Plasmodium falciparum polypeptides released during in vitro cultivation*

Bulletin ofthe World Health Organization, 61(1): 105-112 (1983) ©) World Health Organization 1983 Plasmodium falciparum polypeptides released during in vitro cultivation* L. RODRIGUEZ DA SILVA,1 M. LOCHE, R. DAYAL, & L. H. PERRIN,3 -Synchronous cultures of Plasmodium falciparum were successively labelled with(3S)-methionine and both the supernatants and the pellets of infected red blood cells were collected. The release of TCA-precipitable material in the culture supernatants was low during the development of ringforms and trophozoites, increased during schizogony, and was maximum at the time of schizont rupture and merozoite reinvasion. Analysis of the supernatants by SDS- PAGE and autoradiography showed that both polypeptides common to the various developmental stages of the parasite and schizont/merozoite- specific polypeptides were released. Polypeptides of relative molecular mass 140000, 82000 and, to a lower degree, 41000 were present in high amounts in the culture super- natants. These polypeptides have been shown to be the target of monoclonal antibodies that are able to inhibit the growth of P. falciparum cultures, and may be involved in protective immunity. The released polypeptides may also be used as target antigens in immunodiagnostic tests aiming at the detection of malaria infection. The sera of individuals with an acute Plasmodium falciparum infection are known to contain soluble malaria antigens (1, 2). These antigens have been detected by double diffusion in gels using, as antisera, the sera of adult individuals who had repeatedly been exposed to P. falciparum infection (1, 2). Several malaria antigens have been recognized and they have been classified on the basis of their thermostability (3). Some of these antigens have also been characterized by their relative molecular mass and susceptibility to proteolytic enzymes (4). Other studies, using short-term in vitro cultures of P. knowlesi and P.falciparum, showed that malaria antigens are released during the schizont develop- mental stage and/or at the time of schizont rupture and merozoite invasion of new erythrocytes (5, 6). Some of the polypeptides released by the parasite in the supernatant of short-term P. falciparum cultures in vitro, have the same immunological reactivity as antigens detected in the sera of infected individuals (5). The precise origin of the released antigens has not been elucidated. * From the WHO Immunology Research and Training Centre, Geneva Blood Centre, Department of Medicine, Geneva University Hospital, 1211 Geneva 4, Switzerland. ' Assistant. 2 Post-Doctoral Fellow. 3Research Associate. The aim of the present study was to determine and quantify the sequential release of P. falciparum antigens during the asexual development of the parasite and to identify at the molecular level the antigens released in the culture medium. MATERIALS AND METHODS Culture conditions and synchronization of the culture P. falciparum (SGE1 strain) was adapted to in vitro culture by using blood taken from a European patient who had a first acute P. falciparum infection after returning from Senegal. The adapted isolate was propagated in group A red blood cells (RBC) by the candle jar method (7). This isolate did not produce gametocytes under the culture conditions used. Synchronized cultures were initiated and the multinucleated forms of the parasite were separated from ring forms and normal RBCs by flotation in Physiogel (8). Over 75%o of the cells floating in Physiogel were parasitized RBCs. These cells were mixed in a ratio of 1 to 3 with fresh uninfected RBCs. The reinvasion period was limited to 5 h, after which the remaining mature forms were lysed with 5%/o mannitol (9). The procedure was repeated 425 -105- L. RODRIGUEZ DA SILVA ET AL. over a second replication cycle to increase synchrony and parasitaemia. In the experiment reported here (the experimental protocol was repeated three times with similar results), a pellet of 0.7 ml RBC with a parasitaemia of 2307o (93.7% ring forms) was diluted in 28 ml of culture medium and divided in seven Petri dishes (60 mm diameter). The culture medium was changed every 6 h and the course of the erythrocytic development was assessed micro- scopically by using Giemsa-stained thin smears. Labelling conditions The synchronized cultures were labelled successively every 6 hours as follows: 0-6, 6-12, 12-18, 18-24, 24-30, 30-36, and 36-42 hours. Successive cultures were washed once in methionine- free MEM (minimum essential medium), supple- mented with 10%7o human serum, 2 mmol of glutamine and 25 mmol of HEPES buffer, and resuspended in 3.5 ml of the same medium containing 1.48 x 104 Bq (40 ACi) per ml of L-(35S)-methionine (specific activity 3.84x 102 Bq (10.39 mCi)/ml). After 6 h of labelling, the parasitized RBC were pelleted by centrifugation at 1000 g for 10 min and then the pellets and the supernatants were treated separately. The pelleted cells were washed twice in cold phosphate-buffered saline (PBS), lysed by the addition (to the last pellet) of 0.6 ml of TNE (Tris- NaCl-EDTA buffer, pH 8) containing 0.50%o of Nonidet P40 (NP40) and kept in aliquots at - 75 'C. The supernatants were centrifuged at 20 000 g for 30 min at 4 'C and dialysed overnight at 4 'C against PBS and stored at - 75 'C. Analysis of the labelled material Measurement of radioactivity associated with infected RBCs and released in culture medium. Duplicate 10 Al aliquots of the various infected RBC lysates and 100 pl aliquots of the supernatants were incubated for 15 min at 37 'C in 0.5 ml of 1 mol/l NaOH containing 25 Al of hydrogen peroxide (30 volumes) and precipitated with 3 ml of cold 257o trichloroacetic acid (TCA). The precipitates were filtered on 0.2 A millipore filters and, after several washings with 5% TCA and alcohol, dissolved in Readysol scintillation fluid and counted in a Beck- man LS20 $-counter. The radioactivity associated with macromolecules in the infected RBCs and culture supernatants was expressed as a percentage of the total radioactivity (counts per minute (cpm) of (35S)-methionine added to individual cultures at the beginning of each labelling). Immunoprecipitation assay. Immunoprecipitation was carried out as described by Kessler (10). Aliquots of culture supernatants containing TCA- precipitated polypeptides (0.5 x 106 cpm) were brought to a final concentration of 0.5/o NP40 and a final volume of 400 Al by the addition of TNE buffer. These samples were were incubated for 1 h at room temperature with 10 Al of each individual serum. A volume of 50 Al of a 10o suspension of formalin-treated Staphylococcus aureus ("Pan- sorbin" from Behring) was added to the antigen- antibody mixture. After 15 min of incubation, the mixture was centrifuged (2000 g for 10 min), and the precipitates washed five times. The bound antigen- antibody complexes were eluted with 50 Al of sodium dodecyl sulfate (SDS) polyacrylamide gel electro- phoresis (PAGE) sample buffer at 60 °C for 15 min. The pellets of labelled infected RBCs were solubi- lized in TNE buffer, pH 7.2, containing 0.5% NP40 and then centrifuged at 20 000 g for 30 min at 4 'C. The supernatants containing soluble polypeptides (accounting for 90% of the measured cpm) were used for immunoprecipitation, following the same procedure as for the culture supernatants. Three sera from adults living in a malaria endemic area (in Kenaba, the Gambia) and a serum from a European who had never suffered from malaria were used for the immunoprecipitation assay. SDS-PAGE analysis and autoradiography. Electrophoresis was carried out in 10% slab gel, as described by Laemmli (11). All samples were equilibrated in Tris-HCI-buffer, pH 6.8, containing iWo SDS and 2% mercaptoethanol. The gels were calibrated with standard protein markers (Pharmacia Fine Chemicals), fixed, stained with 0.1% Coomassie blue, dried, and autoradiographed on X-ray films (Kodak) for 5-10 days at - 75 'C. RESULTS A good synchrony of P.falciparum cultures was achieved by using the methodology described (Fig. 1). The incorporation of (35S)-methionine into parasitized RBCs varied during the course of the asexual cycle. As ring forms matured into tropho- zoites, the incorporation of (35S)-methionine increased, reaching its highest level in mature tropho- zoites. A decline of incorporation was later observed in schizonts. The measure (cpm) of TCA-precipitable polypeptides in the supernatants of cultures was used to quantify the amount of parasite polypeptides released into the supernatants. Low counts were obtained from supernatants of cultures containing ring forms and trophozoites. The highest counts were obtained with the supernatants of cultures containing mature schizont segmenters and new ring forms after reinvasion, at which time the TCA-precipitable polypeptides in the supernatant represented 7% of the 106 P. FALCIPARUM POLYPEPTIDE RELEASE IN VITRO Sequentiel incorporation and release of 35S-Methionine labelled P falciparum polypeptides in synchronised culture 1 r- age of culiureihours) 0 parasitemia: 22. 9 ring torms'. 9 3. 7 t rophozoi tes: 6.3 schizonts cpm incorporated in IRBC cpm released in culture medium - 6 12 18 24 30 36 42 22.4 23.54 23.23 23.83 63.75 1 5.0 9.2 - 36.05 80 96.0 49.2 - - 6.9 50.8 24.52 30.05 5.4 23.0 10.3 4 831 72.6 Table 1. Percentage of (35S)-methionine counts incor- porated in infected RBCs and released in culture super- natants % counts' % counts' Labelling incorporated released intervals Developmental in infected in culture (hours) stages RBCs medium 0-6 Early rings 5.1 0.06 6-12 Rings 5.8 0.07 12-18 Rings + 11 5 0-07 trophozoites 18 24 Rings+ 15.1 0.07trophozoites 24-30 Trophozoites + 11.4 0.1 5 schizonts 30-36 Schizonts 10.2 0.36 36-42 Schizonts + 3.4 0.48 rings e 100% represents the total cpm added to each culture at the start of the labelling. Fig. 1. Incorporation of (35S)-methionine in infected RBCs (IRBC) and release of radiolabelled material in culture supernatants during the asexual erythrocytic cycle. Each of the successive individual cultures was incubated for 6 hours with a constant amount of (35S)-methionine. At the bottom of the figure, the percentage of each erythrocytic stage of the parasite is indicated at various time points during the experiment. Non-infected RBCs were also used as a control under identical conditions: there was less than 0.5 x 104 cpm incorporated in normal RBCs and less than 0.2 x 104 incorporated in the supernatant. radioactivity (as cpm) incorporated in the infected RBCs (Fig. 1, Table 1). Both the parasitized RBCs (collected successively during the asexual cycle) and the corresponding supernatants were analysed by SDS-PAGE. Fig. 2A presents autoradiographs of successive samples of parasitized RBCs applied on SDS-PAGE. All the labelled polypeptides were parasitized gene products since mature RBCs are depleted of their gene pool. Most of the P. falciparum polypeptides are synthesized throughout the asexual cycle, as reported previously (12). However, some polypeptides are syn- thesized or processed only, or mainly, at the time of schizogony. Their localization on the gel is indicated by arrows (Fig. 2A) and their relative molecular masses (M,) are respectively > 200 000, 160 000, 140 000, 105 000, 82 000, 55 000 and 41 000. Super- natants recovered from each of the successive cultures have been analysed under identical conditions (Fig. 2B). As would be expected from the results shown in Table 1, parasite-specific polypeptides are released in small amounts in the supernatants before schizogony and the number of detectable polypeptides increases at the time of schizont rupture, which is when the supernatant contains most of the schizont-specific polypeptides. In addition, four polypeptides of Mr 180 000, 76 000, 60 000 and 45 000 that are common to the three developmental stages are clearly detectable. Based on the intensity of the radiolabelled bands, polypeptides of Mr > 200 000, 180 000, 140 000, 82 000, 60 000 and 45 000 were identified as the major components released (Fig. 2B and Fig. 3, second band). To further identify the labelled polypeptides as parasite-specific products and assess their immuno- genicity, the supernatants were used as a source of malaria antigens and immunoprecipitated by sera of individuals living in endemic areas, as shown in Fig. 3 (bands Is, 2s, 3s). Lysates of unsynchronized, labelled infected cells were also used as a source of antigens and immunoprecipitated by the same sera (Fig. 3, bands lc, 2c, 3c). The results demonstrate, on the basis of the intensity of the polypeptide bands detected on the autoradiographs, that some malaria antigens are present in higher concentration in the culture supernatants than in the lysate of infected RBCs. This difference is particularly evident for the schizont- and merozoite-specific polypeptides of Mr 140 000, 82 000 and 41 000. Another schizont- specific polypeptide of Mr 160 000 is precipitated similarly from both antigenic sources, whereas the Mr 200 000 polypeptide is detected in higher concen- tration in the immunoprecipitates obtained by using the infected cells as a source of malaria antigens. 500- x 100- 50- 10- 5- 10.7 L. RODRIGUEZ DA SILVA ET AL. DISCUSSION This study shows that, during the in vitro culture of asexual erythrocytic stages of P.falciparum, parasite- specific polypeptides are released in the culture medium. The results also confirm previous data demonstrating that the release of parasitic material is very low during the maturation of ring forms and trophozoites, increases at the end of schizont maturation, and is maximum at the time of schizont rupture and reinvasion of RBCs by merozoites (4, 5). All the radiolabelled polypeptides are parasite- specific since mature human RBCs do not contain nucleic acids. In addition, the radiolabelled poly- peptides released are precipitated by sera from immune individuals living in malaria endemic areas. In this study, the relative molecular masses of the released polypeptides were determined, two groups being identified: polypeptides common to the various developmental stages of the parasites (with Mr 180 000, 76 000 and 45 000, for example) and polypeptides that are synthesized or processed during the schizont developmental stage. The polypeptides with low Mr migrate with the front of the gels and probably account for most of the TCA-precipitable material detected in the supernatants of cultures containing ring forms and early trophozoites. This material probably consists of small polypeptide chains that leak from infected RBCs. The exact origin of the released polypeptides is difficult to specify. They probably do not arise from lysis of schizonts that fail to mature or from lysis of merozoites that fail to invade the erythrocytes, since some of the major polypeptides of schizonts are not represented in the supernatants, and since the culture conditions have been optimized. One possibility is that the released antigens represent residual parasitic material that is not incorporated into schizonts. Alternatively, they may be parasite antigens associated with the knobs present at the surface of erythrocytes containing schizonts, or antigens associated with the coat of merozoites. The parasite polypeptides common to the various developmental stages probably represent parasite material that is not incorporated into the merozoites. The schizont- specific polypeptides have previously been identified and are shown in Fig. 2A (12, 13). They are good candidates for expression at the surface of schizonts and/or merozoites since polypeptides expressed late in sporogony and schizogony have been shown in other plasmodia species to be expressed at the surface of sporozoites and merozoites and to be involved in protective immune responses (14, 15). The apparent M, of the more intensively labelled schizont-specific polypeptides are respectively about 200 000, 160 000, 140 000, 105 000, 82 000 and 41 000. Some of these polypeptides are present in high concentration in the supernatant of P.falciparum cultures at the time of reinvasion, notably polypeptides of A(r 140 000, 82 000 and, to a lesser extent, 41 000. Interestingly, these polypeptides have been shown to be the target of monoclonal antibodies that are able to inhibit the in vitro growth of P.falciparum cultures (16). These monoclonal antibodies are known to react with schizonts and merozoites, as demonstrated by the indirect immunofluorescence technique (13, 16). It has been shown by electron microscopy that mero- zoites release their coat during the invasion of RBCs (17). There is evidence here to suggest that the released polypeptides ofMr 140000, 82000 and 41000 are part of the merozoite coat and may be compo- nents of the merozoites. In addition, the Mr 82000 polypeptides may be identical with the knob- associated protein described by Kilejian (18). This is on the basis of its relative molecular mass and stage specificity. It has not been elucidated whether or not these polypeptides are intact polypeptides or frag- ments of a polypeptide of higher Mr, as shown in other malaria species (15). Comparison of the poly- peptide bands precipitated, using either infected RBC lysate or culture supernatants, indicates that there is no (or only limited) additional degradation of malaria antigens in the culture supernatants, with the possible exception of the polypeptide with Mr > 200 000. The importance of soluble antigens released by haemosporidia during in vitro culture has been emphasized by the recent demonstration that vaccination with soluble B. bovis antigens derived from cultures can induce protective immunity in cattle (19). These experiments and other vaccination experiments, using soluble antigens of various plasmodia (20, 21), suggest that the supernatants of P.falciparum cultures may be a good source of malaria antigens that are suitable for inducing protec- tion. Soluble parasite antigens may also be of impor- tance for immunodiagnosis, since they may be used as target antigens in serodiagnostic assays that are designed to detect current or recent malaria infection. ACKNOWLEDGEMENTS This work was financed by the World Health Organization, the UNDP/World Bank/WHO Special Programme for Tropical Diseases, and the Swiss National Foundation (grant No. 3.890-0.82). The authors thank Ms E. Ramirez for her expert technical help and Ms G. Fiaux for her excellent secretarial services. 108 A Pellet of cultures 240 67 43 30- 6 12 18 24 30 3.6 42 age of cultures(hours) Fig. 2A. Autoradiographs (by SDS-PAGE analysis) of in- fected RBCs collected from successive cultures of P. falciparum, metabolically labelled with (35S)- methionine at 6-hour intervals. The arrows point to the schizont-specific polypeptides with Mr> 200 000, 160 000, 140 000, 105 000, 82 000, 55 000 and 41 000. -O. 0 E B 240- Culture supernatant 94- 67- 43 - 30- 6 12 I8 24 30 36 42 Pf age of cultures(hours) Fig. 2B. Autoradiographs (by SDS-PAGE analysis) of successive culture supernatants of P. falciparum, meta- bolically labelled with (35S)-methionine. The culture supernatant corresponds to the infected cells analysed in Fig. 2A. Identical volume of culture supernatant (8 Al) was applied on each lane of the gel. Pf: unsynchronized P. falciparum-infected RBC preparation labelled with (35S)-methionine. The arrows point to polypeptides of M, > 200 000, 140 000, 82 000 and 45 000. C,) 30 0 E 220 - o 194 - 67- E 43 - 36 - C S ic is 20 25 30 35S NHS Fig. 3. Autoradiographs (by SOS-PAGE analysis) of immunoprecipitates obtained by using immune sera and either P. falciparum-infected RBCs (bands ic, 2c, 3c) or culture supernatants (bands is, 2s, 3s). Abbreviations: C = P. falciparum-infected RBCs; S = culture super- natant; NHS = normal human serum. P. FALCIPARUM POLYPEPTIDE RELEASE IN VITRO 111 RESUME CARACTERISATION DES POLYPEPTIDES LIBERES PAR PLASMODIUM FALCIPARUM LORS DE LA CULTURE IN VITRO Des antigenes paludiques ont e mis en evidence dans le serum de malades souffrant de paludisme a P.falciparum. Certains de ces antigenes ont et retrouves dans le milieu employe pour les cultures continues in vitro des formes erythrocytaires asexuees de P.falciparum. Pour les experiences presentees ici, des cultures syn- chrones successives de P.falciparum ont et marquees pendant 6 heures A la (35S)-methionine. On a constate que l'incorporation de cet acide amine etait maximale a la fin du stade trophozoite. L'etude des surnageants des cultures a montre que tres peu de polypeptides marques etaient liberes durant la maturation des formes annulaires et des tropho- zoites. Assez marquee A la fin de la maturation des schi- zontes, cette liberation devient importante au moment de la rupture des schizontes et de l'invasion de nouveaux globules rouges. On deduit l'origine parasitaire des polypeptides marques trouves dans le milieu de culture du fait qu'il n'y a pas d'incorporation de (35S)-methionine en presence de globules rouges normaux non parasites et que les polypep- tides marques liberes dans le milieu de culture sont precipites selectivement par des serums humains d'individus partielle- ment immuns. Les surnageants de culture et les globules rouges parasites ont et analyses par SDS-PAGE, suivi d'autoradiographie. Il apparait que les polypeptides liberes appartiennent A deux groupes principaux: le premier comprend des polypeptides qui sont synthetises et exprimes a tous les stades de deve- loppement &rythrocytaire asexue du parasite; le second comprend des polypeptides synthetises ou exprimes A la fin de la schizogonie; ces derniers sont donc schizonto- ou merozoito-specifiques. On peut envisager que les polypep- tides liberes communs aux divers stades de developpement erythrocytaire (principalement polypeptides de Mr egale A 180 000, 76 000, 60 000, et 45 000) representent du materiel parasitaire non incorpore dans les merozoites; ce materiel serait libere par l'eclatement des schizontes. Les polypep- tides schizonto/merozoito-specifiques dont la masse molaire relative (Mr) est de 140 000 ou 82 000 et, dans une moindre mesure, ceux dont la Mr est de 41 000, sont particu- lierement abondants dans le surnageant des cultures. II est interessant de relever que ces polypeptides sont reconnus par des anticorps monoclonaux qui ont la capacite d'inhiber la croissance de P.falciparum in vitro. Ces anticorps reagissent avec les merozoites en immunofluorescence indirecte. I1 apparait donc que le surnageant des cultures de P.falciparum peut representer une source de materiel interessante pour la purification d'antigenes paludiques susceptibles de jouer un role dans la reponse immune de l'h6te. REFERENCES 1. McGREGOR, I. A. ET AL. Soluble antigens in the blood of African patients with severe Plasmodiumfalciparum malaria. Lancet, i:881-884 (1968). 2. WILSON, R. J. M. ET AL. Antigens associated with Plasmodium falciparum infections in man. Lancet, fi: 201-205 (1969). 3. WILSON, R. J. M. ET AL. The stability and fractionation of malarial antigens from the blood of Africans infected with Plasmodium falciparum. International journal of parasitology, 3: 511-520 (1973). 4. WILSON, R. J. M. The production of antigens by Plasmodium falciparum in vitro. International journal of parasitology, 4: 537-547 (1974). 5. WILSON, R. J. M. & BARTHOLOMEW, R. K. The release of antigens by Plasmodium falciparum. Parasitology, 71: 183-192 (1975). 6. MCCOLM, A. A. & TRIGG, P. I. Release of radio- isotope-labelled antigens from Plasmodium knowlesi during merozoite re-invasion in vitro. Parasitology, 81: 199-209 (1980). 7. TRAGER, W. & JENSEN, J. B. Human malaria parasites in continuous culture. Science, 193: 673-676 (1976). 8. REESE, R. T. ET AL. Isolation of stages of the human parasite Plasmodium falciparum from culture and from animal blood. Bulletin of the World Health Organization, 57 (Suppl. 1): 53-61 (1979). 9. LAMBROS, C. & VANDERBERG, J. P. Synchronisation of Plasmodium falciparum erythrocytic stages in culture. Journal ofparasitology, 65: 418-420 (1979). 10. KESSLER, S. W. Rapid isolation of antigens from cells with a staphylococcal protein H-antibody absorbant: parameters of the interaction of antibody-antigen complexes with protein A. Journal of immunology, 115: 1617-1624 (1975). 11. LAEMMLI, U. K. Clearage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-682 (1970). 12. KILEJIAN, A. Stage-specific proteins and glycoproteins of Plasmodium falciparum: identification of antigens unique to schizonts and merozoites. Proceedings of the National Academy of Sciences (USA), 77: 3695-3699 (1980). 13. PERRIN, L. H. & DAYAL, R. Immunity to asexual erythrocytic stages of Plasmodium falciparum: role of defined antigens in the humoral response. Immunological reviews, 61: 245-269 (1982). 14. POTOCNJAK, P. ET AL. Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection. Journal of experimental medicine, 151: 1504-1513 (1980). 112 L. RODRIGUEZ DA SILVA ET AL. 15. HOLDER, A. A. & FREEMAN, R. R. Immunisation against blood-stage rodent malaria using purified parasite antigens. Nature (London), 294: 361-364 (1982). 16. PERRIN, L. H. ET AL. Inhibition of P. falciparum growth in human erythrocytes by monoclonal anti- bodies. Nature (London), 289: 301-302 (1981). 17. AIKAWA, M. ET AL. Erythrocyte entry by malarial parasites. Journal of cell biology, 77: 72-82 (1978). 18. KILEJIAN, A. Characterisation of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum. Proceedings of the National Academy of Sciences (USA), 76: 4650-4653 (1979). 19. SMITH, R. D. ET AL. Bovine babesiosis: protection of cattle with culture-derived soluble Babesia bovis antigen. Science, 212: 335-338 (1981). 20. JERUSALEM, C. Active immunization against malaria (Plasmodium berghei). 1. Definition of antimalarial immunity. Zeitschriftfur Tropenmedizin und Parasito- logie, 19: 171-181 (1969). 21 TODOROVic, R. ET AL. Immunogenic properties of serum antigens from chickens acutely infected with Plasmodium gallinaceum. Annals of tropical medicine and parasitology, 61: 117-124 (1967).