Molecular basis of antigenic variation in trypanosomes

49

TIBS- March 1978

lower risk to the experimentor than less familiar isolates. This is fortunate as, for the biochemist, T. brucei has one overwhelming advantage over other species: it causes acute infections in laboratory rodents and high yields can be obtained. After a 3-day infection, T. brucei can account for 30", of the packed cell volume. Furthermore, due to charge differences, T. brucei may be rapidly and completely separated from all host blood cells

Molecular basis of antigenic variation in trypanosomes G. A. M. Cross Sequential expression of alternative antigens enables African tr)'panosomes to evade the immune response. Characterisation of surface glycoproteins exposes the molecular basis of antigenic variation. The trypanosoma and members of related genera form a ubiquitous group of unicellular spindle-shaped parasitic protozoa characterised by four principal structural features: a single flagellum, a complex microtubular skeleton underlying the plasma membrane, a single but often highly branched giant mitochondrion and an unusually structured mass of mitochondrial DNA - the so-called kinetoplast. There are several species responsible for the various forms of animal and human trypanosomiasis ('sleeping sickness') in Africa. In contrast to the situation in the South American form of trypanosomiasis (Chagas' disease), where the causative agent (Trypanosoma cruzi) multiplies inside the cells of its human host, the African trypanosomes have no known intracellular stage. They multiply by binary fission in the vascular system and the tissue interstices. Transmission of African trypanosomes from one mammalian host to another is mediated principally by members of the genus Glossina (tsetse). Other bloodsucking insects may be implicated as vectors in some circumstances. It is generally believed that many animals in the wild can tolerate trypanosome infections without overt pathological complications but most domestic livestock are extremely susceptible to disease. Trypanosomiasis is a severe veterinary problem affecting around 4 million square miles of Africa. The three species primarily responsible are Trypanosoma congolense, vivax and brucei. G.A.M.C. moved recently from the MRC Biochemical Parasitology Unit in Cambridge to head the Department of Immunochemistry at the Wellcome Research Laboratories. Beekenham. Kent. U.K.

It is an understatement of some magnitude to say that the drugs currently available for treating animal and human trypanosomiasis are far from ideal Human trypanosomiasis is less prevalent than the animal disease but nevertheless constitutes a serious risk to the health of 35 million people [1]. The names of the causative agents, T. rhodesiense and T. gambiense, reflect the apparent geographical dominance of acute and chronic forms in East and West Africa respectively. T. rhodesiense and T. gambiense cannot yet be distinguished with certainty from T. brucei. Until the basis for host range specificity or an entirely reliable differential test has been established, animal trypanosomes of the brucei type must be regarded as potentially pathogenic to humans. Despite this caveat, some isolates of brucei-type trypanosomes have been loosely handled in laboratories for many years without experiencing infection and can therefore be considered to present a

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More natural trypanosome infections may run for several months and are often characterised by distinct waves of parasitaemia (Fig. l) in which successive parasite populations are antigenically distinct. Antigenic variation appears to be the primary mechanism for parasite survival in an immunocompetent host. Variable antigens eharacterised Antigenic variation can be largely if not entirely explained by the sequential expression of alternative genes specifying a major class of surface glycoproteins. The repertoire of alternative glycoproteins which may be expressed by a single trypanosome clone is so far undetermined but seems likely to exceed 50. The total number of variants circulating in Nature could be very much higher. For this reason, empirical approaches to vaccination have been unsuccessful. Cell surface labelling techniques facilitated the definitive identification and purification of variable antigens [5,6]. Following simple mechanical disruption in the absence of each antigenically distinct and homogeneous detergents, population of T. brucei yields a characteristic soluble glycoprotein which is the major if not the sole component of the surface coat. The surface coat (Fig. 2) is an additional characteristic feature of the mammalian stage of the life cycle of African trypanosomes [7]. Removal of the surface coat with trypsin is paralleled by release of glyco-

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50

TIBS- March 1978

Fig. 2. Tran.~verse section through the hod)' and Ilagellum O[ T. brucei illustrating surlace structure. The bar represents 0.2 Ira1.

protein fragments without resulting in immediate cell lysis [8,9]. The glycoprotein accounts for about 10",, of total cell protein - a massive amount compared to the major surface antigens of other cells. The variant glycoproteins appear to comprise a single polypeptide chain of 65000 apparent molecular weight on SDSpolyacrylamide gels. Glycoprotein antigens purified from a range of variants representing several clones or isolates of T. bruceido not appear to differ significantly •in molecular weight. This may not be true for other species. Preliminary results reported for T. evansi [6] suggest a higher molecular weight, a subunit structure and lower solubility. We must be cautious where lower molecular weights are reported, owing to the particular sensitivity of some variant glycoproteins to proteo~ytic degradation. The C-terminal end of the polypeptide appears to be especially susceptible to degradation [9,10]. Sialic acids are absent from the variable glycoproteins. The oligosaccharide components differ from one variant glycoprotein to another, both in total amount (7-17°, by weight), in their attachment sites on the polypeptide backbone and in the proportions of the four sugar components - galactose, glucose, mannose and glucosamine [I 1]. Periodate oxidation of sugars, whilst destroying the precipitability of antigens (in agar gels) by concanavalin A, did not inhibit precipitation by antisera [9]. Carbohydrate therefore does not appear to be the major antigenic determinant, although it would be exlJected to display some antigenicity. Preliminary structural studies strongly indicate that the immunological uniqueness of each glycoprotein is a reflection of immense variation in amino acid sequence. The isoelectric points and amino acid compositions of immunologically distinguishabl,, glycoproteins differ greatly [5,6]. An exlensive study of amino acid sequences hasbeen initiated. Preliminary results [10], comparing the N-terminal amino acid sequences of antigens from four variants isolated during a 3-week period from a rabbit infected with a clone of T. brucei, are reproduced in Fig. 3. These sequences,

which represent only 5". of the polypeptide, show no homology and the amino acids are present in proportions which represent the overall glycoprotein compositions. Variation appears not to be restricted to the N-terminal end but it is too early to say whether regions of conserved structure exist elsewhere in the molecule. This is the focus of current studies. Antigen organisation on the cell surface The surface area of T. brucei was experimentally estimated to be similar to that of an erythrocyte [5], suggesting that a 65000 molecular weight glycoprotein comprising 10",, of the total cell protein (7-10 x 106 molecules/cell) could form a closelypacked monolayer covering the entire trypanosome surface. This hypothesis is supported by experiments which suggest that the surface coat is sufficiently dense to prevent significant penetration of a molecule the size ofconcanavalin A [8,9]. I have previously suggested [8,12] that a major function of the variable glycoprotein coat could be to act as a barrier preventing immunoglobulins or other secondary components of the immune response from attacking underlying invariable integral membrane antigens. The variable antigens themselves elicit a very strong immune response. Experiments in which concanavalin A binding to cells was quantitated by radioactive methods and localised by cytochemical techniques suggested that an essential fraction of the carbohydrate is at1 1

tached to an area of the glycoprotein close to the membrane attachment site [8,9]. The mechanism of glycoprotein attachment to the cell surface is not yet apparent but seems likely to involve non-covalent binding to a membrane receptor rather than deep penetration into the lipid bilayer [9]. Other mechanisms are possible but their discussion at this stage could only amount to speculation. Self aggregation of glycoproteins, which occurs with certain bacterial surface components, did not occur in our preliminary experiments but should perhaps be further investigated. Could there be any significance in our observation that approx. 20"0 of the 65000 molecular weight surface glycoprotein is not solubilised on cell breakage? The entire 65 000 component is however destroyed by trypsinisation of intact cells. So far, no one has looked at trypanosome surface components other than the variant glycoprotein. The predominance of this component makes it impossible to pick out any significant additional bands from straightforward cell labelling experiments but it should not be difficult to devise alternative approaches. The overall structural simplicity of T. brucei suggests'it should be a relatively simple matter to purify surface membranes. Control of antigen expression The genetic basis for antigenic variation is unexplored [12]. The only certainty is that variation is primarily phenotypic and each cell contains structural genes specifying a large number of variant glycoproteins. The unusual aspects of trypanosome mitochondrial (kinetoplast) DNA have, until recently, eclipsed any interest in nuclear DNA, but this situation may soon be reversed. Isolation of trypanosome antigen genes should present no special technical problems. A recent report described the first steps in isolating purified messenger RNA and its translation into antigen in vitro [13]. Antigenic variation is a feature of the bloodstream stages ofthe trypanosome life 10

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Fig. 3. N-terminal amino aeid ~equences O[four variant antigens [10].

T I B S - March 1978 cycle, When bloodstream trypanosomes are ingested by Glossina, or transferred to in vitro culture at 250-28 `' C, variant antigens are not expressed and the surface coat is absent. Infectivity for the mammalian host is lost. After a period of multiplication in the insect midgut, trypanosomes invade the salivary glands where they regain the surface coat and their infectivity. Current debate on control mechanisms is centred on two questions. Firstly, is the sequence of antigen expression constant within one clone and does reversion to a definitive initial variant type occur during cyclical transmission through Glossina? Secondly, what is the mechanism for triggering variation and what role does the immune response play? These two points have been discussed elsewhere in more detail [12,14] and I am reluctant to speculate further in the absence of fresh and strictly .controlled experimental data. Available evidence is consistent with the hypothesis that variant genes may be activated in sequence but that, at any point in an infection, the phenotypically predominating variant may be determined by several interacting aspects of the host-parasite relationship which combine to determine the relative virulence ofdifferent variants. Antigenic variation is minimal when trypanosome clones are maintained in severely immunosuppressed mice [14]. This observation does not resolve the basic question of whether intrinsically programmed variation occurs constantly at a low level in trypanosome populations and the immune response merely exerts a selective effect by successively eliminating the predominant variant, or whether antibodies (or other extrinsic factors) may induce individual trypanosomes to undergo antigenic variation. New approaches to the study of cellular control mechanisms will be possible following a recent breakthrough which allows bloodstream-type T. brucei expressing the variant antigens to be cultured in vitro for the first time [15]. Antigenic variation is a fascinating and far from academic problem. It is acknowledged [I] that a detailed understanding of this phenomenon is essential to a rational evaluation of the feasibility of vaccination and the development of improved diagnostic techniques. References I De Raadt, P. (1976) World Health Organization Documentation for Special Programme for Research and Training in Tropical Diseases

[TDR/WP/76.12] 2 Lanham, S.M. (1968) Nature (London) 218, 1273-1274. 3 Lanham, S.M. and Godfrey, D.G. (1970) Exp. ParasitoL 28, 521-534 4 Ross. R. and Thomson, D. (1910) Proc. R. Sac. Land. Ser. B 82, 411-415 5 Cross, G.A.M. (1975) Parasitology 71,393-417

51 6 Cross, G.A.M. (1977) Ann. Sac. Beige Med. Trap. in press 7 Vickerman, K. (1969) J. Cell. Sci. 5, 163-194 8 Cross, G. A. M. and Johnson, J.G. (1976) in Biochemistry of Parasites and Host-Parasite Relationships (Van den Bossche, ed.) pp. 413-420

9 Johnson, J.G. and Cross, G.A.M., manuscripts in preparation 10 Bridgen, P.J., Cross, G.A.M. and Bridgen, J. (1976) Nature (London) 263, 613-614 11 Johnson, J.G. and Cross, G. A. M., J. Proto'.ool. in press

12 Cross, G.A.M., Proc. R. Sac. Land. Set. B in press 13 Eggin, M.J., Tappenden, L. and Brown, K.'N. (1977) Parasitology75, 133-141 14 Doyle, J.J. (in press) in Blood-borne Parasitic Diseases (Pino, J., Miller, L. and MeK¢lvey, J., eds) Plenum Press, New York 15 Hirumi H., Doyle, J.J. and Hirumi, K. (1977) Science 196, 992-994

Cholesterol in membranes: studies with mycoplasmas Shmuel Razin and Shlomo Rottem Mycoplasmas, unique among the prokaryt~tes in requir&g cholesterol./or growth, ore most usefitl too[s .]'or studyhlg the tran.~'f'er of cholesterol./'ram serum lipoprote&s to the cell membrane, the IDealization o[ cholesterol h7 the membrane attd its role as a reguhttor o f membrane fhtidit3'. The role played by cholesterol in biological membranes and the factors controlling its incorporation are currently subjects of great interest. A recent hypothesis [I] suggests that the increased incorporation of cholesterol into the plasma membrane of the arterial intima cells triggers the formation of the atherosclerotic lesion. However, the factors which cause cholesterol accumulation and the resulting damage to membrane function are ill-defined. Studies with artificial membrane systems made of phospholipid-cholesterol mixtures have advanced our knowledge of the interactions of cholesterol with phospholipids and provided some clues to the possible role of cholesterol in biological membranes [2]. Yet, valid conclusions must depend on studies with the more complex biological membrane systems, and of these the mycoplasmas appear to be the simplest. Properties of mycoplasmas The mycoplasmas belong to the class Mollicutes (soft-skin micro-organisms) established in 1967 to include all the wallless prokaryotes. Though these prokaryotes are prevalent in Nature, only a small fraction of them has been cultivated, identified and classified. Presently, the class Mollicutes contains only one order, Mycoplasmatales, and three established families, Myeoplasmataceae, Spiroplasmataceae, and Acholeplasmataceae. The organisms included in the first two families Tile authors are tit the Biomemhraoc Research Lahoratorl'. Oepal'DllCtlt of C/ilti~'al MicrohiohJg.v. The Hehre w Lhlirersit.v-Hu&tssah Medical Schm~l. Jerusalent. Israel

and classified in the genera Mycoplasnla, Ureaplasma, and Spiroplasma require cholesterol for growth, whereas those in Acholeplasmataceae do not share this requirement [3]. The trivial term 'mycoplasmas' used in this review includes all the organisms classified in the order Mycoplasmatales. The mycoplasmas are the smallest known organisms capable of autonomous growth and reproduction, with the diameter of some of the coccoid cells being as small as 0.3 pm [3]. These organisms are bound by a plasma membrane only and, unlike other prokaryotes, lack cell walls and mesosomes. This is most useful in membrane studies, for once the plasma membrane is isolated, it is not contaminated by other membrane types. Due to the lack of cell walls, mycoplasmas resemble bacterial protoplasts and erythrocytes in their sensitivity to osmotic lysis. This gentle technique can thus be employed to lyse the cells and isolate the membranes in large quantities and in a very pure form. Roughly speaking, two-thirds of the mass of the isolated mycoplasma membrane is composed of protein and the balance is almost entirely lipid [3]. The structural simplicity of mycoplasmas is accompanied by a relative biochemical simplicity in that these minute organisms, all of which are parasites of animals, plants, and insects, lack many of the biosynthetic systems found in the more familiar bacteria such as Escherichia coll. This biosynthetic deficiency may be correlated to the finding that the genome of mycoplasmas is only one-fifth to two-fifths the size of that of E. coll. Mycoplasmas require complex growth media, therefore,