin (data not shown).
DISC U SSIO N
E
pimastigotes o f the two parasite populations analyzed presented very similar values o f surface negative charge. These values are closely related to those observed for T. (S.) c r u z i or T rypa
n o s o m a (S ch iz o try p a n u m ) vespertilion is and distinct from T ry p an osom a (S ch iz o try p a n u m ) d io n is ii or Try
p a n o s o m a (S h iz o try p a n u m ) m yoti (Souto-Padró n et a l., 1990).
On the contrary, the lectins used clearly discriminated the two stocks o f trypanosom es from each other.
Indeed, these two populations are also different in their E coR.1 k-DNA digestion products and glucose phos
phate isom erase (GPI) isoenzymatic patterns (Teixeira et al., 1993).
Probably the most interesting finding in our study was the agglutination o f trypomastigotes from both para
site populations with the D-GlcNAc-binding lectins, WGA and BSII. These lectins did not react with T. (S.) c r u z i trypomastigotes obtained in similar conditions (Pereira et al., 1980). Additionally, the isoenzymatic pat
terns o f these two stocks studied are different from those o f T. (S.) c r u z i Z l, Z2, Z3 and ZB T. (S.) cru z i reference zymodemes (Teixeira et al., 1993).
Carbohydrate specificity and lectins
Minimum concentration required to agglutinate (pg/ml)
Epimastigotes T rypomastigotes
M5 stock M29 stock M5 stock M29 stock
D-GlcNAc
WGA (Weat germ agglutinin) 7.8 62.5 31.2 31.2
BS II (B an d eiraea sim plicifolia II) ND 31.2 125.9 250.0
D-GalNAc
WFH (W istaria flo ribu n d a ) 0.12 0.24 0.12 0.12
SBA (Soybean agglutinin) 7.8 31.2 15.6 31.2
HP (Helix p om atia) 250.0 500.0 250.0 500.0
DBL (Dolichos biflorus) 500.0 1,000.0 1,000.0 1,000.0
D-Gal
PNA (Peanut agglutinin) 500.0 250.0 1.000.0 1,000.0
RCAI (Ricinus com m unis) 250.0 7.8 31.2 15.6
D-Man a n d sim ilar
Con A (C an avalia ensiform is) 0.49 0.98 0.98 0.49
LCL (Lens culinaris) 250.0 250.0 500.0 500.0
ND = not determined.
Table I. — Activity o f various lectins for parasites o f the subgenus Schizotrypanum isolated from the bat Phyllostomus hastatus in Minas Gerais, Brazil.
Parasite, 1996, 3, 143-146
Mémoire 145
« É P O A S. TEL\I-.1 K \ 1. F.M.. SOLTO-PADRÔN T & AN DRADI' \.T:.] î
Chagas’disease remains a serious public health problem in Latin America w here bats w ere frequently found infected by trypanosomes o f the subgenus S chizotry- p a n u m (Pinto and Costa Bento, 1986). More extensive studies are required to characterize surface components o f flagellates o f this subgenus. In particular, isolates from bats collected over a wider geographic area need to be exam ined. Perhaps, improved agglutination tests with trypomastigotes using the D-GlcNAc-b inding lec
tins, mainly WGA, may be useful tool to distinguish T. (S.) c r u z i from others S cb iz o try p a n u m isolate from bats in endem ic areas.
analysis o f trypanosom es o f the subgenus S ch izo - try p a n n u m from the bat. P a r a s ito lo g y R esearch , 1983, 79, 497-500.
Reçu le 27 octobre 1995 Accepté le 10 janvier 1996
ACKNOWLEDGEMENTS
T
his work was supported by grants from CNPq, FAPEMIG and Pro-Reitoria de Pesquisa da Uni- versidade Federal de Minas Gerais, Brazil. We thank Dr. Michel Tibayrenc for the “resu m e” revision.REFERENCES
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146 Mémoire Parasite, 1996, 3, 143-146
