Malaria proteases and red blood cell invasion

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Malaria Proteases and Red Blood Cell Invasion C. Braun Breton and L.H. Pereira da Silva Studies of malaria proteases have focused on two general groups, corresponding to activities specific to malaria parasites: (1) proteases involved in hemoglobin degradation which are active in thefood vacuole and which exhibit optimal activity at low pH; and (2) proteases specific to schizonts and/or merozoites which are involved in merozoite maturation and red blood cell invasion and which exhibit optimal activity at neutral pH. In this paper, Catherine Braun Breton and Luis H. Pereira da Silva will focus on those activities necessaryfor the releaseof infectious merozoites and the entry of the parasite into its host cell. The invasion of red blood cells by malaria merozoites is a multi-step process comprising erythrocyte recognition, attachment, cell-membrane invagination and parasite entry. Our understanding of invasion essentially relies on light- and electron-microscopy studies ~,2. First, the merozoite adheres to the erythrocyte at any point of its surface but then re-orients itself to make contact with the host cell at its conical, apical end. This contact leads to the formation of a distinctive tight junction zone between the two cells. When this happens, the contents of two apical organelles, the rhoptries, are released at the junction between the erythrocyte and the merozoite. This is followed by the invagination of the red blood cell membrane, forming a parasitophorous vacuole membrane in which the parasite is internalized into the host cell. Although microscopy has added a lot to our understanding of invasion, the biochemical events involved in its different steps are, as yet, poorly understood 3. Erythrocyte-binding molecules that might participate in the specific recognition of the host cell and the tight binding of the merozoite to the erythrocyte plasma membrane, have been identified at the surface of malaria merozoites4. However, none of these erythrocyte-binding molecules was clearly localized in the short and long filaments extending from the merozoite surface to the red blood cell during the initial step of contact2,s. Invasion only proceeds if the apical end of the merozoite binds to the erythrocyte surface. Unfortunately, no significant molecular difference was found to be associated with this conical end of the merozoite which might be involved in merozoite reorientation. Only a 60 kDa molecule, merozoite capping protein 1 (MCP-1), has been located at the moving junction between the parasite and the red Catherine Braun Breton and Luis H. Pereira da Silva are at the Unit of Experimental Parasitology, Institut Pasteur, 75724 Paris Cedex 15, France.

blood cell surface6. During invasion, MCP-1 moves from the apical end around to the posterior of the merozoite, suggesting a role for this protein in facilitating attachment or movement of the junction along the parasite surface coat. A combination of inhibitor studies and invasion assays have implicated parasite proteases in the maturation and release of fully invasive merozoites as well as in subsequent steps of red blood cell invasion 7-11. A serine-protease activity involved in erythrocyte invasion has been characterized in Plasmodiumfalciparum and P. chabaudi merozoites n,12. This activity participates in a biochemical cascade that might mediate efficient cell invasion, in response to merozoite-erythrocyte contact. The involvement of parasite proteases in the process of red blood cell invasion is reviewed here.

Diversity of malaria proteases Since the description of the first malaria proteolytic activity in 1946, at least 25 proteases of all four classes (serine, cysteine, aspartate and metallo proteases) have been described in different malaria species 13,14. We have been able to catalog P. falciparum proteases from the schizont and merozoite stages by their ability to degrade gelatin (zymograms 15) following separation by electrofocalization in a pH gradient (pH 3-10) in a Rotofor cell (Biorad) (Fig. 1). Proteolytic activities from pI 4 to pI 9.5 were detected and at least ten are specific for parasite infection. The addition of different, class-specific inhibitors to the zymograms allowed us to assign these proteases to the general classes of proteases as indicated. Only two natural substrates for the many proteolytic activities described have been identified. Goldberg et alJ 6 have described a hemoglobin-degrading activity and we have recently shown that the merozoite-specific P. chabaudi serine protease degrades erythrocyte band 3 protein n. Processing of merozoite surface proteins Many prokaryotic and eukaryotic proteins are synthesized as pre- or pre-pro-enzymes, which undergo proteolytic maturation both intra- and extracellularly 17. In P. falciparum blood-stage parasites, the major merozoite surface protein, MSP-1, and a protein released in the blood at the time of schizont rupture (known as p126 or SERP1) have been shown to undergo a limited number of processing steps 18A9. Due to its position on the merozoite surface and its use as a potential vaccine, MSP-1 has been the subject of intense study 20. This protein is synthesized as a © 1993, Elsevier Science Publishers [ td, (UK)

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high-molecular-weight precursor in mature trophozoites and schizonts and processed at, or just prior to, the release of merozoites ~8. The precise timing of processing is unknown. On the surface of merozoites, even when processed, the protein remains as a complex of non-covalently associated polypeptides. Although the intact MSP-1 is polymorphic, the pattern of degradation products is conserved 2°. This observation suggests that the enzymes involved in the processing are conserved in different malaria strains and species. Two steps in the processing of MSP-1 are observed. The first step generates fragments of 83, 42 and 38 kDa. The amino-terminal sequences of these fragments have been determined by direct amino acid sequencing of the purified polypeptides and localized within the sequence of the protein determined by gene cloning and DNA sequencing 21-23. None of these cleavages could be easily assigned to a given class of protease. This first processing step is partially sensitive to sulfhydryl reagents, suggesting that a cysteine protease is responsible for at least one of these cleavages 24. Serine-protease and aspartate-protease inhibitors do not affect the first processing step of MSP-1 (Ref. 25). Therefore only cysteine a n d / o r metallo proteases, or a protease of an unusual type, appear to be responsible for this process. The second step of MSP-1 processing corresponds to the degradation of the 42 kDa fragment (MSP-la2) to processing products of 33 and 19 kDa. The MSP-119 polypeptide remains on the merozoite surface after invasion, while MSP-133 seems to be shed from the merozoite surface prior to erythrocyte invasion 26,27. Both cleavage of MSP-142 and shedding of MSP-133 were not detected prior to merozoite release 27. Since the processing of MSP-142 takes place at the merozoite surface in the blood and thus, in an environment hostile to proteolytic activities, the enzyme should exhibit a specificity different from that of host proteases. The cleavage of MSP-142 is clearly due to a serine protease since it is sensitive to general serine protease inhibitors such as di-isopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF)28. Moreover the cleavage site sequence is suggestive of a chymotryptic cleavage site. The processing is also sensitive to calcium chelators 28. This suggests that the protease responsible for the second step of processing of MSP-1 is a Ca 2+ dependent serine protease. None of the gelatin degrading activities detected on zymograms of P. falciparum mature schizonts appeared to be both Ca2÷-dependent and PMSF- or DFP-sensitive. Like the p76 serine protease 12, this activity might be only detected in mature merozoites. Alternatively, it might be highly specific and not degrade gelatin. Autocatalytic processing might be invoked for the parasitophorous vacuole antigen p126, or SERP1. This protein is processed into 56, 47 and 18 kDa fragments. SERP147 and SERP11s are linked by disulfide bonds to form a 73 kDa complex. Recently, the amino-terminal sequences of the different products have been determined 29. Two putative cleavage sites have been identified: Glu376--Thr377 between SERP147 and SERP156 and Gln873-Asp874 between SERP156 and SERPll8. Since the sites are different, two proteolytic activities might be involved in this processing but they have not yet been identified. SERP156 undergoes a second

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pl'l kDa 2

Fig. I. Isoelectric focusing of a P. falciparum schizont extract (0.5% Triton X-100, 0.5% Sulfobetaine SBI4, Serva) performed in a ROTOFOR cell (Biorad) in the presence of 2% Pharmalytes pH 3-10 (Fluka). The 20 fractions were analysed by zymogram Is. The pH of each fraction is indicated. The activities are arrowed: red blood cell proteases (GR); parasite activities, assigned to metalloproteases (M), serine proteases (S), and cysteine proteases (C). Aspartate proteases were detected only in trophozoite and merozoite extracts (not shown).

processing into a 50 kDa product. This cleavage seems to be located at the carboxy-terminus of SERP156 and the sequence of the cleavage site is not yet known. This last processing step is inhibited by leupeptin 3°, suggesting that a serine protease is responsible for this cleavage, although leupeptin also inhibits most cysteine proteases 31. Moreover, trypsin can mimic the processing of SERP156 into SERPlso, suggesting that a trypsin-like protease is involved 30. Computer analysis of the SERP gene has revealed the presence of consensus sites for a cathepsin L-like cysteine protease 32. However, the sequence appeared more reminiscent of a serine protease with a cysteineprotease conformation33. Therefore, SERP1 might be homologous to viral proteases with a serine replacing the cysteine of a classical cysteine-active-center enzyme. These consensus sites seem to be located in SERP156 and the putative activity might be responsible for its autoprocessing into SERPlso. Although an elegant observation, it remains theoretical, as no proteolytic activity has been reported, to date, for SERP1. Since SERP1 is processed at the time of schizont rupture, the enzymes involved, as for the processing of MSP-1, seem to act extracellularly. It should be noted that, if SERPls~ is active as a protease, it might be involved in late processing events of merozoite maturation and / or release. Release of invasive merozoites

The release of merozoites from lysed erythrocytes has been shown to be sensitive to protease inhibitors as. When P. falciparum schizonts were grown

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in culture in the presence of a mixture of protease inhibitors (leupeptin, chymostatin, pepstatin and antipain), they were able to mature and lyse normally, but merozoites remained clustered around pigment aggregates and were surrounded by an invisible erythrocyte membrane. These results suggested at least two steps in merozoite release: a first step of red blood cell and parasitophorous vacuole lysis which is insensitive to serine, aspartate and cysteine protease inhibitors; followed by a second step involving protease(s) of the serine, aspartate a n d / o r cysteine type and yielding free invasive merozoites. Lysis of the erythrocyte and parasitophorous vacuole membranes might be mediated by several different enzymatic activities such as phospholipases, hemolysins a n d / o r proteases. Indeed, the degradation of erythrocyte cytoskeletal proteins has been reported in P. lophurae- and P. chabaudi-infected erythrocytes34,35, and more recently in P. falciparum-infected red blood cells 13. Two Plasmodium proteolytic activities able to degrade erythrocyte cytoskeletal proteins have been described 36,37. The P. falciparum 37 kDa protease has been purified and appears to be a cysteine protease with an optimal activity at pH 5.0 (Ref. 37). Since the enzyme has a peculiar inhibitor pattern (sensitivity to some cysteine protease inhibitors and to some serine protease peptidic inhibitors), it might not have been inhibited by the inhibitor mixture used 25, and therefore might be involved in merozoite release. Cellular localization of the enzyme should help in understanding its role within the parasite life cycle. A stage-specific phospholipase Ac~ activity has been recently described in P. falciparum and P. chabaudi mature schizonts and merozoites38. However, since the activity appeared higher in merozoites than in schizonts, it may be involved in merozoitespecific processes, such as erythrocyte invasion. No hemolysin-like molecule has yet been described in malaria parasites which would allow escape from the host cell as has been described for the escape of invasive organisms like Listeria monocytogenes or Trypanosoma cruzi, from a phagocytic vacuole into their host celP9,40. The second step of merozoite release clearly involves a proteolytic activity. The nature of the invisible membrane surrounding the clustered merozoites has not been determined. Although it seems to react with antibodies raised against erythrocyte membranes 25, it might be of an unusual composition. One hypothesis is that megadalton proteins synthesized and translocated by the parasite to the erythrocyte surface41 might form a rather rigid coat which has to be removed by proteolysis to allow merozoite release. The enzyme(s) should act extracellularly. Serum molecules are probably not required for merozoite release, since successful invasion has been observed, in vitro, in cultures of P. falciparum schizonts, containing only Hepes-buffered RPMI medium and 0.5 mg m1-1 defatted bovine serum albumin (C. Braun Breton, unpublished). Thus the protease(s) involved should be released by the infected erythrocyte and act extracellularly. Both erythrocyte proteases and parasite proteases, possibly present as inactive zymogens in the erythrocyte cytoplasm or in the parasitophorous vacuole, might be involved. The putative SERPls6 protease could play such a role.

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Since the exact timing of MSP-1 processing has not been determined, it is not known whether released merozoites already have processed MSP-1 on their surface. This processing has even been considered as artefactual degradation by some investigators 4. However, the processing pattern of MSP-1 is highly conserved within Plasmodium species and strains and, furthermore, only the MSP-119 fragment appeared to enter the erythrocyte during invasion 27. These results strongly suggest that the processing is highly specific and necessary for successful invasion. The presence of EGF-like domains in MSP-119, as well as in many proteins involved in receptor binding, cell-surface interactions, protein adhesion or signalling, led Blackman et al. to propose that the purpose of the proteolytic processing of MSP-1 is to reveal these motifs during red blood cell invasion 42. Successful invasion might also depend on the processing of erythrocyte surface molecules. Alteration of the red blood cell surface

The involvement of a parasite chymotrypsin-like serine protease in the process of red blood cell invasion by P. knowlesi merozoites was first reported in 1981 (Ref. 7). When free P. knowlesi merozoites were first isolated and used in invasion assays, Hadley et al. 8 showed that chymostatin inhibits invasion without affecting attachment of the parasite to the host cell. These observations were confirmed and extended for P. falciparum when it was shown that pretreatment of human red blood cells with chymotrypsin partly reversed the inhibition of P. falciparum invasion by chymostatin9,1°. This result indicated that the penetration of the parasite into the host cell requires the proteolytic degradation of a red blood cell surface molecule. Only the P. falciparum gp76 and its P. chabaudi gp68 analogue have been reported to be merozoite-specific serine proteases n,12. The gp76 was shown to be glycosylphosphatidylinositol (GPI) anchored in schizonts and partially recovered as a soluble form in osmotically lysed merozoites43. The merozoite soluble form of gp76 has characteristics of a phospholipase Ccleaved glycolipid-anchored protein 43 and a GPIspecific phospholipase C activity has been detected in merozoites, which could be responsible for the solubilization of gp76 (Ref. 38). Both the merozoite soluble p76 and the p76 form released from schizont membranes by an exogenous phosphatidylinositol-specific phospholipase C exhibit a serine-protease activity 12. The glycolipid-anchored membrane form of gp76 has no detectable proteolytic activity, suggesting that solubilization of the protein is correlated with its detection as an enzyme. However, the membrane form has a preformed active site since it binds tritiated DFP, a specific inhibitor of serine proteases 44. Taken together, these results suggest a novel mechanism of enzyme regulation: gp76 is stockpiled as a catalytically inert membrane protein in schizonts and newly formed merozoites, and solubilized/secreted as an active enzyme by mature merozoites, presumably at the parasite-erythrocyte junction. Investigation of the precise role of gp76 in P. falciparum blood stages was not easy since P. falciparum purified merozoites usually do not invade human red blood cells in vitro. For this reason, we turned to

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P. chabaudi merozoites, which we have been able to purify in a form that retains 50 to 90% infectivity n. In addition, we have established an invasion assay, in vitro, of mouse red blood cells by purified P. chabaudi merozoites. Invasion, but not attachment, is inhibited by serine protease inhibitors like DFP, Pefabloc SC [4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride} and chymostatin. Treatment of erythrocytes with chymotrypsin prior to invasion prevents the inhibition of parasite entry by serine-protease inhibitors. Plasmodium chabaudi exhibits both a merozoitespecific serine-protease activity n, p68, and a glycosylphosphatidylinositol-specific phospholipase C 38. p68 is the main target of DFP at the surface of merozoites and might therefore be the parasite serine protease involved in erythrocyte invasion. It is present in schizonts as a GPI-anchored, catalytically inert membrane protein. The enzyme has been purified from merozoites. Treatment of red blood cells with purified p68, prior to invasion, is:more efficient than chymotrypsin treatment to prevent inhibition by DFP or Pefabloc. It suggests that p68 is involved in erythrocyte invasion by carrying out the required specific degradation of a red blood cell surface molecule. Interestingly, erythrocyte band 3 is a substrate for the purified parasite enzyme, acting extracellularly at the erythrocyte surface. Two degradation products of 80 and 65 kDa derived from mouse erythrocyte band 3 have been detected ~1. Three pieces of evidence have previously implicated the trans-membrane anion transporter, erythrocyte band 3, in invasion: (1) invasion is inhibited by liposomes harbouring band 3 (Ref. 45); (2) band 3derived carbohydrates also affect the efficiency of invasion46; and (3) a monoclonal antibody to Rhesusmonkey erythrocyte band 3 inhibits invasion, but not attachment, of P. knowlesi merozoites in vitro 47. The first two observations are consistent with a role for band 3 in at least the binding of merozoites to the red blood cell surface, but the last observation suggests that band 3 is actually involved in the process of parasite entry into the host cell. It is noteworthy that band 3 is a trans-membrane protein, with an external domain that could be implicated in parasite binding and a cytoplasmic domain interacting with cytoskeletal proteins like spectrins and ankyrin4S,49. The degradation of band 3 by the merozoite p68 protease could achieve the modifications of the red blood cell cytoskeleton necessary for the formation of the parasitophorous vacuole and the successful entry of the parasite into the host cell. A search for potential cleavage sites of the p68 protease within band 3 was based on its ability to degrade various fluorogenic peptides. Numerous potential sites were identified, including several in the amino-terminal cytoplasmic domain, three within internal loops and three within external loops of band 3. Only the last three sites are consistent with the observed molecular weights of the degradation products, and with the role of purified p68 in the process of in vitro invasion. The observation of band 3 degradation by purified gp68, in vitro, does not exclude the possibility that, in vivo, the enzyme has another target, such as a minor

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surface molecule. Invasion-inhibition studies with band 3-based peptides, corresponding to the identified potential cleavage sites, should help in answering this question. Another observation suggests that the invasion process may be more complicated. When the serine-protease inhibitor, chymostatin, was introduced into resealed ghosts, invasion was efficiently inhibited ~°. This suggested that a parasite enzyme might act at the cytoplasmic side of the erythrocyte plasma membrane. Since chymotrypsin and p68 were able to reverse inhibition of invasion when added extracellularly, one might imagine that two distinct chymostatin-sensitive protease activities are involved: an enzyme acting extracellularly (p68 for example) to prepare parasite entry, and a second enzyme acting intracellularly as invasion proceeds and achieving the necessary modifications of the erythrocyte cytoskeleton. Again, the P. falciparum spectrindegrading, chymostatin-sensitive 37 kDa cysteine protease 37 might play this role.

Concluding remarks Only recently have experimental studies focused on parasite enzymes necessary for erythrocyte invasion. Although some of these activities have been identified, their precise targets and, consequently, their precise role in invasion have yet to be determined. Surface proteases clearly seem to be involved in merozoite surface protein maturation and red blood cell invasion. The observation that parasite enzymes are active in the serum has two implications: (1) the parasite enzymes are probably accessible to specific antibodies, some of which should be inhibitory; and (2) these enzymes should be different from host proteases since they are active in an environment where 10% of the proteins are protease inhibitors whose function is to limit the half-lifes of host proteases. This makes it theoretically possible to design specific inhibitors that will not affect host physiological processes but will prevent red blood cell invasion by malaria parasites. References 1 Dvorak, I.A. et al. (1975) Science 187, 748-779 2 Bannister, L.H. and Dluzewski, A.R. (1990) Blood Cells 16, 257-292 3 Wilson, R.J.M. (1990) Blood Cells 16, 237-252 4 Barnwell, J.W. and Galinski, M.R. (1991) Res. hnmunol. 142, 666-672 5 Aikawa, M. and Seed, T.M. (1980) in Malaria (Kreier, J.P., ed.), pp 285-344, Academic Press 6 Klotz, F.W. et al. (1989) Mol. Biochem. Parasitol. 36, 177-186 7 Banyal, H.S. et al. (1981) J. Parasitol. 67, 623-626 8 Hadley, T. et al. (1983) Exp. Parasitol. 55, 306-311 9 Dejkriengkraikhul, P. and Wilairat, P. (1983) Z. Parasitenkd. 69, 313-317 10 Dluzewski, A.R. et al. (1986) Exp. Parasitol. 62, 416-422 11 Braun Breton, C. et al. (1992) Proc. Natl Acad. Sci. USA 89, 9647-9651 12 Braun Breton, C. et al. (1988) Nature 332, 457-459 13 Schrevel, J. et al. (1990) Blood Cells 16, 563-584 14 Barale, J.C. et al. (1991) Res. Immunol. 142, 672-681 15 Heussen, C. and Dowdle, E.B. (1980) Anal. Biochem. 102, 196-202 16 Goldberg, D.E. et al. (1991)J. Exp. Med. 173, 961-969 17 Birch, N.P. and Peng Loh, Y.P. (1989) in Proteolytic Enzymes: a Practical Approach (Rickwood, D. and Hames, B.D., eds), pp 211-230, IRL Press 18 Freeman, R.R. and Holder, A.A. (1983) 1. Exp. Med. 158, 1647-1652 19 Delplace, P. et al. (1987) Mol. Biochem. Parasitol. 23, 193-201

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20 Holder, A.A. (1988) in Malaria Immunology (Perlmann, P. and Wigzell, H., eds), pp 72-97, Karger 21 Holder, A.A. et al. (1985) Nature 317, 270-272 22 Strych, W. et al. (1987) Parasitol. Res. 73, 435441 23 Heidrich, H.G. et al. (1989) Mol. Biochem. Parasitol. 34, 147-154 24 Pirson, P.J. and Perkins, M.E. (1985) J. Immunol. 134, 1946-1951 25 Lyon, J.A. and Haynes, J.D. (1986) J. Immunol. 136, 2245-2251 26 Blackman, M.J. et al. (1990)J, Exp. Med. 172, 379-382 27 Blackman, M.J. et al. (1991) Mol. Biochem. Parasitol. 49, 35-44 28 Blackman, M.J. et al. (1992)J, Cell Biochem. 16A, 120 29 Debrabant, A. et al. (1992) Mol. Biochem. Parasitol. 53, 89-96 30 Debrabant, A. and Delplace, P. (1989) Mol. Biochem. Parasitol. 33, 151-158 31 Beynon, R.J. and Salvesen, G. (1989) in Proteolytic Enzymes: a Practical Approach (Rickwood, D. and Harnes, B.D., eds), pp 241-249, IRL Press 32 Higgins, D.G. et al. (1989) Nature 340, 604 33 Mottram, J.C. et al. (1989) Nature 342, 132 34 Weidekamm, E. et al. (1973) Biochim. Biophys. Acta 323, 539-546

The Use of Electrophoretic Profile of RNA to Differentiate Trypanosoma brucei from T. congolense One of the major objectives in the control of African trypanosomiasis is the development of simple and specific diagnostic tests that can easily differentiate between subgenera or closely related parasite species and subspecies. The precise identification of the type of parasite involved in the infections is sometimes essential as it may strongly influence the treatment to be administered. Most efforts have been devoted to immunological tests such as the indirect immunofluorescent antibody test (IFAT), the enzyme-linked immunosorbent assay (ELISA) and the card agglutination test for trypanosomiasis (CATT); and to DNA technology diagnostics like the recombinant DNA probe hybridization and the polymerase chain reaction (PCR) amplification of the

Are Antibodies Important in Mice Infected with

Plasmodium yoelii ? A recent article by Sayles and Wassom ~ draws attention to the antibody response of mice infected with Plasmodium yoelii. It is stressed that strikingly different results may be obtained by using various combinations of inbred mouse strains and P. yoelii isolates. Thus, caution must be exercised in extrapolating findings from such studies to other models and to human malarias when attempting to propose a universally applicable mechanism for protective immunity to blood-stage malaria parasites. This can be exemplified by work on the two subspecies ofP. chabaudi. It has been shown both by ourselves2 using T-cell clones, and by Langhorne et o/.~ using

35 K6nigk, E. and Mirtsch, S. (1977) Tropenmed. Parasitol. 28, 17-22 36 Sherman, I.W. and Tanigoshi, L. (1983) Mol. Biochem. Parasitol. 8, 207-226 37 Deguercy, A. et al. (1990)Mol. Biochem. Parasitol. 38, 233--244 38 Braun Breton, C. et al. (199l) Exp. Parasitol. 74, 452-462 39 Portnoy, D.A. et al. (1992) Infect. Immun. 60, 1263-1267 40 Andrews, N.W. (1990) Exp. Parasitol. 71, 241-244 41 Mattei, D. et al. (1992) Parasitology Today 8, 426-428 42 Blackman, M.J. et al. (1991)Mol. Biochem. Parasitol. 49, 29-34 43 Braun Breton, C. et al. (1990) Res. Immunol. 141,743-745 44 Braun Breton, C. and Pereira da Silva, L.H. (1988) Biol. Cell 64, 223-231 45 Okoye, V.C.N. and Bennett, V. (1985) Science 227, 169-171 46 Friedman, M.J. et al. (1985) Science 228, 75-77 47 Miller, L.H. et al. (1983) J. Clin. Invest. 72, 1357-1364 48 Bennett, V. (1983) Methods Enzymol. 96, 313-324 49 Davies, K.A. et al. (1990) in Cellular and Molecular Biology of Norreal and Abnormal Erythroid Membranes (Cohen, C. and Palek, J., eds), pp 27-41, Alan R. Liss

parasite DNA using parasite species-specific primers or arbitrary primers (reviewed in Ref. I). Although there is sometimes a need for sophisticated technologies to differentiate between trypanosomes, some inter-genera genetic differences can be visible at the level of the molecular karyotypes 2 or even simply from the electrophoretic profile of total RNA. We have found that Tryponosoma congolense, on the one hand, and T brucei brucei, T brucei gambiense and T brucei rhodesiense, on the other, can be differentiated by comparison of the electrophoretic profile of their RNAs. This electrophoretic profile presents polymorphism of the large ribosomal RNAs that are characteristic for T. congolense and for T brucei type parasites, whereas no significant difference is observed between T brucei and the T wvax species (other species were not tested in this study because of the non-availability of the material). This approach is simple, and can be useful for discrimination between T congolense and the common African

limiting dilution cultures that both antibodydependent and antibody-independent effector mechanisms operate in immunity to P. chGbaudi chabaudi (incorrectly stated as P. chabdudi adami in Rett I). The relative contributions of these distinct pathways reflect the dynamic balance between the two subsets of CD4+ T cells at any given time. It has been argued4 that soon after challenge, when T. I cells predominate, a major mechanism for parasite destruction is via toxic mediators secreted by activated macrophages. Later on, as the proportion of T.2 cells increases, antibody-mediated immunity begins to operate, so facilitating resolution of patent infection. In this model, therefore, first T. I cells activate various non-specific immune mechanisms, such as nitric oxide production (A.W. Taylo~ Robinson etd., unpublished), to control a rapidly escalating acute parasitaemia (so precipitating the phenomenon of parasite

T brucei, since both the RNA purification 3 and gel electrophoresis (R. Peil~ and N.B. Murphy, unpublished) can be performed in less than three hours. (NB RNA prepared using either hot phenol or cesium chloride methods yielded similar results4.)

Acknowledgement I wish to thank Henrie Gathuo for his technical assistance. References

I Majiwa, P.A.O. et al. AgBiotech News and Information (in press) 2 Majiwa, PA.O. et al. (1986) Parasitology 93, 29 I- 304 3 Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156 159 4 Maniatis,T. et al. (1982) Molecular Cloning:A Laboratory Manual, Cold Spnng Harbor Laboratory Press Roger Pell~

InternationalLaboratoryfor Researchon Animal Diseases PO Box 30709, Nairobi Kenya

'crisis'). This paves the way for the T.2 promoted specific immunoglobulin response to effectively clear the blood infection. The dichotomy of response during a primary P. chdbdudi chabaudi infection is not matched by other murine malaria infections. Notably, immunity to P. chabaudl adami is characterized by a classical antibody-independent responseS. Recent reconstitution experiments using severe combined immunodeficient (SOD) and nude mice have demonstrated that T cells, but not B cells, are required for the resolution of P. chabaudi adami infection (data of H. Van tier Hyde, cited in Ref. 6). There is little or no apparent B-cell involvement. This contrasts with P. chabaudi chabaudi challenge of SCID mice7, which eliminate infection only after reconstitution with immune B cells. The dual nature of the host immune response to P. chabaudi chdbaudi infection