Aogenw1iy of Rypanosoines E C C

ARCHIV IDRC-132e LOSOS Aogenw1Iy of rypanosoines E C C

Proceedings of a workshop held at Nairobi, Kenya, 20 23 November 1978

litors: George Losos and Amy Chouinard IDRC- 132e

Pathogenicity of Trypanosomes Proceedings of a workshop held at Nairobi, Kenya, 20-23 November 1978

Editors: George Losos1 and Amy Chouinard2

Sponsored by Veterinary Research Department, Kenya Agricultural Research Institute, Muguga, Kenya

in collaboration with International Development Research Centre, Ottawa, Canada, International Laboratory for Research on Animal Diseases, Nairobi, Kenya, and Canadian International Development Agency, Ottawa, Canada

1IDRC project coordinator, Veterinary Research Department, Muguga. Kenya. 2Editor, Communications Division, IDRC, Ottawa, Canada. The International Development Research Centre is a public corporation created by the Parliament of Canada in 1970 to support research designed to adapt science and technology to the needs of developing countries. The Centre's activity is concentrated in five sectors: agriculture, food and nutrition sciences: health sciences: information sciences: social sciences: and communications. 1DRC is financed solely by the Government of Canada: its policies, however, are set by an international Board of Governors. The Centre's headquarters are in Ottawa, Canada. Regional offices are located in Africa, Asia, Latin America, and the Middle East.

Losos, G. Chouinard, A. Kenya Agricultural Research Institute, Veterinary Research Dept., Muguga KE IDRC, Ottawa CA International Laboratory for Research on Animal Diseases, Nairobi KE CIDA, Ottawa CA IDRC-132e Pathogenicity of trypanosomes: proceedings of a workshop held at Nairobi,

Kenya, 20-23 November 1978. Ottawa, Ont., IDRC, 1979. 216 p. :ill.

!IDRC publication!. Compilation of workshop papers on !trypanosomiasis/ particularly in /Africa south of Sahara! - discusses the !metabolism/ of the trypanosome !parasite!s, mechanisms of !disease transmission!, effects on !blood! and !serum/ !protein! levels in !cattle,', ,'immunology!cal aspects, !disease resistance!.

UDC: 616.937 ISBN: 0-88936-214-9

Microfiche edition available

2 Contents

Participants 5

Foreword B.L. Nestel 11 introduction Welcoming addressW. Masiga 13 Opening addressJ. Muliro 14 Vote of thanksB.L. Nestel 15 Theme and objectives of the conferenceL. Goodwin 16 The Organism The metabolism of African trypanosomes in relation to pathogenic mechanisms B.A. Newton 17 Biology and ultrastructure of trypanosomes in relation to pathogenesis K. Vickerman and L. Tetley 23 Biochemistry of variant antigensG.A.M. Cross 32 Cross-reacting determinants in trypanosome surface antigens A.F. Barbet, T.C. McGuire, A.J. Musoke, and H. Hirumi 38 Mechanisms of antigenic variation in salivarian trypanosomes J.J. Doyle, H. Hirumi, and A.L.W. de Gee 44 Genetic basis of antigenic variationR.O. Williams 46 Cyclical transmission and antigenic variationL. Jenni 49 Antigenic heterogeneity of bloodstream and metacyclic forms of T.hrucei J.D. Barry and S.L. Hajduk 51 Discussion summaryB.A. Newton and K. Vickerman 57 infections Infections caused by pathogenic African trypanosomesG.J. Losos 59 Rodent trypanosomiasesP. A. D'Alesandro 63 Parasitemia and host susceptibility to African trypanosomiasis M. Murray and W.I. Morrison 71 Immunity in the bovine to T.con golenseinduced by self-cure or chemotherapy B.T. WeIlde, W.T. Hockmeyer, R.M. Kovatch, andM.S.Bhogal 82 Trypanosomiasis of game animalsR. Olubayo 87 Discussion summaryF.E.G. Cox and G.A.M. Cross 89 Mechanisms of Cellular Injury: Blood and Circulatory System Is the anemia in bovine trypanosomiasis caused by immunologic mechanisms? H. Tabel, F.R. Rurangirwa, andG.J.Losos 91 Complement in experimental trypanosomiasis K.H. Nielsen, t.R. Tizard, and J. Sheppard 94

3 Biologically active lipids generated by autolysis of T. con golense I.R. Tizard, K.H. Nielsen, A. Mellors, and R.K.G. Assoku 103 Pharmacologically active substances in T. vivax infections D. Zwart and G.H. Veenendaal 111 Pharmacologically active substances in T. hrucei infections P.F.L.Boreham 114 Discussion summaryP.F.L. Boreham and F.E.G. Cox 120 Blood and Hematopoietic Tissue Responses Anemia of bovine African trypanosomiasis: an overviewM. Murray 121 Erythropoietic response in bovine trypanosomiasisJ.D. Dargie 128 Pancytopenia in bovine trypanosomiasis M.G. Maxie and V.E.O. Valli 135 Effect of bovine trypanosomiasis on hematopoiesis G.P. Kaaya, G.J. Losos, M.G. Maxie, and V.E.O. Valli 137 Effects of T. con golense and T. hrucei on the circulatory volumes of cattle J.D. Dargie 140 Hemodilution in bovine trypanosomiasis M.G. Maxie and V.E.O. Valli 145 Discussion summaryJ.D. Dargie and P.A. D'Alesandro 149 Lymphoid Tissue Responses Serum protein changes in bovine trypanosomiasis: a reviewH. Tabel 151 Lymphoid changes in African trypanosomiasis W.I. Morrison and M. Murray 154 Changes in the immune system during experimental African trypanosomiasis T.W. Pearson, G. Roelants, and W.I. Morrison 161 Immunosuppression of humoral immune response in bovine trypanosomiasis F.R. Rurangirwa, H. Tabel, and G.J. Losos 165 Discussion summaryL. Karstad and V.E.O. Valli 169 Tissue Lesions Pathogenesis of tissue lesions in T. brucei infections W.I. Morrison, M. Murray, and P.D. Sayer 171 Organ and tissue weights in diseases caused by T. vivax and T. congolense G.J. Losos and P.M. Mwambu 178 Pathology of T. con golense in calves V.E.O. Valli, C.M. Forsberg, and J.N. Mills 179 Ultrastructural changes in blood vessels of tissues of cattle experimentally infected with Trypanosoma congolense and T. vivax: a preliminary report P.M. Mwambu and G.J. Losos 184 Discussion summaryV. Houba and G.J. Losos . 186 Conclusions The trypanosome revisited: a summary of the conferenceL. Goodwin 187 References 189

4 The Organism

Biology and ultrastructure of trypanosomes in relation to pathogenesis K. Vickerman and L. Tetley

Department of Zoology, University of Glasgow, Scotland

Abstract. The powerfullocomotory flagellumandmicrotubule-reinforced body of the trypanosome endow it with the ability to inflict mechanical damage on its host by penetrating through or between cells. Pinocytosis from the flagellar pocket deprives the host of plasma proteins. Destructive lysosomal enzymes and the residual bodies of autophagosomes are discharged into the flagellar pocket. Antigenantibody complexes, which are believed to be at the root of much trypanosome pathogenesis, are produced when a host's antibody combines with either common antigens, which are released from residual bodies and from lysed trypanosomes, or variable antigens, which compose the surface coat of the trypanosome. The ability to replace one surface coat with another (antigenic variation) made of a different glycoprotein antigen enables the salivarian trypanosomes to avoid the host's immune responses.

We shall here consider the trypanosome asa are detectable in the host's blood, the peak and destructive piece of machinery, examining the remission of each wave being characterized by an working of its parts in relation to the different ways increasing percentage of nondividing short stumpy in which it harms its host. This parasite deprives its forms.Unlike theslender forms,the stumpy host of essential substances while burdening it with trypanosomes and those of intermediate morpholo- unwanted wastes;itinflicts mechanical damagegy are poorly infectiveto other mammals by through its incessant physical activity; and it goads syringe transfer but if ingested by the tsetse fly will the host into responding to its presence and then initiate the cycle in the vector. The differences in evades the backlash. These activities will be set infectivity accompanying trypanosome pleomor- against the background of trypanosome biology. phism correlate with changes in the trypanosome's single mitochondrion andcorrelated energy metabolism (Vickerman 1965, 1971). The stumpy Trypanosomes in Mammals forms partially activate the repressed mitochondrion and so become preadapted to life in the tsetse Incyclicallytransmitted trypanosomes(i.e., fly, where proline and other amino acids replace where the parasite has undergone multiplication glucose as the parasite's principal source of energy. and development in the vector), infection of the Chronic infectionsin the mammal are made mammal is initiated by the metacyclic stage, the possible by antigenic variation. Each parasitemic trypanosomes being injected with the saliva of the wave represents a population composed of trypano- tsetse fly (Salivaria) or contaminating the ruptured somes carrying a different surface antigen from epidermis from vector hindgut contents(Ster- those of the previous wave. Parasitemic remission coraria).In the tsetse-bome trypanosomes, the is induced by the host's immune response (Vicker- metacyclic trypanosomes develop further in the man et al.1976) to a particular variable antigen chancre that forms in the host's skin at the site of type (VAT). The stumpy forms are more resistant the bite. T. brucei metacyclics transform into longto immune assault than their slender progenitors thin slender forms that invade the local lymph(Balber 1972), but the small minority of slender vessels and then the bloodstream where theyforms that bear a different surface antigen - the multiply by binary fission. From the vascularheterotypes - evade the immune system's attack system the trypanosomes may invade the connec- altogether and survive to continue the infection. We tive tissues. Waves of trypanosome multiplication do not know what halts trypanosome division and

23 induces genesis of the stumpy forms, but theAlthough slender and broad forms have been transformation occurs in the absence of a detectable observed among bloodstream forms of T. cruzi, immune response (Balber 1972; Vickerman et al. evidence that the former may carry on the infection 1976). in the mammal and the latter serve to infect the InT. vivax and T. congolense, pleomorphismvector (Brener 1976), paralleling the situation in and distinct dividing and nondividing stages may beT. hrucei,isinsufficient at present.Strangely, present in the mammal host (Nantulya et al. 1978), respiratory differences between the different stages but mitochondrial changes have not yet been in its life cycle are quantitative rather than qualita- detected. Both trypanosomes are believed to betive (Gutteridge, Cover, and Gaborak 1978). hematic, i.e., confined to the vascular system and Demonstration of a common basis to the life not prone to invade secondarily the connective cycles of the trypanosomes of mammals isstill tissues (Losos and Ikede 1972). T. congolense lacking. The claim that T. brucei has anoccult'' certainly has an extensive primary developmental dividing amastigote (or spheromastigote) stage in phase in connective tissue of the chancre (Luckins the mammalian host during which antigenic varia- and Gray 1978), with distinct morphological forms tion occurs (Ormerod and Venkatesan 1972) has (reminiscent of the fly proventricular stage) (see not been confirmed. The brain-inhabiting forms of Roberts, Gray, and Gray 1969). The bloodstream T. hrucei that survive chemotherapy (Jennings et trypanosomes are attached to the endothelium ofal. in press) may simply be slender trypanosomes small vessels (Banks 1978; Büngener and Mullerlocated beyond the blood-brain barrier. The amas- 1976) according to a circadian rhythm (Hawking tigote and multinucleate flagellates may actually 1978). T. vivax is not known to be localized in this have littleroleinthelife cycle,as they are way. Antigenic variation is a feature of infectionsfrequently the products of degeneration or aberrant with both species. morphogenesis. Ruthless selection of viable forms Pleomorphism is rare in the mechanically trans- is probably a feature of all protozoan life cycles. mitted T. evansi and unknown in the venereally transmitted T. equiperdum. which is almost exclu- sivelya parasite of the connective tissuesin Functional Anatomy of a Trypanosome equines.Evidence from morphology and geo- graphicaldistributionsuggeststhatbothare The principalmorphologicalfeatures of a descendants of T. brucei (Hoare 1972); like the generalized salivarian bloodstream trypanosome parent species both undergo antigenic variation. are revealed by the electron microscope (Fig.1 and In contrast, stercorarian trypanosomes illustrate 2).Cortical (pellicular) microtubules, longitudi- the alteration of dividing and nondividing phases. nally disposed, lie immediately beneath the plasma T. lewisi undergoes multiple fission in the epimas- membrane of the flagellate's body and maintain its tigote form lodged in capillary vessels for 5 days elongate shape. The single flagellum arises from a following infection by metacyclics. Nondividing basal body (kinetosome) submerged in the floor of broad trypomastigotes are released into the blood the flask-shaped flagellar pocket. After leaving the but shortly afterwards are usually killed by aneck of the flask, the flagellum is attached to the trypanolytic antibody. A few of the trypomastigotes body between two cortical microtubules to become resist the attack and survive to infect the vector, but free at the flagellate's anterior extremity; there is no they are later demolished by a second trypanolytic projecting free flagellum in T. con golense nor in antibody. The peculiar immunoglobulin ablastin the advanced stumpy form ofT. brucei. The single (see D'Alesandro p. 63) is responsible for checking mitochondrion is associated with the locomotor division of T.lewisiin the blood, and, as inapparatus through a capsular expansion of the T. brucei, respiratory and mitochondrial changes former which houses the kinetoplast (Fig. 2). accompany the gross morphological changes of the The trypanosome's ability to move and its ability trypanosome (reviewed by Vickerman 1971). to alter its shape go hand in hand. Beating of the Whereas T. lewisi is capable of limited antigenic flagellum, and hence propulsion of the trypano- variation (implicit in the failure of the first lytic some, may proceed in either direction but usually antibody to erase all trypanosomes), T. cruzi does the waves pass from tip to base of the flagellum: not appear to have the capability at all, having ahence theflagellartipmarkstheorganism's pattern of development thatrendersvariation anterior end. A series of junctional complexes of unnecessary. In T. cruzi, division takes place in the desmosome type (see Brooks 1978) attach the intracellular amastigotes in muscle, mononuclear flagellum to the body and the body is deformed by phagocyte or nerve,sheltered from the host'sthe beating flagellum to give the characteristic immune assault. Nondividing trypomastigotes are undulating membrane appearance.Powerful released into the blood from the ruptured host cell. swimming movements combined with the Ion-

24 Flagellum-host attachment

Surface coat on body, flagellum, and plasmanemes

Flagellum attached Pellicular microtubules to body maintain shape and surface/volume relationship Lipid Glycosome

Polyphosphate

Coat synthesizing machinery Mitochondrion (rough er'doplasmic reticulum and Golgi apparatus)

Primary lysosome Nucleus

Exocytotic pathway (export of coated Heterophagy membrane - digestion of host to flagellar plasma proteins by pocket) lysosomal enzymes

Autophagy (digestion of isolated cystoplasmic islands by lysosomal enzymes and residual bodies voided into flagellar pocket; source of common antigens and excreted hydrolases)

Endocytotic pathway (pinocytosis of plasma proteins from flagellar pocket)

Plasmaneme (source Kinetoplast of exoantigen)

Fig. 1. Principal structures of a salivarian trvpanosome and their functions in relation to pathogenesis.

25 migratoryactivitiesinthe mammalian body trypanosomes can split apart junctional complexes binding endothelial cells, actively penetrate the endothelial cell membrane, or induce the cell to phagocytose and then exocytose them,isnot known. T.brucei procyclics appear to invade Glossina midgut epithelial cells (Evans and Ellis 1978) by activepenetration.T.cruzi blood trypomastigotes induce even "nonprofessional" phagocytes to engulf them (Nogueira and Cohn 1976), and the ability of T. cruzi actually to penetratecell membranes (e.g.,thatof the parasitophorous vacuole) is well established (Kip- nis, Calich, and DaSilva 1979). One uniquefeatureofthetrypanosomatid flagellum is its ability to form attachments to host surfaces. The ultrastructural details of the attachments to the chitin-lined regions of the vector's gut are now well known (reviewed by Molyneux 1977). Not so well characterized is the attachment of T. congolense to the walls of small (10-30 im diameter) blood vessels (Banks 1978). Electron micrographs of the attachment (Büngener and Muller 1976) have not as yet revealed the characteristic hemidesmosome-like arrangement of fila- ments (Vickerman 1973) converging on the flagellate's membrane in the attachment region, but interdigitations of the membranes of parasite and host have been reported. Attachment of trypano- somestophagocyticcellsalso seemstobe mediated initially by the flagellum (Stevens and Moulton 1978; Takayanagi, Nakatake, and Enriquez 1974). Macrophage cytophilic antibody induces attachment in vitro, but opsonizing antibody is necessary for engulfment of the parasite (Vasquez, Cattan, and Herbert 1975). When attached to host surfaces, the trypanosome flagellum continues to beat, and its movement may prevent stagnation of the medium surrounding the parasite in the vector. Its role in the free-swimming Fig. 2. Electron micrograph of longitudinal section of trypanosome is less clear. One likely possibility is posterior half of stumpy bloodstream form ofT. brucei: the flagellum (F) arising from the flagellar pocket (FP), that it circulates the contents of the flagellar pocket its basal body (BB) apposed to the kinetoplast (K)-bearing (Vickerman and Preston 1976). The pocket is an region of the single mitochondrion (M), the nucleus (N) important site of entry and exit of materials in bulk with itslarge nucleolus (NUC), rough endoplasmic (Fig.1), as the pellicular microtubules preclude reticulum (RER), the Golgi apparatus (G), and partially these activities elsewhere on the flagellate's sur- formed autophagosomes (AP). The body is supported by face. pellicular microtubules (PM), the flagellar pocket con- Pinocytosis of proteins by trypanosomes has tains exocytosed residual bodies (RB). Adjacent trypano- been observed by investigators using ferritin and some profiles show a polyphosphate vacuole (PV) and other electron-dense tracers that can be located by plasmanemes (PL). Fixation in glutaraldehyde, followed electron microscopy. Details of the relationships by osmium tetroxide, staining with uranyl and lead. between pinocytosis vesicles and lysosomes con- gitudinal reinforcement of the slim body by pel- taining digestive enzymes (recognized by their licular microtubules and lateral deformability en- positivereactioninGomori stainingfor acid able the trypanosome to penetrate between cells and phosphatase) have been elucidated by Langreth and into cells. Details of how penetration is effected, Balber (1975). Ferritin is taken up in spiny vesicles however, are surprisingly sparse. Whether in their (Fig.I) pinched off from theflagellar pocket

26 lining.These vesiclesfuse with an elaborate (Langreth and Balber 1975). The cytoplasm to be network of smooth-membraned cisternaethat destroyed is first walled off by membrane (probably appear to store the protein. Primary lysosomes from supplied by the adjacent Golgi complex) to form an the Golgi complex fuse with parts of this network autophagic vacuole (Fig. 2) with which primary presumably digesting its contents. In some stages lysosomes fuse. Amino acidsreleased from of the life cycles of certain trypanosomes, e.g., the autophagic vacuoles may be utilized as an energy intracellular amastigote and culture epimastigotesource in the stumpy form, which is activating its forms of T.cruzi,but never Salivaria, a specialized proline oxidase system. cytostome and cytopharynx are present to mediate The undigested remains of heterophagic and such "heterophagy." Theprotein sequesteringautophagic vacuoles are cast out into the flagellar system of T.bruceiis so well developed that thepocket along with their accompanying lysosomal uptake of host serum protein might be envisaged as enzymes (Fig.I,2, and 3). This exocytosis of taking place on a massive scale in relation to the residual bodies may be of considerable significance size of the organism. Serum albumin has long beenin relation to pathogenesis. Trypanosome lyso- recognized as a prolonger of the activelife ofsomes contain acid proteases, leucine aminopep- bloodstream trypanosomes in vitro and might betidase, phosphatases,phosphodiesterases, expected to be consumed avidly in vivo. Quantita- deoxyribonucleases, -mannosidase,/3-hex- tive estimates of its uptake, however, have provedosaminidase, and lipase (Venkatesan, Bird, and surprisingly low - 700 ng/h/mg tiypanosomeOrmerod 1977: Steiger 1975) as well as phos- protein for monomorphic (slender) bloodstream T. pholipases (Tizard et al.1977). Their destructive rhodesiensethough this figure was double for a effects have not been fully investigated, but Tizard pleomorphic line (A.H. Fairlamb personal com- et al.p.l03 have explored phospholipase and its munication), implying greater activity on the part role in the generation of lytic fatty acids. of the stumpy trypanosomes. The residual bodies exocytosed by T.bruceimay Autophagy the digestion of isolated islands of be quite largeinrelationtothesize of the cytoplasm containing unwanted organelles also trypanosome (Fig.3).They may embody a appears to be more active in the stumpy forms mechanism for release of common (i.e., nonvari-

Fig. 3. Electron micrograph of longitudinal section of the distended flagellar pocket (FP) of T. brucei (bloodstream stumps' form) containing large residual body (RB), which has the structure of a liposome, i.e., several concentric membrane layers. Surface coat on the membrane lining the pocket and covering the residual body as well as the trvpanosome surface increases the apparent thickness of the membrane at this low resolution. Other labeled structures are the basal body (BB). pellicular microtubules (PM), and the kinetoplast (K).

27 able) antigens before the wholesale liberation ofthe plasma membrane properisoverlaid by a these agents, which occurs when trypanosomes are surface coat of glycoprotein, 12-15 nm thick as destroyed by lytic antibody at a crisis. Discharge of seen in electron micrographs of sections (Vicker- these bodies as thick streamers from the flagellar man1969).CompactanddenseinT. canal has been described by Ellis, Orrnerod, andbrucel and T. congolense, this coat is somewhat Lumsden (1976). diffuse in T.ivax. An even more diffuse coat is The functional significance of many trypano- present in the stercorarian trypanosomes such as T. some cytoplasmicinclusionsstilleludesus. lewisi and T. cruzi bloodstream forms. The coat Trypanosomes have no means of storing energy asappears to be an adaptation to life in the mam- carbohydrate. These flagellatessequestertri- malian host, for it is discarded in the vector until it glyceride (Dixon, Ginger, and Williamson 1971),is reacquired at the metacyclic stage of develop- but it seems quite probable that, in the absence of ment.In the Salivaria,itcontains the variable an exogenous supply of respirable substrate, pro- antigen (VA) of the trypanosomes (Vickerman tein (of either internal or external origin) provides I969b: Vickerman and Luckins 1969; Cross 1975; the only source of energy. Our recent X-ray Fruit et al.1977), and antigenic variation occurs microanalysis studies (Vickerman and Tetley 1977) when one coat is replaced with another of different of dense inclusionsthatlieto one side of a antigenic type (reviewed by Cross 1978a: Vicker- thick-membraned vacuole (Fig. 2 and 3) in try-man 1978). The way in which replacement is panosome cytoplasm suggest thattheseare instigated eludes us, but we believe that the change polyphosphate,buttheir rolein trypanosome isphenotypic(i.e.,geneticmutationisnot physiology is uncertain. Although they may func- required), that it occurs in only a small number of tion as phosphagens, i.e., to store phosphate bond individuals (heterotypes)in a given population energy when ATP production exceeds demand, (approximately 1in 10 000), and that antibody is they probably serve as phosphate reserves. The notnecessaryforinduction of theswitchto calcium and zinc associated with these granules expressing a new VA glycoprotein. suggest that they play a partin regulation of Cytochemical,especiallylectin-binding, divalent cation concentrations in the cytoplasm - a techniques indicate that in T. brucei the carbohy- function that can be vitally important in unicellular drate moieties attached to the single polypeptide organisms. The mitochondrial cycle of the trypano- chain of the variable antigen glycoprotein are somes may be related to sequestration and release located close to the membrane proper (Wright and of divalent cations, as the ability of mitochondria to Hales 1970; Steiger 1975: Cross and Johnson 1976; performthisfunctioniswell known. The Seed, Seed, and Brindley 1976; Renwrantz and pathological significance of polyphosphates dis- Schottelius 1977) and may even play a part in charged from the trypanosome on lysis deserves attachment of the molecule tothe membrane. investigation,asthese polyanionsareutilized Whether inallvariable antigen types of the commercially as detergents. subgenus Trypanozoon it is unexposed and plays no One cytoplasmic organelle whose function has part in hostparasite interactions remains controv- recently come to light is the glycosome (previously ersial (Baltz, Baltz, and Pautrizel 1977). In T. referred to as a microbody or peroxisome-likecongolense (Rovis, Barbet, and Williams 1978: organelle by Vickerman and Preston 1976). Opper- Jackson, Honigberg, and 1-bIt1978), T.vivax does and Borst (1978) have shown thatin (Tetley unpublished observations), and the ster- bloodstream T. brucei, it houses enzymes for the corarian trypanosomes (Dwyer 1976: Dwyer and breakdown of glucosetoglycerol-3-phosphate D'Alesandro 1976), carbohydrate groups appear to (G-3-P) and 3-phospho-glycerate (3-PG), which be exposed on the surface coat rather than con- pass out into the cytoplasm. G-3-P is oxidized by cealed. molecular oxygen in the outer membrane of the The ease with which variable antigenis dis- mitochondrion and the dihydroxyacetone phosphate sociated from the surface of the trypanosome in produced is shipped back to the glycosome for vitro suggests that itis a peripheral protein, i.e., recycling. The 3-PG is converted to pyruvate in the has weak attachment to the membrane. In addition, cytoplasmic matrix, with linked phosphorylation ofthe VA molecules appear to be mobile over the ADP, and the pyruvate is excreted by the trypano- surface of the trypanosome in that they can be some. The contribution of excreted pyruvate tocapped by homologous antibody in the indirect pathogenesis is discussed in Newton p.17. immunofluorescence reaction (Barry in press) and One of the most important trypanosome compo- are lost uniformly from the surface as shown by nents in any discussion of pathogenesisisthe electron microscopy and immunofluorescence surface membrane the interface between parasite studies on the trypanosomes transforming to culture and host. In bloodstream African trypanosomes, forms in vitro (Barry and Vickerman in press). The

28 '4 ' w$.

Fig. 4. Electron micrograph of replica offreeze-fractured Tbrucei(cultured procyclic form). The fracture plane has passed along the centre of the plasma membrane of the body (right) and the attached flagellum (left), revealing intramembranous particles (IMPs, integral proteins or lipoprotein complexes) that are embedded in the lipid bilayer. The extracellular face (EF) of the body membrane is shown and the protoplasmic face (PF) of the flagellar membrane. The clustering IMPs (a band running the length of the PF) represent the desmosome-like attachments of the flagellum to the body. Arrow indicates direction of shadowing of the replica. strong antigenic character of the VA may be due to or'filopodium-like appendages'' (Wright, its release from the trypanosome either on its entire Lumsden, and Hales 1970; Vickerman and Luckins surface, perhaps after combination with antibody, 1969). These fine extensions of the body and or on those enigmatic structures the plasmaneines flagellum (Fig. 1) are readily observed on trypano-

29 4

I Fig. 5. Electron micrograph of a replica ofa freeze-fractured T. vvax (bloodstream form), which has been subjected to etching before shadowing. The protoplasmic face (PF) of the trypanosome body and the extracellular face (EF) of the flagellum plasma nembrane are visible with their scattered intramembranous particles. In the etching process some of the covering ice is sublimed away to disclose the true surface of the membrane adjacent to the internal faces exposed by fracturing. The rough surface coat (SC) is visible because of the retreat of the ice table (IT). somes in vitro by light and electron microscopy, blood cells would provide a major source of but definite proof of their formation in circulating antigenantibody complexes to account for vas- blood is still lacking. Plasmanemes themselves are cular damage. highly immunogenic (Herbert and MacAdam Organized surface glycoproteins on flagellates 1971), and their attachment to endothelial or red are usually secreted by an active rough endoplasmic

30 reticulum-Golgi complex system, with externaliza- electron microscope level and the freeze-fracture tion of new surface membrane bearing the secretionreplica technique. Differentiated regions of the close to the flagellar bases. The existence of such a surface of salivarian trypanosomes may prove to be well developed secretory apparatus between the discernible using similar methods. nucleus and flagellar pocket (Fig. 1 and 2) strongly In the freeze-fracture technique, frozen cells are suggests that a similar pathway exists in trypano- cleaved; their membranes tend to split along the somes (Vickerman 1969). Owing to presence of the middle of the lipid bilayer, the fracture plane pellicular corselet of microtubules, it is difficult to passing over and under integral proteins. The see where new coated membrane could be inserted proteinsthenstandoutas intramembranous into the surface other than in the flagellar pocket. particles" (IMPs) when metal-shadowed replicas The possibility thatsecretioniscontrolled by are made of the exposed fracture faces (Fig. 4 and lysosomal enzymes (asin the coccolithophorid5). The true surface of the membrane for flagellates)(Pienaar 1971) also exists. Secretion ofexample the surface coat of bloodstream trypano- a mitogen by salivarian trypanosomes has been somes can be revealed by freeze-etching, i.e, suggested (Esuruoso 1976: Mansfield, Craig, andsubliming away surface ice adjacent to the fractured Stelzer 1976) and that variant antigen itself has aregion of membrane (Fig. 5). No regular packing of mitogenic effect has been postulated. coat glycoprotein units has been observed. The Thereis now convincing evidence that thedensity of intramembranous particles visible (sum- soluble "exo-antigen" believed by Weitz (1960) toming counts made from both faces of the split be a secretion is surface coat material liberatedmembrane) is - 3 x l03/zm2 for bloodstream from the trypanosomesintothesurroundingT. brucei (Vickerman and Tetley 1977), whereas medium (Allsopp, Njogu, and Humphreys 1971).the density of coat glycoprotein molecules was Whether the material is also liberated in vivo fromcalculated by Cross (1975) to be 105Im2 so that viable trypanosomes is a matter of considerable coat molecules outnumber IMPs 20 to I. interest to pathologists who are investigating com- The proteins(or lipoprotein complexes) rep- plement activation by antigenantibody complexesresented by IMPs are probably heterogeneous in and its role in inflammatory disease (see Boreham function as well as size. Most noticeable of the p.114). Musoke and Barbet (1977) found thatIMPs are those thatliein clusters along the purified (soluble) variable antigen of T. brucei flagellum-body junction (Fig. 4) the clusters pre- activated complement via the classical pathway,sumably corresponding with the desmosomal but a particulate fraction from the same clone ofattachments of the flagellum (for further details see trypanosomes activated the alternate pathway of Smith etal.1974: Hogan and Patton1976; complement fixation.Thisfractionprobably Vickerman and Tetley 1977) and the particles with included thesurface membrane proper of the the intramembranous portions of molecules that trypanosome.Uncoated trypanosomes,asrep- bridge the gap, i.e., protrude beyond the surface resented by culture forms, are susceptible to lysis coat. There may be similar membrane differentia- by normal serum. In T. cruzi lysis appears to be due tions, for example, where T. con golense attaches to to the activation of complement via the alternatehost endothelium or where non VAT-specific pathway (Nogueira, Bianco, and Cohn 1975). It cytophilic antibody binds the tiypanosome to a seems likelythatthe trypanosome membranemacrophage. We are confident that in the not too proper has complement-activating components that distant future the combination of freeze-etching in the intact flagellate are inaccessible owing to the with techniques for the labeling of specific mem- barrier presented by the surface coat (see Nielsen, brane components will result in significant infor- Tizard, and Sheppard p.94). mation on the organization of the trypanosome In the short term, the glycoprotein coat protects surface and, indirectly, lead to a deeper under- vulnerable membrane proteins (such as substrate standing of the intricate relationship between para- transport sites) from complement components and site and host. in the long term the coat provides a replaceable surface enabling the trypanosome to evade the host's immune response. But that the variable antigens are the only proteins exposed on the Acknowledgments surface of the bloodstream trypanosomesis unlikely. De Souza and colleagues (1978) have We wish to thank the U.K. Ministry of Overseas demonstrated differentiated regions on the surface Development for the research support grants and Maureen of T.cruzi using cytochemical staining at the Gardner for skilled electron microscope assistance.