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The surface coat of bloodstream forms of trypanosomamusculi from mice
THE SURFACE COAT OF BLOODSTREAM FORMS OF TRYPlQNOSOK4 MUSCULI FROM MICE and M. J. HOWELL*
N. A. SAMARAWICKREMA
Department of Zoology, Australian National University, P.O. Box 4, Canberra, A.C.T. 2601, Australia (Received 19 ~e&ember 1989; accepted27 June 1990) Abstract-SAMARnwrcKREMAN. A. and HOWELL M. I. 1990. The surface coat of bloodstream forms of Trypanosoma musculi from mice. International Journalfor Parasitology 20: 1055-1062. Iodination and immunoprecipitation techniques together with indirect fluorescent antibody tests identified two polypeptides (SP) of molecular weights 88,OOt%92,000and 66,000-70,000 in the surface coat of bloodstream forms of the mouse trypanosome, Trypanosoma musculi. As parasites multiply and enter the early plateau phase of infection the 88,000-92,000 SP is present while the 66,000-70,000 SP is only detectable after the mid-plateau phase. Western blotting of parasite extracts showed that the 88,00&92,000 SP was present throughout the course of infection, but it appears to become masked by the 66,000-70,000 SP or possibly immunoglobulin from about I6 days after infection. Based on results when Western blots of parasite extracts were probed with antibodies affinity purified against the 88,000-92,000 SP, the two SP appear to be immunolo~cally related and the smaller may be a cleavage product of the larger. This would explain why affinity purified antibodies to each SP bound to trypanosomes collected 8 days after infection, when only the 88,~92,0~ is detectable in parasite extracts. However, the failure of antibodies affinity purified against the 66,00&70,000 SP to bind to the 88,000-92,000 SP in Western blots suggests that the smaller SP has some epitopes that are immunologically distinct from those of the larger SP. INDEX KEY WORDS: Trypanosoma musculi; mouse; trypanosome; surface coat; surface antigens; iodination, immunoprecipitation; Western blots; affinity purified antibodies; fluorescent antibody tests.
INTRODUCTION THE major surface glycoproteins
of African (Cross, 1975; Turner, 1982, 1985a, b) and American (Snary, 1985a,b) trypanosomes have been identified and purified and their properties extensively analysed. In the case of African forms, the variable nature of the glycoproteins throughout infection is well established and the probable importance of this phenomenon in avoiding the consequences of host immune responses is clearly recognized. Thus, studies of the surface coats of these parasites can be particularly revealing about aspects of the host-parasite relationship. The presence of surface molecules on the rodent trypanosome Tr~~~nos~~a lewisi has been investigated by a number of workers (Battaglia, Zani, Del Bue, Ponzi & Birago, 1983; Sturtevant & Balber, 1983; Giannini & D’Alesandro, 1984). As many as 13 molecular species have been identified, some of which are sensitive to proteolytic cleavage. While it is difficult to compare these studies closely with each other because of differences in the methods used, it seems clear that the surface molecules of T. lewisishow a
* To whom all correspondence should be addressed.
greater degree of complexity than those of other trypanosom~s. Moreover, there is evidence that antigenie changes occur in the surface proteins during the course of infection (D’AIesandro, 1976). The present study was undertaken on a related stercorarian-T. musculiwhich infects mice. A distinct surface coat is present on parasites from the blood of mice but is lacking or more sparse in vector and culture forms (Vickerman, 1969; Roger, Garzon, Stryowski & Viens, 1987). Dwyer C D’Alesandro (1976a,b) showed that treatment of bloodstream forms with trypsin removed the surface coat indicating that it consisted of, at least in part, protein. Moreover, staining with polycationic dyes revealed a moderate density of carbohydrate moieties in the surface coat. Thus, the surface of T. muscufi, like other trypanosomes, appears to be covered by glycoprotein. In the present investigation, the surface coat of T. musculi bloodstream forms was characterized throughout the course of infection in mice. Surface coat components were labelled by iodination and their antigenicity demonstrated by immunoprecipitation and Western blotting. In addition, confirmation of the surface location of iodinated molecules was sought by exposure of living parasites to trypsin, and the use of immuno~obulins eluted from intact parasites for fluorescent antibody tests.
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N.A. MATERIALS
SAMARAWICKREMA~~~
AND METHODS
Animals. Male CBA:Ca/H inbred mice, between 6 and 10 weeks old, were used. Parasites. A Trypanosoma musculi stabilate was originally obtained from the London School of Hygiene and Tropical Medicine, University of London (LUMP 1189 strain) and maintained by syringe passage in mice. The pattern of infection-comprising growth, plateau and elimination phases over a period of about 3 weeks-is described by Viens, Targett, Leuchars & Davies (1974) and has been verified many times in our laboratory. Parasites were harvested by centrifuging whole blood from infected mice on a gradient of Ficoll-Paque (Pharmacia) and passing the parasite-white cell layer through a cellulose CF-I1 (Whatman) column to remove white cells (Samarawickrema & Howell, 1988). Collection of serum.Mice under deep ether anaesthesia were bled out by severing the axillary vessels. The blood was allowed to clot. centrifuged at 3000 rr for 15 min at 4°C and the serum collected and stored at -- 20°C until required. Serum collected from mice 25 days after infection is referred to as immune serum. Normal serum was collected from uninfected mice at the same age. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) of parasite polypeptides. Samples for electrophoresis were prepared by suspending approximately 10” parasites in 100 ~1 of PBS (pH 7.6), 100 ~1 of 2 x SDSPAGE sample buffer [6% SDS, 20% (w/w) glycerol, 0.008% bromophenol blue, 0.125 M-Tris-HCI (pH 6.8)] containing 10% Pmercaptoethanol and boiled for 2 min. Electrophoresis was carried out on 10% polyacrylamide gels (14 x 14 x 0.15 cm) under reducing conditions at a constant current of 40 mA for 34 h (Laemmli, 1970). Molecular weight protein standards were run on each gel. Gels were prepared for autoradiography using standard procedures (Irving & Howell, 1981). Isolation of surface polypeptides. The technique developed by Cross (1975) to isolate surface glycoproteins of African trypanosomes was applied to T. musculi. Briefly, 2 x lo9 parasites were subjected to sonication, centrifugation and dialysis and the products analysed by SDS-PAGE. Iodination of trypanosome surface components. Parasites were collected 12, 15 and 18 days after infection and iodinated using the Iodogen method (Fraker & Speck, 1978). Iodination of samples of 10’ parasites were carried out in glass tubes previously coated with Iodogen (1,3,4,6tetrachloro-3 alpha, 6 alpha-diphenylglycouril) (Sigma) in 50 ~1 of chloroform and reacted with 8-10 ~1 of carrier-free YNaI (Amersham) (about 18.5 MBq) for 5 min on ice and 5 min at room temperature. Parasites treated with trypsin accordine to the method of Dwver & D’Alesandro (1976a) were al&-processed in a similar manner. Iodinated parasites were washed three times in PBS (pH 7.6) and resuspended in 50 ~1 PBS. Half the sample was stored at - 20°C to be used for immunoprecipitation experiments. The remainder of the sample was spun, resuspended in 25 pl of 0.5% Non-Idet P40 (Bio-Rad) in PBS, left for 5 min at room temperature, spun for 5 min and the supernatant discarded. The pellet was dissolved in 25 ~1 of 2 x SDS-PAGE sample buffer containing 2.5 ~1 of pmercaptoethanol, then boiled for 2 min and subjected to SDS-PAGE and autoradiography as described above. Immunoprecipitation of iodinated surface components. The remaining half of the iodinated parasite sample from above was made up to 50 ~1 with PBS, 25 ml ofwhich was allowed to react with antibody by adding 25 ~1 of immune serum and incubating overnight at 4’C. The remainder of the sample (25
M.J.HOWELL
~1) was similarly incubated with normal serum. Immune complexes were absorbed onto 250 ~1 of heat-killed, formaldehyde-fixed Staphylococcus aureus Cowan I strain (Kessler, 1975) at room temperature for 30 min. Each sample was washed in 500 ~1 of 0.05% Non-Idet P-40 in NET buffer (0.1 M-NaCl. 0.005 M-EDTA. 0.01 M-Tris-HCl. DH 7.5) and ieft for 15 min at room temperature. The pellet fas further washed in NET buffer, then dissolved in 50 ~1 of 2 x SDSPAGE sample buffer containing 10% pmercaptoethanol and boiled for 5 min. Undissolved material was pelleted by centrifugation for 5 min, and samples ofthe supernatant were analysed by SDS-PAGE and autoradiography. Western blotting (immunoblotting) of parasite samples. Samples subjected to SDS-PAGE as above were transferred onto nitrocellulose membranes (Schleicher and Schull) in a vertical electrophoresis tank (Bio-Rad) using Western blot transfer buffer (20 mh+Tris, 150 mM-glycine with 20% methanol) to renature the proteins. Elcctrophoresis was carried out at 30 V/l50 mA overnight. Filters were blocked with 5% (w/v) non-fat skim milk powder (Diploma) in Trisbuffered saline (TBS) for 30 min then treated with antibody diluted in TBSiskim milk for 1-2 h and washed in 0.05% Tween-20 (Bio-Rad)/TBS with several changes for 30 min. Filters were treated with goat anti-mouse IgG (H+ L) coniueated with horseradish peroxidase (HRP, Bio-Rad) at a dil&n of 1:4000 in TBS/skjm milk for‘1 h. Following’this, the filters were washed in Tween/TBS and treated with HRPcolour development reagent (60 mg of 4-chloro-1-napthol in 20 ml methanol) and 0.1% 100 vol. H,O, for 2G30 min. The filters were then washed in deionized water and air dried. Biotinylated SDS-PAGE molecular weight standards (BioRad) were electrophoresed at the same time as the samples, transferred onto nitrocellulose and blocked with 3% gelatine in TBS, washed with 0.05% Tween-20 in PBS, treated with 0.3% Avidin-HRP (Bio-Rad) in 1% gelatine in TBS for 1 h and washed again with Tween/TBS before reacting with HRP-colo~r development reagent. Afinity purification of antibodies to antigenic polypeptides. Parasite samples were electrophoresed in duplicate, transferred onto nitrocellulose and one lane was probed with immune serum (I:5 dilution) as above in order to determine the location of antigenic polypeptides in the extracts. Strips corresponding to each of the antigenic bands on the remaining lane were cut out, then treated individually. Each was blocked with 5% skim milk in TNT (10 mr+Tris, 150 mM-NaC1,0.05% Tween-20, pH 8.0) for 1h, washed in TNT alone for 30 min and incubated for 3 h with immune serum diluted 1:5 in TNT. The filters were washed in TNT for 1 h with several changestreated with 100 mM-boric acid/500 mMNaCl (pH 8.0) for 30 min and PBS for 30 min. Any antibody bound to each filter was eluted by incubation in 200 ml of 100 mmglycine/l50 mM-NaCl (pH 2.6) for 3 min after which the pH was immediately brought to 8.0 by adding 20 ~1 of 2 MTris (pH 8.0) (Beall & Mitchell, 1986). Eluates were stored at - 20°C until required. Elution of surface immunoglobulin Ig from parasites.About lo* live parasites from day 16 to day 18 after infection in I ml PBS were incubated in 9 ml of 25 mM-glycine_HCI in 2 MNaCl (pH 2.5) for 15 min on ice to elute surface Ig. Following ccntrifugation at 3000 g for 15 min at 4 “C, the pH of the supernatant was restored to pH 8.0 by adding an equal volume of 100 mr+borate buffer in PBS (pH 8.3). The eluate was concentrated to 1 ml by dialysis against 30% w/v polyethylene glycol in the borate buffer as above. After dialysis against several changes of PBS, the concentrated sample was stored at -20°C until required for Western
Surface coat of T. musculi blotting. Indirect fluorescent antibody tests. Blood smears were prepared from mice 8 days after infection, a time when surface Ig cannot be detected on the parasites (Samarawickrema & Howell, unpublished). Smears were fixed with acetone for 5 min and allowed to air dry. The slides were treated with affinity purified antibody samples for l-2 h at room temperature and washed thoroughly in PBS for 15 min. The slides were then reacted with FITC (fluorescein isothiocyanate) labelled rabbit anti-mouse IgG (Silenus) at a dilution of 1:20 in PBS for 1 h, and examined by fluorescence microscopy as wet mounts with a Leitz U.V. photomicroscope using a BG-12 excitation filter and K530 and AUS barrier filters. RESULTS
Analysis of parasite extracts SDS-PAGE of unlabelled extracts of parasites prepared 16 days after infection by the method of Cross (1975) indicated the presence of a single broad band of about 66,OO(r70,000 mol. wt (Fig. 1). The SDS-PAGE profile of total parasite extracts is also shown (Fig. 2). Most noticeable is a prominent band of about 66,000 mol. wt which is absent from the trypsintreated parasite extract. Iodination of surface molecules Autoradiographic patterns resulting from the electrophoresis of ‘Z51-labelled parasite extracts are shown in Fig. 3. A single polypeptide of between 88,000 and 92,000 mol. wt was identified in parasites obtained 10 and 12 days after infection (Fig. 3a). It appeared as a broad band, and exhibited slightly different mobihties between gels; prior treatment of parasites with trypsin reduced the intensity of its labelling (Fig. 3b). A number of labelled polypeptides were detected in parasites iodinated 15 days after infection (Fig. 3~); a prominent species of 66,00& 70,000, a less prominent yet distinct polypeptide of 88,000-92,000 and a number of minor ones (80,000, 60,000, 50,000 and 25,000). In parasites iodinated 18 days after infection (Fig. 3d), a prominent band of 66,000-70,000 was detected and the 88,000-92,000
1057
band was not discernible. Polypeptides of about 80,000, 60,000 and 50,000 were also labelled but less prominently than the 66,00@70,000 species. In the day 18 sample iodination of polypeptides was abolished by prior trypsin treatment (Fig. 3e). Immunoprecipitation The results of immunoprecipitating ‘ZSI-labelled polypeptides of T. musculi by immune and normal sera are illustrated in Fig. 4a-d. Immune serum precipitated polypeptides of 88,00&92,000 and 45,00& 50,000 from parasites obtained 12 days after infection (Fig. 4a). Normal serum did not precipitate polypeptides from these samples (Fig. 4b). From samples prepared 18 days after infection, immune serum precipitated polypeptides of 66,00@-70,000 and 45,00&50,000 (Fig. 4c). Normal serum precipitated a 45,000-50,000 polypeptide (Fig. 4d). Western blotting Unlabelled parasite extracts probed with immune serum highlighted several antigenic polypeptides (Fig. 5a-d). Many high molecular weight bands were detected, particularly in extracts prepared in the later stages of infection. Most noticeable was the presence of a polypeptide of about 88,00&92,000 at all stages of infection. A 66,000-70,000 polypeptide only appeared in parasite samples prepared 16 and 18 days after infection. Present from day 12 onwards were polypeptides of about 45,000 and 50,000. The control filter probed with normal serum recognized the 50,000 polypeptide, but very weakly, in samples prepared 16 and 18 days after infection (Fig. 5e, f). Western blots of unlabelled parasite extracts probed with a#inity purzfied antibody A Western blot probed with antibody that was affinity purified against the 88,000-92,000 polypeptide in parasite extracts prepared 18 days after infection (Fig. 6aad) recognized a 88,000-92,000 polypeptide consistently through infection. The same antibody also recognized a prominent 66,OO(r70,000 poly-
FIG. 1. SDS-PAGE gel stained with Coomassie blue of surface molecules of T. musculi (from 16 days after infection), purified as described by Cross (1975). Molecular weight markers: 92,000 phosphorylase B; 66,000 bovine serum albumin; 45,000 ovalbumin; 31,000 carbonic anhydrase. FIG. 2. Coomassie
blue stained SDS-PAGE gel comparing protein profiles of trypsin treated parasites parasites (lane b). Molecular weight markers as for Fig. 1.
(lane a) with untreated
FIG. 3. Autoradiograph of SDS-PAGE gel following electrophoresis of ‘z51-labelled parasite extracts obtained various days after infection. Lane (a): 12 days, untreated parasites; lane (b): 12 days, trypsin treated parasites; lane (c): 15 days, untreated parasites; lane (d): 18 days, untreated parasites; lane (e): 18 days, trypsin treated parasites. Molecular weight markers as for Fig. 1. FIG. 4. Autoradiograph of SDS-PAGE gel comparing immunoprecipitates of ‘251-labelled parasite extracts. Lane (a): immunoprecipitate obtained with immune serum and parasite extract prepared 12 days after infection; lane(b): immunoprecipitate obtained with normal serum and parasite extract prepared 12 days after infection; lane (c): immunoprecipitate obtained with immune serum and parasite extract prepared 18 days after infection; lane(d): immunoprccipitate obtained with normal serum and parasite extract prepared 18 days after infection. Molecular weight markers as for Fig. 1.
N. A. SAMARAWICKREMAand M. J. HOWELL
1058
1 a_
-“‘(’ .^‘
6
4%
&-
b
Surface coat of T. musculi
-_I
1*-__
-92 -66
-45
e
FIGS. 5-9.
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N.A.
SAMARAWICKREMA
and M. J. HOWELL
peptide in samples prepared 16 and 18 days after infection; this pol~pt~dew~ barely det~tablein day 8 and 12 samples. A poly~ptide of about 50,000 was evident in day 12,16 and I8 samples but not in the day 8 sample. A few polypeptides were also present between the more prominent species of 66,000-70,000 and 88,000-92,000. A Western blot probed with antibody affinity purified against the 66,000-70,000 polypeptide recognized a 66,000-70,000 polypeptide in parasite samples, especially from days 16 and 18 after infection (Fig. 6e-h). Some minor polypeptides of higher molecular weight (about 80,000) were faintly detectable. A 50,000 polypeptide was detected in all samples from day 12 of infection onwards. A Western blot probed with Ig eluted from the surface of parasites 16 days after infection highlighted a polypeptide of 66,~-70,~ in parasite extracts (Fig. 9).
polypeptide with a molecular weight of about 66,00070,~. That this polypeptide is exposed on the surface was deduced from the fact that it was absent from the polypeptide profile derived from live parasites that were treated with trypsin. Moreover, antibody eluted from the surface of parasites 16 days after infection reacted with a 66,00@-70,000 SP in Western blots. The molecular mass of this SP of T. musculi is close to that of African trypanosomes (Cross, 1975). Iodination of cell surface components of T. musculi demonstrated the presence of at least two major polypeptides of 88,000-92,000 and 66,00&70,000 mol. wt during the course of infection. Parasites obtained from the growth and early plateau phases (days 10, 12) possess a 88,0~-92,000 SP. By the midplateau phase of infection (day 15) the second ~lypeptide (66,0~70,~0) is also present on the surface of parasites, and as the infection progresses to late-plateau and elimination phases only that polypeptide appears to persist. Thus, the nature of the coat appears to be less complex than in related Fluorescent antibody tests Antibodies affinity purified against the 66,OOO- stercorarians T. cruzi and T. lewisi where numerous surface glycoproteins are present (Snary, 1985a, b; 70,000 and 88,000-92,000 mol. wt parasite extracts Battaglia et al., 1983; Giannini & D’Alesandro, 1984; prepared 18 days after infection each bound to the surface of intact parasites in blood films prepared 8 Sturtevant & Balber, 1983). Surface iodination used in conjunction with imdays after infection (Figs. 7 and 8). The intensity of munoprecipitation confirmed that the labelled fluorescence in each case was similar. Control parasites polypeptides of T. musculi were of parasite origin and treated with fluorescein-labelled anti-mouse fg alone present on the surface. Immune serum precipitated the did not fluoresce. 88,00~92,~ species early in the infection (day 12). However, from the mid-plateau phase onwards, DISCUSSION immune serum recognized the 66,000-70,000 species The present study has provided information on the only. The few minor polypeptides that became iodinnature and dynamics of surface coat components of ated (for example 80,000, 60,000) but which were not Trypanosoma musculi throughout the course of precipitated by immune serum may be host material that becomes lodged in the parasite’s surface coat. infection in mice. Application of the method of Cross The present results are open to two interpretations. (1975), for isolating surface glycoproteins of African Firstly the parasite may alter the composition of its trypanosomes, to T. musculi collected 16 days after infection demonstrated the presence of a single surface coat by ceasing synthesis of the 88,000-92,000
FE. 5. Western blot showing parasite extracts probed with mouse serum (I 5 dilution) followed by goat anti-mouse IgG-HRP. Lanes (a), (b), (c) and (d) consisted of parasite extracts obtained on days 8, 12, 16 and I8 after infection, respectively and probed with immune serum. Lanes e and f replicated lanes c and d but were probed with normal serum (the nitroceIlulose was damaged in processing). Molecular weight markers as for Fig. 1. FIG. 6. Western blot showing parasite extracts probed with antibodies affinity purified against surface polypeptides from parasites obtained 18 days after infection, followed by goat anti-mouse IgG-HRP. Lanes (a), (b), (c) and (d) consisted of parasite extracts obtained on days 8,12,16 and 20 after infection, respectively and probed with affinity purified antibody to the 88,000-92,000 polypeptide; lanes (eHh) replicate lanes (a)-(d) but were probed with affinity purified antibody to the 66,OO(t 70,000 polypeptide. Molecular weight markers as for Fig. 1. FIG.7. Fluorescence micrographs ofparasites 88,000-92,000 surface polypeptide
obtained followed
FIG. 8. Fluorescence micrograph of parasites 66,~~70,~0 surface polypeptide
obtained 8 days after infection treated with antibody affinity purified against the followed by FITC-labelled rabbit anti-mouse IgG. Scale bar, 10 pm.
FIG. 9. Western parasite surface
8 days after infection treated with antibody affinity purified against the by FITC-labelled rabbit anti-mouse IgG. Scale bar, 10 pm.
blot of extracts from parasites obtained I6 days after infection and probed with antibody eluted from the (16 days after infection) followed by goat anti-mouse IgG-HRP. (Molecular weight marker bovine serum albumin, 66,000.)
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Surface coat of T. musculi
SP and commencing synthesis of the 66,~~70,000 SP from about the mid-plateau phase Qf infection. This may account for the iodination of both SP in day 15 parasites. On the other hand, it is possible that the 88,000-92,000 SP continues to be synthesized and expressed on the parasite’s surface throughout the course of infection, but becomes masked (either by the 66,00@-70,000 SP or by antibody which progressively accumulates on the parasite’s surface) and cannot be iodinated. In connection with the presence of antibody, the appearance of iodinated polypeptides of about 45,000-50,000 in the autoradiographic profiles of day 15 and 18 samples examined directly or following immunoprecipitation is suggestive of the presence of Ig heavy chains on the parasite’s surface. A similarly sized poly~ptide was also detected in Western blots using both normal and immune serum followed by anti-mouse IgG as second antibody; this is indicative of the presence of Ig heavy chains in parasite extracts. By the use of Western blotting techniques it was found that the 88,000-92,000 SP is present in parasite extracts throughout the course of infection. On the other hand, the 66,000-70,000 SP is apparently stage specific, and present only after the mid-plateau phase of infection (from about day IS), a result in line with the iodination experiments. Thus, the Western blotting results support the masking hypothesis referred to above in relation to the failure of the 88,00~92~~0 SP to become iodinated at the later stages of infection. However, a further explanation is also possible. Late in infection, the 88,00~92,0~ SP may not be translocated from the cell interior to the surface coat. Instead, it may be post-translationally modified into the 66,000-70,000 species which then becomes exposed on the parasite’s surface. That the 88,00092,000 SP did not co-purify with the 66,OOO-70,000SP when day 16 parasites were extracted by the method of Cross (1975) supports the view that it may be confined to the cytoplasm at the later stages of infection. Antibodies affinity purified against the 88,00092,000 SP from day 18 parasites reacted with both the 88,00~92,~0 and 66,00~70,0~ SP (plus some minor components that may represent degraded products of the 88,0~92,000 species) in Western blots of parasite extracts. On the other hand, affinity purified antibodies to the 66,00&70,000 SP reacted only with the 66,000-70,000 SP. The first result suggests that these two SP are immunologically related; the second is difficult to reconcile with that hypothesis. It may be that the 66,000-70,000 SP has unique epitopes to which antibodies bind preferentially later in the infection, a view supported by the observation that antibodies eluted from parasites at day 16 bound only to the 66,00@-70,000 SP in Western blots of parasite extracts. That antibodies afEnity purified against each of the two SP from day 18 parasite extracts bound to parasites in indirect fluorescent antibody tests supports the view that both polypeptides are dispersed
on the surface of the parasite. However, the parasites used were from day 8 post-infection, a time at which the 88,~~92,~0 is present but the 66,~~70,0~ SP cannot be detected in the parasite by iodination, immunoprecipitation or Western blotting. These results can be readily explained by assuming the two SP are immunologically related, but as noted above evidence for this is equivocal. From the results of the study a model for SP dynamics in T. musculi can be presented. It is evident that the 88,000-92,000 polypeptide is expressed on the surface of parasites as they enter the early plateau phase of infection (about day 10) and probably even earlier (day 8). Although this SP appears to be present throughout the subsequent stages of infection it is unlikely that it is exposed on the parasite’s surface after the mid-plateau phase of infection because it is not detected from that time onwards by iodination. From the mid-plateau phase of infection a second polypeptide of 66,000-70,000 is expressed on the parasite’s surface. The appearance of immunoglobulins on the surface shortly before the appearance of the 66,000-70,000 SP at day 15 suggests that antibody may play a role in influencing the expression of this polypeptide. If the 66,000-70,000 SP is immunologically related to the 88,000-92,000 species, cleavage or some other post-translational modification of the latter may give rise to it. The 88,00092,~O SP may then become either obscured or remain internalize over the later stages of infection. The change that occurs in the surface coat a short time before parasites are cleared from the circulation may be a preadaptation to life in the vector since it does not appreciably extend the survival of parasites in the bloodstream of the mouse. While not on the scale seen in the African trypanosomes, some antigenic variation in surface coat molecules appears to occur in T. musculi. In the related species T. lewisi, antigenic variation has also been detected in which dividing forms differ from non-reproducing ‘adult’ forms (D’Alesandro, 1976). Further work is necessary to determine if variation of this kind occurs in T. musculi also. REFERENCE BATTAGLIA P. A., ZANIB., DEL BUE M., PONZIM. & BIRACOC. 1983. The glycoproteins of the complex surface coat of Trypanosoma lewisi. Cell Biology International Reports 7: 755-762. BEALL J. A. & MITCHELLG. F. 1986. Identification of a
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1975. Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypa~osa~ brucei. PurasifaZogy 71: 393-417. D’ALESANDROP. A. 1976. The relation of agglutinins to antigenic variation of Trypartosoma Protozoology 23: 256-261.
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SAMARAWICKREMA N. A. & HOWELL M. J. 1988. Interactions between peritoneal cells and Trypanosoma musculi in mice. International Journalfor Parasitology 18: 69-73. SNARY D. 1985a. The cell surface of Trypanosoma cruzi. Current Topics in Microbiology and Immunology 117: 7592. SNARY D. 1985b. Biochemistry of surface antigens of Trypanosoma cruzi. British Medical Bulletin 41: 144-148. S~URTEVANTJ. & BALBER A. E. 1983. Externally disposed membrane polypeptides of intact and protease-treated Trypanosoma lewisi correlated with sensitivity to alternate complement pathway-mediated lysis. Infection and Immunity 42: 869-875. TURNER M. J. 1982. Biochemistry of the variant surface glycoproteins of salivarian trypanosomes. Advances in Parasitology 21: 69-153. TURNER M. J. 1985a. Antigens of African trypansomes. Current Topics in Microbiology and Immunology 120: 14lL 155. TURNER M. J. 1985b. The biochemistry of the surface antigens of the African trypanosomes. British Medical Bulletin 41: 137-143. VICKERMAN K. 1969. On the surface coat and flagellar adhesion in trypanosomes. Journal of Cell Science 5: 163193. VIENS P., TARGETTG. A. T., LEUCHARSE. & DAVIESA. J. S. 1974. The immunological response of CBA mice to Trypanosoma musculi. I. Initial control of the infection and the effect of T cell deprivation. Clinical and Experimental Immunology 16: 279-294.
N. A. SAMARAWICKREMA
Department of Zoology, Australian National University, P.O. Box 4, Canberra, A.C.T. 2601, Australia (Received 19 ~e&ember 1989; accepted27 June 1990) Abstract-SAMARnwrcKREMAN. A. and HOWELL M. I. 1990. The surface coat of bloodstream forms of Trypanosoma musculi from mice. International Journalfor Parasitology 20: 1055-1062. Iodination and immunoprecipitation techniques together with indirect fluorescent antibody tests identified two polypeptides (SP) of molecular weights 88,OOt%92,000and 66,000-70,000 in the surface coat of bloodstream forms of the mouse trypanosome, Trypanosoma musculi. As parasites multiply and enter the early plateau phase of infection the 88,000-92,000 SP is present while the 66,000-70,000 SP is only detectable after the mid-plateau phase. Western blotting of parasite extracts showed that the 88,00&92,000 SP was present throughout the course of infection, but it appears to become masked by the 66,000-70,000 SP or possibly immunoglobulin from about I6 days after infection. Based on results when Western blots of parasite extracts were probed with antibodies affinity purified against the 88,000-92,000 SP, the two SP appear to be immunolo~cally related and the smaller may be a cleavage product of the larger. This would explain why affinity purified antibodies to each SP bound to trypanosomes collected 8 days after infection, when only the 88,~92,0~ is detectable in parasite extracts. However, the failure of antibodies affinity purified against the 66,00&70,000 SP to bind to the 88,000-92,000 SP in Western blots suggests that the smaller SP has some epitopes that are immunologically distinct from those of the larger SP. INDEX KEY WORDS: Trypanosoma musculi; mouse; trypanosome; surface coat; surface antigens; iodination, immunoprecipitation; Western blots; affinity purified antibodies; fluorescent antibody tests.
INTRODUCTION THE major surface glycoproteins
of African (Cross, 1975; Turner, 1982, 1985a, b) and American (Snary, 1985a,b) trypanosomes have been identified and purified and their properties extensively analysed. In the case of African forms, the variable nature of the glycoproteins throughout infection is well established and the probable importance of this phenomenon in avoiding the consequences of host immune responses is clearly recognized. Thus, studies of the surface coats of these parasites can be particularly revealing about aspects of the host-parasite relationship. The presence of surface molecules on the rodent trypanosome Tr~~~nos~~a lewisi has been investigated by a number of workers (Battaglia, Zani, Del Bue, Ponzi & Birago, 1983; Sturtevant & Balber, 1983; Giannini & D’Alesandro, 1984). As many as 13 molecular species have been identified, some of which are sensitive to proteolytic cleavage. While it is difficult to compare these studies closely with each other because of differences in the methods used, it seems clear that the surface molecules of T. lewisishow a
* To whom all correspondence should be addressed.
greater degree of complexity than those of other trypanosom~s. Moreover, there is evidence that antigenie changes occur in the surface proteins during the course of infection (D’AIesandro, 1976). The present study was undertaken on a related stercorarian-T. musculiwhich infects mice. A distinct surface coat is present on parasites from the blood of mice but is lacking or more sparse in vector and culture forms (Vickerman, 1969; Roger, Garzon, Stryowski & Viens, 1987). Dwyer C D’Alesandro (1976a,b) showed that treatment of bloodstream forms with trypsin removed the surface coat indicating that it consisted of, at least in part, protein. Moreover, staining with polycationic dyes revealed a moderate density of carbohydrate moieties in the surface coat. Thus, the surface of T. muscufi, like other trypanosomes, appears to be covered by glycoprotein. In the present investigation, the surface coat of T. musculi bloodstream forms was characterized throughout the course of infection in mice. Surface coat components were labelled by iodination and their antigenicity demonstrated by immunoprecipitation and Western blotting. In addition, confirmation of the surface location of iodinated molecules was sought by exposure of living parasites to trypsin, and the use of immuno~obulins eluted from intact parasites for fluorescent antibody tests.
1055
1056
N.A. MATERIALS
SAMARAWICKREMA~~~
AND METHODS
Animals. Male CBA:Ca/H inbred mice, between 6 and 10 weeks old, were used. Parasites. A Trypanosoma musculi stabilate was originally obtained from the London School of Hygiene and Tropical Medicine, University of London (LUMP 1189 strain) and maintained by syringe passage in mice. The pattern of infection-comprising growth, plateau and elimination phases over a period of about 3 weeks-is described by Viens, Targett, Leuchars & Davies (1974) and has been verified many times in our laboratory. Parasites were harvested by centrifuging whole blood from infected mice on a gradient of Ficoll-Paque (Pharmacia) and passing the parasite-white cell layer through a cellulose CF-I1 (Whatman) column to remove white cells (Samarawickrema & Howell, 1988). Collection of serum.Mice under deep ether anaesthesia were bled out by severing the axillary vessels. The blood was allowed to clot. centrifuged at 3000 rr for 15 min at 4°C and the serum collected and stored at -- 20°C until required. Serum collected from mice 25 days after infection is referred to as immune serum. Normal serum was collected from uninfected mice at the same age. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) of parasite polypeptides. Samples for electrophoresis were prepared by suspending approximately 10” parasites in 100 ~1 of PBS (pH 7.6), 100 ~1 of 2 x SDSPAGE sample buffer [6% SDS, 20% (w/w) glycerol, 0.008% bromophenol blue, 0.125 M-Tris-HCI (pH 6.8)] containing 10% Pmercaptoethanol and boiled for 2 min. Electrophoresis was carried out on 10% polyacrylamide gels (14 x 14 x 0.15 cm) under reducing conditions at a constant current of 40 mA for 34 h (Laemmli, 1970). Molecular weight protein standards were run on each gel. Gels were prepared for autoradiography using standard procedures (Irving & Howell, 1981). Isolation of surface polypeptides. The technique developed by Cross (1975) to isolate surface glycoproteins of African trypanosomes was applied to T. musculi. Briefly, 2 x lo9 parasites were subjected to sonication, centrifugation and dialysis and the products analysed by SDS-PAGE. Iodination of trypanosome surface components. Parasites were collected 12, 15 and 18 days after infection and iodinated using the Iodogen method (Fraker & Speck, 1978). Iodination of samples of 10’ parasites were carried out in glass tubes previously coated with Iodogen (1,3,4,6tetrachloro-3 alpha, 6 alpha-diphenylglycouril) (Sigma) in 50 ~1 of chloroform and reacted with 8-10 ~1 of carrier-free YNaI (Amersham) (about 18.5 MBq) for 5 min on ice and 5 min at room temperature. Parasites treated with trypsin accordine to the method of Dwver & D’Alesandro (1976a) were al&-processed in a similar manner. Iodinated parasites were washed three times in PBS (pH 7.6) and resuspended in 50 ~1 PBS. Half the sample was stored at - 20°C to be used for immunoprecipitation experiments. The remainder of the sample was spun, resuspended in 25 pl of 0.5% Non-Idet P40 (Bio-Rad) in PBS, left for 5 min at room temperature, spun for 5 min and the supernatant discarded. The pellet was dissolved in 25 ~1 of 2 x SDS-PAGE sample buffer containing 2.5 ~1 of pmercaptoethanol, then boiled for 2 min and subjected to SDS-PAGE and autoradiography as described above. Immunoprecipitation of iodinated surface components. The remaining half of the iodinated parasite sample from above was made up to 50 ~1 with PBS, 25 ml ofwhich was allowed to react with antibody by adding 25 ~1 of immune serum and incubating overnight at 4’C. The remainder of the sample (25
M.J.HOWELL
~1) was similarly incubated with normal serum. Immune complexes were absorbed onto 250 ~1 of heat-killed, formaldehyde-fixed Staphylococcus aureus Cowan I strain (Kessler, 1975) at room temperature for 30 min. Each sample was washed in 500 ~1 of 0.05% Non-Idet P-40 in NET buffer (0.1 M-NaCl. 0.005 M-EDTA. 0.01 M-Tris-HCl. DH 7.5) and ieft for 15 min at room temperature. The pellet fas further washed in NET buffer, then dissolved in 50 ~1 of 2 x SDSPAGE sample buffer containing 10% pmercaptoethanol and boiled for 5 min. Undissolved material was pelleted by centrifugation for 5 min, and samples ofthe supernatant were analysed by SDS-PAGE and autoradiography. Western blotting (immunoblotting) of parasite samples. Samples subjected to SDS-PAGE as above were transferred onto nitrocellulose membranes (Schleicher and Schull) in a vertical electrophoresis tank (Bio-Rad) using Western blot transfer buffer (20 mh+Tris, 150 mM-glycine with 20% methanol) to renature the proteins. Elcctrophoresis was carried out at 30 V/l50 mA overnight. Filters were blocked with 5% (w/v) non-fat skim milk powder (Diploma) in Trisbuffered saline (TBS) for 30 min then treated with antibody diluted in TBSiskim milk for 1-2 h and washed in 0.05% Tween-20 (Bio-Rad)/TBS with several changes for 30 min. Filters were treated with goat anti-mouse IgG (H+ L) coniueated with horseradish peroxidase (HRP, Bio-Rad) at a dil&n of 1:4000 in TBS/skjm milk for‘1 h. Following’this, the filters were washed in Tween/TBS and treated with HRPcolour development reagent (60 mg of 4-chloro-1-napthol in 20 ml methanol) and 0.1% 100 vol. H,O, for 2G30 min. The filters were then washed in deionized water and air dried. Biotinylated SDS-PAGE molecular weight standards (BioRad) were electrophoresed at the same time as the samples, transferred onto nitrocellulose and blocked with 3% gelatine in TBS, washed with 0.05% Tween-20 in PBS, treated with 0.3% Avidin-HRP (Bio-Rad) in 1% gelatine in TBS for 1 h and washed again with Tween/TBS before reacting with HRP-colo~r development reagent. Afinity purification of antibodies to antigenic polypeptides. Parasite samples were electrophoresed in duplicate, transferred onto nitrocellulose and one lane was probed with immune serum (I:5 dilution) as above in order to determine the location of antigenic polypeptides in the extracts. Strips corresponding to each of the antigenic bands on the remaining lane were cut out, then treated individually. Each was blocked with 5% skim milk in TNT (10 mr+Tris, 150 mM-NaC1,0.05% Tween-20, pH 8.0) for 1h, washed in TNT alone for 30 min and incubated for 3 h with immune serum diluted 1:5 in TNT. The filters were washed in TNT for 1 h with several changestreated with 100 mM-boric acid/500 mMNaCl (pH 8.0) for 30 min and PBS for 30 min. Any antibody bound to each filter was eluted by incubation in 200 ml of 100 mmglycine/l50 mM-NaCl (pH 2.6) for 3 min after which the pH was immediately brought to 8.0 by adding 20 ~1 of 2 MTris (pH 8.0) (Beall & Mitchell, 1986). Eluates were stored at - 20°C until required. Elution of surface immunoglobulin Ig from parasites.About lo* live parasites from day 16 to day 18 after infection in I ml PBS were incubated in 9 ml of 25 mM-glycine_HCI in 2 MNaCl (pH 2.5) for 15 min on ice to elute surface Ig. Following ccntrifugation at 3000 g for 15 min at 4 “C, the pH of the supernatant was restored to pH 8.0 by adding an equal volume of 100 mr+borate buffer in PBS (pH 8.3). The eluate was concentrated to 1 ml by dialysis against 30% w/v polyethylene glycol in the borate buffer as above. After dialysis against several changes of PBS, the concentrated sample was stored at -20°C until required for Western
Surface coat of T. musculi blotting. Indirect fluorescent antibody tests. Blood smears were prepared from mice 8 days after infection, a time when surface Ig cannot be detected on the parasites (Samarawickrema & Howell, unpublished). Smears were fixed with acetone for 5 min and allowed to air dry. The slides were treated with affinity purified antibody samples for l-2 h at room temperature and washed thoroughly in PBS for 15 min. The slides were then reacted with FITC (fluorescein isothiocyanate) labelled rabbit anti-mouse IgG (Silenus) at a dilution of 1:20 in PBS for 1 h, and examined by fluorescence microscopy as wet mounts with a Leitz U.V. photomicroscope using a BG-12 excitation filter and K530 and AUS barrier filters. RESULTS
Analysis of parasite extracts SDS-PAGE of unlabelled extracts of parasites prepared 16 days after infection by the method of Cross (1975) indicated the presence of a single broad band of about 66,OO(r70,000 mol. wt (Fig. 1). The SDS-PAGE profile of total parasite extracts is also shown (Fig. 2). Most noticeable is a prominent band of about 66,000 mol. wt which is absent from the trypsintreated parasite extract. Iodination of surface molecules Autoradiographic patterns resulting from the electrophoresis of ‘Z51-labelled parasite extracts are shown in Fig. 3. A single polypeptide of between 88,000 and 92,000 mol. wt was identified in parasites obtained 10 and 12 days after infection (Fig. 3a). It appeared as a broad band, and exhibited slightly different mobihties between gels; prior treatment of parasites with trypsin reduced the intensity of its labelling (Fig. 3b). A number of labelled polypeptides were detected in parasites iodinated 15 days after infection (Fig. 3~); a prominent species of 66,00& 70,000, a less prominent yet distinct polypeptide of 88,000-92,000 and a number of minor ones (80,000, 60,000, 50,000 and 25,000). In parasites iodinated 18 days after infection (Fig. 3d), a prominent band of 66,000-70,000 was detected and the 88,000-92,000
1057
band was not discernible. Polypeptides of about 80,000, 60,000 and 50,000 were also labelled but less prominently than the 66,00@70,000 species. In the day 18 sample iodination of polypeptides was abolished by prior trypsin treatment (Fig. 3e). Immunoprecipitation The results of immunoprecipitating ‘ZSI-labelled polypeptides of T. musculi by immune and normal sera are illustrated in Fig. 4a-d. Immune serum precipitated polypeptides of 88,00&92,000 and 45,00& 50,000 from parasites obtained 12 days after infection (Fig. 4a). Normal serum did not precipitate polypeptides from these samples (Fig. 4b). From samples prepared 18 days after infection, immune serum precipitated polypeptides of 66,00@-70,000 and 45,00&50,000 (Fig. 4c). Normal serum precipitated a 45,000-50,000 polypeptide (Fig. 4d). Western blotting Unlabelled parasite extracts probed with immune serum highlighted several antigenic polypeptides (Fig. 5a-d). Many high molecular weight bands were detected, particularly in extracts prepared in the later stages of infection. Most noticeable was the presence of a polypeptide of about 88,00&92,000 at all stages of infection. A 66,000-70,000 polypeptide only appeared in parasite samples prepared 16 and 18 days after infection. Present from day 12 onwards were polypeptides of about 45,000 and 50,000. The control filter probed with normal serum recognized the 50,000 polypeptide, but very weakly, in samples prepared 16 and 18 days after infection (Fig. 5e, f). Western blots of unlabelled parasite extracts probed with a#inity purzfied antibody A Western blot probed with antibody that was affinity purified against the 88,000-92,000 polypeptide in parasite extracts prepared 18 days after infection (Fig. 6aad) recognized a 88,000-92,000 polypeptide consistently through infection. The same antibody also recognized a prominent 66,OO(r70,000 poly-
FIG. 1. SDS-PAGE gel stained with Coomassie blue of surface molecules of T. musculi (from 16 days after infection), purified as described by Cross (1975). Molecular weight markers: 92,000 phosphorylase B; 66,000 bovine serum albumin; 45,000 ovalbumin; 31,000 carbonic anhydrase. FIG. 2. Coomassie
blue stained SDS-PAGE gel comparing protein profiles of trypsin treated parasites parasites (lane b). Molecular weight markers as for Fig. 1.
(lane a) with untreated
FIG. 3. Autoradiograph of SDS-PAGE gel following electrophoresis of ‘z51-labelled parasite extracts obtained various days after infection. Lane (a): 12 days, untreated parasites; lane (b): 12 days, trypsin treated parasites; lane (c): 15 days, untreated parasites; lane (d): 18 days, untreated parasites; lane (e): 18 days, trypsin treated parasites. Molecular weight markers as for Fig. 1. FIG. 4. Autoradiograph of SDS-PAGE gel comparing immunoprecipitates of ‘251-labelled parasite extracts. Lane (a): immunoprecipitate obtained with immune serum and parasite extract prepared 12 days after infection; lane(b): immunoprecipitate obtained with normal serum and parasite extract prepared 12 days after infection; lane (c): immunoprecipitate obtained with immune serum and parasite extract prepared 18 days after infection; lane(d): immunoprccipitate obtained with normal serum and parasite extract prepared 18 days after infection. Molecular weight markers as for Fig. 1.
N. A. SAMARAWICKREMAand M. J. HOWELL
1058
1 a_
-“‘(’ .^‘
6
4%
&-
b
Surface coat of T. musculi
-_I
1*-__
-92 -66
-45
e
FIGS. 5-9.
1059
1060
N.A.
SAMARAWICKREMA
and M. J. HOWELL
peptide in samples prepared 16 and 18 days after infection; this pol~pt~dew~ barely det~tablein day 8 and 12 samples. A poly~ptide of about 50,000 was evident in day 12,16 and I8 samples but not in the day 8 sample. A few polypeptides were also present between the more prominent species of 66,000-70,000 and 88,000-92,000. A Western blot probed with antibody affinity purified against the 66,000-70,000 polypeptide recognized a 66,000-70,000 polypeptide in parasite samples, especially from days 16 and 18 after infection (Fig. 6e-h). Some minor polypeptides of higher molecular weight (about 80,000) were faintly detectable. A 50,000 polypeptide was detected in all samples from day 12 of infection onwards. A Western blot probed with Ig eluted from the surface of parasites 16 days after infection highlighted a polypeptide of 66,~-70,~ in parasite extracts (Fig. 9).
polypeptide with a molecular weight of about 66,00070,~. That this polypeptide is exposed on the surface was deduced from the fact that it was absent from the polypeptide profile derived from live parasites that were treated with trypsin. Moreover, antibody eluted from the surface of parasites 16 days after infection reacted with a 66,00@-70,000 SP in Western blots. The molecular mass of this SP of T. musculi is close to that of African trypanosomes (Cross, 1975). Iodination of cell surface components of T. musculi demonstrated the presence of at least two major polypeptides of 88,000-92,000 and 66,00&70,000 mol. wt during the course of infection. Parasites obtained from the growth and early plateau phases (days 10, 12) possess a 88,0~-92,000 SP. By the midplateau phase of infection (day 15) the second ~lypeptide (66,0~70,~0) is also present on the surface of parasites, and as the infection progresses to late-plateau and elimination phases only that polypeptide appears to persist. Thus, the nature of the coat appears to be less complex than in related Fluorescent antibody tests Antibodies affinity purified against the 66,OOO- stercorarians T. cruzi and T. lewisi where numerous surface glycoproteins are present (Snary, 1985a, b; 70,000 and 88,000-92,000 mol. wt parasite extracts Battaglia et al., 1983; Giannini & D’Alesandro, 1984; prepared 18 days after infection each bound to the surface of intact parasites in blood films prepared 8 Sturtevant & Balber, 1983). Surface iodination used in conjunction with imdays after infection (Figs. 7 and 8). The intensity of munoprecipitation confirmed that the labelled fluorescence in each case was similar. Control parasites polypeptides of T. musculi were of parasite origin and treated with fluorescein-labelled anti-mouse fg alone present on the surface. Immune serum precipitated the did not fluoresce. 88,00~92,~ species early in the infection (day 12). However, from the mid-plateau phase onwards, DISCUSSION immune serum recognized the 66,000-70,000 species The present study has provided information on the only. The few minor polypeptides that became iodinnature and dynamics of surface coat components of ated (for example 80,000, 60,000) but which were not Trypanosoma musculi throughout the course of precipitated by immune serum may be host material that becomes lodged in the parasite’s surface coat. infection in mice. Application of the method of Cross The present results are open to two interpretations. (1975), for isolating surface glycoproteins of African Firstly the parasite may alter the composition of its trypanosomes, to T. musculi collected 16 days after infection demonstrated the presence of a single surface coat by ceasing synthesis of the 88,000-92,000
FE. 5. Western blot showing parasite extracts probed with mouse serum (I 5 dilution) followed by goat anti-mouse IgG-HRP. Lanes (a), (b), (c) and (d) consisted of parasite extracts obtained on days 8, 12, 16 and I8 after infection, respectively and probed with immune serum. Lanes e and f replicated lanes c and d but were probed with normal serum (the nitroceIlulose was damaged in processing). Molecular weight markers as for Fig. 1. FIG. 6. Western blot showing parasite extracts probed with antibodies affinity purified against surface polypeptides from parasites obtained 18 days after infection, followed by goat anti-mouse IgG-HRP. Lanes (a), (b), (c) and (d) consisted of parasite extracts obtained on days 8,12,16 and 20 after infection, respectively and probed with affinity purified antibody to the 88,000-92,000 polypeptide; lanes (eHh) replicate lanes (a)-(d) but were probed with affinity purified antibody to the 66,OO(t 70,000 polypeptide. Molecular weight markers as for Fig. 1. FIG.7. Fluorescence micrographs ofparasites 88,000-92,000 surface polypeptide
obtained followed
FIG. 8. Fluorescence micrograph of parasites 66,~~70,~0 surface polypeptide
obtained 8 days after infection treated with antibody affinity purified against the followed by FITC-labelled rabbit anti-mouse IgG. Scale bar, 10 pm.
FIG. 9. Western parasite surface
8 days after infection treated with antibody affinity purified against the by FITC-labelled rabbit anti-mouse IgG. Scale bar, 10 pm.
blot of extracts from parasites obtained I6 days after infection and probed with antibody eluted from the (16 days after infection) followed by goat anti-mouse IgG-HRP. (Molecular weight marker bovine serum albumin, 66,000.)
1061
Surface coat of T. musculi
SP and commencing synthesis of the 66,~~70,000 SP from about the mid-plateau phase Qf infection. This may account for the iodination of both SP in day 15 parasites. On the other hand, it is possible that the 88,000-92,000 SP continues to be synthesized and expressed on the parasite’s surface throughout the course of infection, but becomes masked (either by the 66,00@-70,000 SP or by antibody which progressively accumulates on the parasite’s surface) and cannot be iodinated. In connection with the presence of antibody, the appearance of iodinated polypeptides of about 45,000-50,000 in the autoradiographic profiles of day 15 and 18 samples examined directly or following immunoprecipitation is suggestive of the presence of Ig heavy chains on the parasite’s surface. A similarly sized poly~ptide was also detected in Western blots using both normal and immune serum followed by anti-mouse IgG as second antibody; this is indicative of the presence of Ig heavy chains in parasite extracts. By the use of Western blotting techniques it was found that the 88,000-92,000 SP is present in parasite extracts throughout the course of infection. On the other hand, the 66,000-70,000 SP is apparently stage specific, and present only after the mid-plateau phase of infection (from about day IS), a result in line with the iodination experiments. Thus, the Western blotting results support the masking hypothesis referred to above in relation to the failure of the 88,00~92~~0 SP to become iodinated at the later stages of infection. However, a further explanation is also possible. Late in infection, the 88,00~92,0~ SP may not be translocated from the cell interior to the surface coat. Instead, it may be post-translationally modified into the 66,000-70,000 species which then becomes exposed on the parasite’s surface. That the 88,00092,000 SP did not co-purify with the 66,OOO-70,000SP when day 16 parasites were extracted by the method of Cross (1975) supports the view that it may be confined to the cytoplasm at the later stages of infection. Antibodies affinity purified against the 88,00092,000 SP from day 18 parasites reacted with both the 88,00~92,~0 and 66,00~70,0~ SP (plus some minor components that may represent degraded products of the 88,0~92,000 species) in Western blots of parasite extracts. On the other hand, affinity purified antibodies to the 66,00&70,000 SP reacted only with the 66,000-70,000 SP. The first result suggests that these two SP are immunologically related; the second is difficult to reconcile with that hypothesis. It may be that the 66,000-70,000 SP has unique epitopes to which antibodies bind preferentially later in the infection, a view supported by the observation that antibodies eluted from parasites at day 16 bound only to the 66,00@-70,000 SP in Western blots of parasite extracts. That antibodies afEnity purified against each of the two SP from day 18 parasite extracts bound to parasites in indirect fluorescent antibody tests supports the view that both polypeptides are dispersed
on the surface of the parasite. However, the parasites used were from day 8 post-infection, a time at which the 88,~~92,~0 is present but the 66,~~70,0~ SP cannot be detected in the parasite by iodination, immunoprecipitation or Western blotting. These results can be readily explained by assuming the two SP are immunologically related, but as noted above evidence for this is equivocal. From the results of the study a model for SP dynamics in T. musculi can be presented. It is evident that the 88,000-92,000 polypeptide is expressed on the surface of parasites as they enter the early plateau phase of infection (about day 10) and probably even earlier (day 8). Although this SP appears to be present throughout the subsequent stages of infection it is unlikely that it is exposed on the parasite’s surface after the mid-plateau phase of infection because it is not detected from that time onwards by iodination. From the mid-plateau phase of infection a second polypeptide of 66,000-70,000 is expressed on the parasite’s surface. The appearance of immunoglobulins on the surface shortly before the appearance of the 66,000-70,000 SP at day 15 suggests that antibody may play a role in influencing the expression of this polypeptide. If the 66,000-70,000 SP is immunologically related to the 88,000-92,000 species, cleavage or some other post-translational modification of the latter may give rise to it. The 88,00092,~O SP may then become either obscured or remain internalize over the later stages of infection. The change that occurs in the surface coat a short time before parasites are cleared from the circulation may be a preadaptation to life in the vector since it does not appreciably extend the survival of parasites in the bloodstream of the mouse. While not on the scale seen in the African trypanosomes, some antigenic variation in surface coat molecules appears to occur in T. musculi. In the related species T. lewisi, antigenic variation has also been detected in which dividing forms differ from non-reproducing ‘adult’ forms (D’Alesandro, 1976). Further work is necessary to determine if variation of this kind occurs in T. musculi also. REFERENCE BATTAGLIA P. A., ZANIB., DEL BUE M., PONZIM. & BIRACOC. 1983. The glycoproteins of the complex surface coat of Trypanosoma lewisi. Cell Biology International Reports 7: 755-762. BEALL J. A. & MITCHELLG. F. 1986. Identification of a
particular antigen from a parasite cDNA library using antibodies affinity purified from selected portions of Western blots. Journal of Immunological Methods 86: 217233. CROSS G. A. M.
1975. Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypa~osa~ brucei. PurasifaZogy 71: 393-417. D’ALESANDROP. A. 1976. The relation of agglutinins to antigenic variation of Trypartosoma Protozoology 23: 256-261.
lewisi.
Journal
of
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DWYER D. M. & D’ALESANDROP. A. 1976a. Thecell surface of Trypanosoma musculi bloodstream forms. I. Fine-structure and cytochemistry. Journal of Protozoology 23: 75-83. DWYER D. M. & D’ALESANDROP. A. 1976b. The cell surface of Trypanosoma musculi bloodstream forms. II. Lectin and immunological studies. Journal of Protozoology 23 : 262271. FRAKER P. J. & SPECKJ. C. 1978. Protein and cell membrane iodinations with a sparingly soluble chloromide, 1,3,4,6 tetrachloro-3q 6a diphenylglycouril. Biochemical and Biophysical Research Communications 80: 849-857. GIANNINI S. H. & D’ALESANDRO P. A. 1984. Isolation of protective antigens from Trypanosoma lewisi by using trypanostatic (ablastic) immunoglobulin G from the surface coat. Infection and Immunity 43: 617-621. IRVING D. 0. & HOWELL M. J. 1981. Characterization of excretory-secretory antigens of Fasciola hepatica. Parasirology 85: 179-188. KESSLER S. W. 1975. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. Journalof Immunology 115: 1617-1624. LAEMMLIU. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 68&685. ROGER M., GARZON S., STRYKOWSKIH. & VIENS P. 1987. Fine structure of the cell surface coat of Trypanosoma musculi bloodstream and metacyclic forms using the thiosemicarbazide-silver proteinate method. Journal of Parasitology 73: 4 154 17.
SAMARAWICKREMA N. A. & HOWELL M. J. 1988. Interactions between peritoneal cells and Trypanosoma musculi in mice. International Journalfor Parasitology 18: 69-73. SNARY D. 1985a. The cell surface of Trypanosoma cruzi. Current Topics in Microbiology and Immunology 117: 7592. SNARY D. 1985b. Biochemistry of surface antigens of Trypanosoma cruzi. British Medical Bulletin 41: 144-148. S~URTEVANTJ. & BALBER A. E. 1983. Externally disposed membrane polypeptides of intact and protease-treated Trypanosoma lewisi correlated with sensitivity to alternate complement pathway-mediated lysis. Infection and Immunity 42: 869-875. TURNER M. J. 1982. Biochemistry of the variant surface glycoproteins of salivarian trypanosomes. Advances in Parasitology 21: 69-153. TURNER M. J. 1985a. Antigens of African trypansomes. Current Topics in Microbiology and Immunology 120: 14lL 155. TURNER M. J. 1985b. The biochemistry of the surface antigens of the African trypanosomes. British Medical Bulletin 41: 137-143. VICKERMAN K. 1969. On the surface coat and flagellar adhesion in trypanosomes. Journal of Cell Science 5: 163193. VIENS P., TARGETTG. A. T., LEUCHARSE. & DAVIESA. J. S. 1974. The immunological response of CBA mice to Trypanosoma musculi. I. Initial control of the infection and the effect of T cell deprivation. Clinical and Experimental Immunology 16: 279-294.