Isolation and immunological analysis of Trypanosoma cruzi glycolipids

Acta Tropica, 48(1991)233-241

233

Elsevier ACTROP 00119

Isolation and immunological analysis of Trypanosoma cruzi glycolipids S. Giovanni De Simone 1'3, R.T. Pinho 2, C.M.M. Vanni 2 and L.C. P o n t e s de C a r v a l h o 2 I Departamento de Bioquimica e Biologia Molecular, 2Departamento de Imunologia, Fundag~o Oswaldo Cruz, Rio de Janeiro, Brazil; 3Departamento de Biologia Celular e Molecular, Universidade Federal Fluminense, Nileroi, Brazil

(Received 22 November 1989; revised version received 6 June 1990; accepted 19 June 1990) A water-soluble glycolipidic fraction from Trypanosoma cruzi was isolated using a mixture of hexane and isopropanol. Analysis by SDS-polyacrylamide gel electrophoresis, after staining by silver and Sudan Black B, showed that the fraction contained one band with a relatively high mobility. Its reactivity and specificity with human chagasic sera and T. cruzi infected mouse sera or with sera from patients with several other pathologies was determined by an immunoradiometric assay. The glycolipid-based radioimmunoassay for the detection of T. cruzi antigens provided a sensitive measure of its activity. However, cross-reactivity with several sera from patients with visceral and cutaneous leishmaniasis was detected. Key words: Trypanosoma cruzi; Anti-glycolipid antibodies; Glycolipids; Hexane-isopropanol

Introduction It is k n o w n that glycoproteins are the m a j o r source o f i m m u n o g e n i c c a r b o h y d r a t e determinants. However, these determinants have also been f o u n d in glycolipids o f several parasites ( H a n d m a n et al., 1984; Ferguson et al., 1985; Slutzky et al., 1985; Weiss et al., 1986) m a k i n g glycolipids a n o t h e r i m p o r t a n t source o f antigens. Besides this, surface lipids are t h o u g h t to play a significant structural role and have p r o b a b l y i m p o r t a n t physiological functions which m a y be linked to proteins (McConville et al., 1987; M e n o n et al., 1988). While their functional significance remains to be elucidated, recent studies have shown i m m u n o g e n i c glycolipids in T. c r u z i ( K a n e d a et al., 1987) and have suggested an association with the i m m u n o p a t h o l o g y o f C h a g a s ' disease (Petry et al., 1987). In this paper, we describe the extraction o f an immunologically active c a r b o h y d r a t e lipid fraction, able to bind to c o m p o n e n t s o f i m m u n e sera from chagasic patients or T. c r u z i infected mice. In addition, we assayed the i m m u n o logical reactivity o f this fraction against both a panel o f sera f r o m patients with different diseases and m o n o c l o n a l antibodies against c a r b o h y d r a t e epitopes o f T. cruzi.

Correspondence address: S. Giovanni De Simone, Departamento de Bioquimica e Biologia Molecular,

Funda~fio Oswaldo Cruz, Av. Brazil 4365, CEP 21040, Rio de Janeiro, RJ, Brazil. 0001-706X/91/$03.50 O 1991 Elsevier Science Publishers B.V. (Biomedical Division)

234 Material and Methods

Human and murine sera Human sera included 10 samples from healthy donors, 53 from chagasic patients (diagnosed clinically and with variable titers of anti-T, cruzi antibodies) and 83 samples from individuals with other infections; namely, cutaneous leishmaniasis (15), visceral leishmaniasis (17), malaria (4), leprosy (13), paracoccidioidomycosis (13), and tuberculosis (21). Sera of Balb/c mice chronically infected with either F strain or Colombian strain were obtained 80-90 days after a boost with 104 blood trypomastigotes. All sera were kept at - 70°C until use. Strongly positive sera for indirect immunoflurescence (1:300 dilution) were selected. Monoclonal antibodies (mAb) The preparation and preliminary characterization of murine mAb against bugderived Colombian strain of T. cruzi were as described (Giovanni De Simone et al., 1987). Preparation of glycolipids T. cruzi Colombian strain epimastigotes (0.2-1 x 1011) were harvested from LIT (Liver infusion tryptose, Difco Laboratories Detroit, MI, U.S.A.) cultures, washed 3 times with PBS and extracted twice (9 ml, 3 ml) with hexane: isopropanol (3:2, HI) according to Radin (1981) with minor modifications. The solubilized material was pooled and concentrated to 1/3 of the volume under a stream of N2. The white precipitate was pelleted by centrifugation, dried under N2, washed twice with chloroform:methanol (2:1) and after a last drying step, dissolved in PBS. From the supernatant the remainder of the HI was evaporated and the lipids were dissolved in chloroform:methanol (2:1) and stored under N z at - 70°C until use. For a radiometric assay, the chloroform:methanol of the lipidic fraction was evaporated and the material dissolved in methanol. Electrophoretic analysis and detection of glycolipids The Laemmli (1970) discontinuous sodium dodecyl sulphate polyacrylamide gel electrophoresis system was used, as modified by Ming and Frash (1982). The glycolipids were also localized, in the gels, with Sudan Black B after or during electrophoresis. The presence of glycolipids in the water-soluble precipitate was detected colorimetrically by the carbocyanine dye 'Stains All' (Sigma Chemical Co., St. Louis, MO, U.S.A.; Slutzky et al., 1985) using an Uvicon 860 spectrophotometer (Kontron Instruments, U.S.A.). Lipopolysaccharide from E. coli (Difco Laboratories, Detroit, MI, U.S.A.) was included in both experiments for comparison. Iodination procedures Protein A from Staphylococcus aureus (Pharmacia Fine Chemicals, Uppsala, Sweden) and afffinity-purified sheep anti-mouse immunoglobulin-F(ab')2 (125I-SAMI)

235 (Amersham Corporation, Arlington Heights, IL, U.S.A.) were iodinated with 125I (Na12SI, Amersham, U.K.) to give specific activities of 8-12 Ci/g with the use of Iodogen (Pierce Chemical Co., U.S.A.) as oxidizing agent (Fraker and Speck, 1978). Anti-glycolipid immunoradiometric assay

The binding of antibodies to glycolipids was measured by solid-phase binding assay (Young et al., 1979) with minor modifications as follows: 20 ~tl of methanol or PBS containing the lipids and glycolipids extracted from T. cruzi epimastigotes (20-30 ~tg ml-1) were added to each well of PVC microtiter plates (Becton Dickinson Co., Oxnard, CA, U.S.A.) and the antigens bound by passive coating for 2-3 h at room temperature. The solution was removed, and additional nonspecific binding sites were blocked by incubation with PBS containing 3% bovine serum albumin and 0.05% NaN 3 (PBS-BSA; 150 p.1 per well) for 1 h at room temperature. After 3 washes with PBS, 50 lal of antibodies (mouse or human sera, or mAb) diluted 1:100 in PBSBSA were added to each well and again incubated for 1 h at 37°C. After 3 washes with cold PBS-BSA, the bound antibodies were detected by the addition of 1251protein A or lzsI-SAMI (60 000 cpm). After 1 h incubation at room temperature, the wells were washed with PBS and the amount of bound radioactivity counted in a gamma counter. Dot assay

Anti-T. cruzi IgG antibodies were affinity purified from sera of chronically infected mice with protein A-Sepharose CI-4B (Pharmacia Fine Chemicals, Uppsala, Sweden). IgM antibodies were purified by chromatography on protamine-Sepharose (Giovanni De Simone et al., 1987) followed by protein A-Sepharose. Glycolipids extracted as described above and corresponding to 106 parasites were spotted on nitrocellulose strips pre-soaked with PBS. After washing in PBS, the remaining adsorption-active sites were blocked with nonfat dry milk (3%) in PBS. The strips were washed 3 times, incubated (1 h at 37°C) with the antibodies and afterwards with 125I-SAMI. After washing, the strips were air dried, and exposed at - 7 0 ° C (16 h) using Kodak X-omat AR films (Eastman-Kodak Co., Rochester, NY, U.S.A.) and intensifying screens.

Results

Partial character&ation of epimastigote glycolipids

The material extracted and concentrated from T. cruzi epimastigotes was a white precipitate which after two washes with chloroform: methanol (2:1) was soluble in PBS. Aliquots from this material were analysed by SDS-PAGE and stained by Sudan Black. A broad band with an Rf value of 0.95 was seen (Fig. 1A). This band was visualized in the gels either by mixing the dye with the glycolipidic solution before the run (data not shown), or by staining the glycolipids after the electrophoresis (Fig. 1A). When the gels were stained with Coomassie blue, this band was not detected, suggesting that this material is not protein (Fig. 1B). In addition, when the

236

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Fig. 1. Electrophoretic profiles of glycolipid fraction of T. cruzi by SDS-PAGE (7-15%) after Sudan Black (A), Coomassie blue (B) and silver staining (C). Lane D represents the LPS of E. coli silver stained. About 50 lag were loaded on each lane.

gels were silver stained, one broad band with a similar Rf value was visualized (Fig. 1C). In contrast to this, several bands were seen with the same concentration in the LPS-bacterial preparations (Fig. 1D). In order to investigate whether the HI extracted material shows characteristics similar to the LPS (Ketteridge, 1978; Goldberg et al., 1983), the material was mixed with 'Stains all' and absorbance curves were taken at 20 nm intervals from 400 to 700 nm. 'Stains all' absorbs maximally at 520 nm, but the dye-glycolipid complexes showed maximal absorption at 460 nm. This effect was similar to that of E. coli LPS (470 nm), LPS-like material isolated from T. cruzi (Ketteridge, 1978) and Leishmania (Slutzky et al., 1985) indicating that the complexes show similar characteristics. LPS-like fraction antigenicity To investigate whether human chagasic sera or sera of T. cruzi-infected mice contain antibodies against both the lipidic and water soluble fractions, several sera were tested by radioimmunoassay. Fig. 2 shows the results of the RIA when the lipidic fraction was used as antigen. A high non-specific binding was observed as seen by the reactivity of the lipidic fraction with normal human sera and sera from tuberculosis patients. However, using the water-soluble fraction as antigen, the non-specific binding was significantly reduced (Fig. 2). To evaluate the specificity of the RIA, sera from patients known to have Chagas' disease and sera from patients with other diseases were used. The results showed that the water-soluble fraction used in the RIA gives a high specificity of binding. However, cross reactivity with several leishmaniasis sera (Kala-azar or cutaneous leishmaniasis) was observed (Fig. 3). On the other hand, sera from mice chronically infected with the Colombian or the F strain (data not shown) of T. cruzi reacted with the glycolipid fraction as much as the respective human sera (Fig. 4). In contrast, no

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reaction was observed using the A5, D11, and D12 monoclonal antibodies, known to recognize carbohydrate epitopes on the cell surface of T. cruzi (Giovanni De Simone et al., 1987).

Specificity of antibody reaction To verify IgM-based response to the HI extracted glycolipids, a dot assay using affinity purified mouse IgM was carried out. The results suggest that IgG antibodies do react with the glycolipids isolated from the Colombian strain but no positive signal was observed wth IgM (Fig. 5), although these antibodies were reactive in the immunofluorescence test (data not shown). When the IgG antibodies against the Colombian strain were tested in the same assay using glycolipids isolated from the F strain, no apparent differences in their reactivity were detected up to the dilution 1:5000 (data not shown). The same positive signal was observed with glycolipids from both T. cruzi strains suggesting the existence of common antigenic determinants.

Discussion

It has been shown that hexane:isopropanol extracted glycolipids from epimastigotes were recognized by a great number of human and murine IgG antibodies. The antigenic determinants recognized are probably conserved in several strains of T. cruzi, because the human sera used were obtained from patients of different regions of Brazil. Since the serum of chagasic patients and infected mice react strongly with

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Fig. 3. Specificity of antigenic epitopes on glycolipids as measured by z25I-protein A radiometric assay. Each microtiter well was coated with glycolipids extracted from 106 epimastogotes of T. cruzi. The open and closed circles, in Leishmaniasis, refer to sera from visceral and cutaneous leishmaniasis patients respectively. Each circle represents one serum. glycolipids as s h o w n by R I A (Fig. 3) it will be o f interest to d e t e r m i n e whether glycolipids a n d / o r g l y c o p r o t e i n s were responsible for eliciting the response to c a r b o h y d r a t e moieties. It seems t h a t glycolipids r a t h e r t h a n n o n - g l y c o l i p i d i c c o n t a m i n a n t s are responsible for the i m m u n o g e n i c i t y since no C o o m a s s i e blue stained b a n d s were seen on the S D S gels. T h e m a t e r i a l stained specifically for c a r b o h y d r a t e moieties. In a d d i t i o n , it was s h o w n t h a t this m a t e r i a l can be retained by p h e n y l - S e p h a r o s e col-

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Fig. 5. Reaction of mouse anti-T, cruzi antibodies with the glycolipid fraction. Nitrocellulose strips containing glycolipid were probed with purified IgM antibodies (A), IgG (B) and sera of chronically infected mice with Colombian (C) and F(D) strain of T. cruzi, followed by incubation with 12SI-sheep antimouse immunoglobulin.

umns, demonstrating the presence of hydrophobic domains (Giovanni De Simone et al., in preparation). It is presently not known which lipid classes carry the antigenic determinants. The glycolipid nature of the antigenic material was further suggested by its interaction with 'Stains All', a carbocyanine dye. In this assay, the glycolipid-dye complex shows an absorption maximum at 460 nm which is different from that observed with proteins, sialic acids and phosphate groups (Campbell et al., 1983). In spite of the difference in the method used by us and others to isolate glycolipids from T. cruzi (Ketteridge, 1978; Goldberg et al., 1983) and Leishmania (Slutzky et al., 1985), samples treated with the dye gave the same spectral shift of the absorption maximum, indicating the presence of glycolipids in the preparation. Recently, the importance of antigenic glycolipids has been determined in several

240

non-parasitic (Brennan, 1981; Hakomori and Kannagi, 1983) and parasitic diseases (Handman et al., 1984; Ferguson et al., 1985; Weiss et al., 1986). In Chagas' disease, glycosphingolipids have been characterized (Barreto-Bergter et al., 1985) and proteins proposed for diagnostic tests have been isolated (Scharfstein et al., 1985). Nevertheless, immunogenic carbohydrate epitopes on easily extracted glycolipids would provide an abundant source of antigens for a sensitive and specific serodiagnostic assay. Unfortunately, the cross-reactivity observed in this work with leishmaniasis sera limits the application for this purpose. In addition, we have observed cross-reactivity between glycolipids isolated from L. major and human Chagasic sera using the same technique (data not shown). As these glycolipids reacted strongly with IgG antibodies, it would be interesting to investigate their involvement in the immunopathology of Chagas' disease, since parasite antigens were observed on the surface of infected cells (Ribeiro dos Santos and Hudson, 1980). However, the identification of processed antigens on the cell surface is difficult since specific reagents for the cell-associated processed antigen do not exist. Therefore, the production o f a mAb or the purification of specific antibodies against these glycolipids would be useful in demonstrating their importance in the pathology of Chagas' disease. While the function of glycolipids in T. cruzi life cycle remains to be elucidated, previous studies with L. major (Elhay et al., 1988) and T. brucei (Doering et al., 1989) suggest that they function as putative anchor precursors. In conclusion, it has been shown that a hexane:isopropanol extraction yields an immunologically active carbohydrate lipid fraction which binds specifically IgG from chagasic patients and T. cruzi-infected mice. The isolation and characterization of glycolipid molecules that stimulate the immune system, may contribute to the understanding of both the immunity process in this disease and the change in the lipid pattern during the transformation of epimastigotes to metacyclic trypomastigotes (Esteves et al., 1989). These aspects and the determination of the chemical composition of this glycolipid fraction are under investigation.

Acknowledgements This work was supported in part by CNPq. We thank Dr. H. Momen and Dr. R. Galler for reviewing this manuscript.

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