differentiation in trypanosoma brucei: host-parasite … · ture of antar 1 (non-infective to man,...

J. Cell Sci. 74, 1-19 (198S)Printed in Great Britain © Company of Biologists Limited 1985

DIFFERENTIATION IN TRYPANOSOMA BRUCEI:

HOST-PARASITE CELL JUNCTIONS AND THEIR

PERSISTENCE DURING ACQUISITION OF THE

VARIABLE ANTIGEN COAT

L. TETLEY AND K. VICKERMAN*Department of Zoology, University of Glasgow, Glasgow G12 8QQ, UJC.

SUMMARY

Acquisition of the variable antigen-containing surface coat of Trypanosoma brucei occurs at themetacyclic stage in the salivary glands of the tsetse fly vector. The differentiation of the metacyclictrypanosome in the gland has been studied by scanning electron microscopy and by transmissionelectron microscopy of thin sections and freeze-fracture replicas. The uncoated epimastigotetrypanosomes (with a prenuclear kinetoplast) divide while attached to the salivary gland epitheliumbrush border by elaborate branched flagellar outgrowths, which ramify between the host cellmicrovilli and form punctate hemidesmosome-like attachment plaques where they are indented bythe microvilli. These outgrowths become reduced as the epimastigotes transform to uncoatedtrypomastigotes (with postnuclear kinetoplast), which remain attached and capable of binaryfission. The flagellar outgrowths disappear but the attachment plaques persist as the uncoatedtrypomastigotes (premetacyclics) stop dividing and acquire the surface coat to become 'nascentmetacyclics'. Coat acquisition therefore occurs in the attached trypanosome and not, as previouslybelieved, after detachment. Coating is accompanied by morphological changes in the glycosomesand mitochondrion of the parasite. Freeze-fracture replicas of the host-parasite junctional com-plexes show membrane particle aggregates on the host membrane but not on the parasite membrane.It is suggested that disruption of the complex occurs when maximum packing of the glycoproteinmolecules has been achieved in the trypanosome surface coat, releasing the metacyclic trypanosomeinto the lumen of the gland.

INTRODUCTION

One of the most crucial stages in the transmission of trypanosome infections is thedevelopment of the metacyclic trypanosome in the vector. In Trypanosoma brucei,the species responsible for human sleeping sickness as well as disease in cattle, thisdevelopment takes place in the salivary gland of the tsetse fly (Glossina spp.). Hereepimastigote trypanosomes (with a prenuclear kinetoplast), attached to the host'sglandular epithelium, transform to trypomastigote metacyclic forms (with apostnuclear kinetoplast); the mature metacyclic trypomastigotes lie free in the glandlumen, ready to be discharged with the saliva when the fly bites a mammal. Thedischarged metacyclic trypanosome resembles all the mammal stages of the parasitein that it possesses a 12-15 nm thick surface coat on both body and flagellum (Vicker-man, 1969). This coat is made up of a monomolecular layer of glycoprotein, which

•Author for correspondence.

Key words: Trypanosoma brucei, flagella, junctional complexes, surface coat.

2 L. Tetley and K. Vickerman

represents the variable antigen of the trypanosome (Vickerman & Luckins, 1970;Cross, 1975; Fruit et al. 1977). By expressing different variable antigen genes thetrypanosome can change the antigenic character of its surface and so evade the host'simmune response (Vickerman, 1978; Borst & Cross, 1982). The coat is absent fromthe epimastigote predecessors of the metacyclic: uncoated trypanosomes are non-infective as they readily fall prey to the mammalian host's non-specific defensemechanisms (Mosser & Roberts, 1982; Ferrante & Allison, 1983). As the metacyclictrypanosomes of a clone infection are heterogeneous with respect to variable antigentype (Le Ray, Barry & Vickerman, 1978), the generation of diversity of variableantigen type must occur during differentiation of the metacyclic forms from theepimastigotes.

Although there has been some chronicling of the ultrastructural events accompany-ing acquisition of the surface coat and infectivity during metacyclic differentiation(Vickerman, 1969; Steiger, 1973), detailed study has been retarded by the difficultiesinherent in securing the tsetse salivary gland stages (as fly infection rates in thelaboratory are notoriously low) and in obtaining satisfactory fixation of this material.These difficulties have now been largely surmounted in this laboratory and we presenthere an account of metacyclic differentiation with emphasis on host-parasite attach-ment in relation to acquisition of the surface coat.

MATERIALS AND METHODS

Infection of tsetse flies with trypanosomesTrypanosomes of the AnTAR 1 and ETAR 1 serodemes were used to infect tsetse flies, Glossina

morsitans morsitans, as described previously (Hajduk, Cameron, Barry & Vickerman, 1981). Flieswere monitored for infection by allowing them to probe saliva onto warm glass slides and recordingthe presence of mature metacyclic trypanosomes. Salivary glands from positive flies were removedinto Cunningham's (1977) medium supplemented with 10% heat-inactivated foetal calf serum andexamined by phase-contrast microscopy to ascertain the extent of infection.

Electron microscopy

For transmission electron microscopy (TEM), heavily infected glands were transferred tofixative at room temperature (20 °C) and immediately cut into four or five segments to facilitateaccess of fixative to the attached trypanosomes. Egress of saliva from the gland segments allowedcollection of unattached forms in fixative and small pellets of these flagellates were detained bycentrifugation (1000# for 10 min) for further processing and ultrastructural comparison withattached stages.

For scanning electron microscopy (SEM) an infected gland was pinned onto 'Sylgard' siliconrubber (Dow Chemicals Ltd) in a pool of Cunningham's medium and slit along its length from theblind end to the salivary duct using a micro-knife fitted with a fractured razor blade; saliva wasflushed gently from the exposed infected epithelium with fresh medium before fixation of the glandin glutaraldehyde.

Primary fixation of gland pieces was in 2-5% glutaraldehyde in 0- lM-phosphate buffer containing10mM-CaCl2 at pH 7-4 for 1-2 h at 20°C. Overnight storage in 0-lM-phosphate buffer containing2 % (w/v) sucrose was followed by several rapid changes of this solution and then gradual introduc-tion of 4 % (w/v) aqueous osmium tetroxide solution to give a final OsC>4 concentration of 2% inthe buffered sucrose solution.

After 1-2 h post-fixation and several changes of distilled water to remove residual osmium ( 3 x 5min), specimens for TEM were stained en bloc with 0-5 % aqueous uranyl acetate for 30 min and

Differentiation in Trypanosoma brucei 3

routinely dehydrated in an ethanol series followed by propylene oxide infiltration and embeddingin Araldite.

Blocks polymerized in silicon rubber moulds for 48 h at 50 °C were cut on an LKB Mkl Ultratometo give sections of 60-80 nm thickness range (as judged by interference colours). These were eithermounted on 300 mesh copper grids or Formvar-coated 3 mm X 1 mm slot grids for serial reconstruc-tions. All sections were contrasted with uranyl acetate and lead citrate and images recorded on IlfordEM4 cut film using an AEI 801 transmission electron microscope operating at 60 kV.

For SEM, infected glands were transferred post-osmication into a retaining chamber comprisingtwo stainless steel meshes separated by a Teflon spacer and housed in a brass fitting to facilitatedehydration in acetone and critical-point drying from CO2. Dried glands were attached to stubs withdouble-sided Sellotape and sputter coated with gold to 20-40 nm thickness. The specimens wereexamined in a Philips 500 SEM operating at 12 or 25 kV.

Freeze-fracture proceduresAfter primary glutaraldehyde fixation, salivary gland pieces were rinsed in 0-1 M-phosphate buffer

and gradually infiltrated over 2-3 h with 25 % glycerol in the same buffer, at 20 °C. Individual piecesmounted in 25 % glycerol on Balzers' copper support plates (flat) were sandwiched in a thin liquidlayer by placing a second support plate over the first and then the assembly was rapidly cooled inliquid propane held at near — 180°C. Such preparations were stored under liquid nitrogen untilrequired.

Fracturing was accomplished at -100 °C and 2 X 10"6 Torr (1 Torr = 133-3 Pa) with a com-plementary replica device (Sleytr & Umrath, 1974) installed in a diffusion-pumped vacuum systemfitted with a liquid nitrogen baffle and a temperature-controlled specimen table, also cooled withliquid nitrogen. Conventional 45° platinum/carbon and 90° carbon evaporation from resistancesources gave replicas, which were cleaned with 40% chromic acid and picked up from distilledwater, after several changes, on 200-mesh copper grids. The matching of complementary replicaswas facilitated by retrieving each on individual referenced grids (Polaron, G200FZ).

Inordertocomparedistributionof/3-hydroxy sterols in the membranes of different developmentalstages, glutaraldehyde-fixed salivary gland pieces were incubated in 50/igml"1 filipin (Upjohn, agift from Dr T. E. Grady) dissolved in dimethylsulphoxide (DMSO) and phosphate buffer (0-5%DMSO final concn) for 4—8h at 20 °C in the dark. After several buffer washes over 30 min thespecimens were infiltrated with 25 % glycerol in phosphate buffer and freeze-fractured as describedabove.

RESULTS

Light microscopy, scanning electron microscopy, and transmission electron micro-scopy of thin sections have enabled us to distinguish four different stages in thedevelopment of T. brucei in the salivary gland of Glossina morsitans. These are: (1)the epimastigote; (2) the premetacyclic trypomastigote; (3) the nascent metacyclic;and (4) the mature metacyclic trypomastigote, which is discharged by the vector withits saliva on feeding. Stages (1) — (3) are attached to the salivary gland epithelium;stage (4) lies free in the gland lumen.

No obvious difference was discerned using any technique between the ultrastruc-ture of AnTAR 1 (non-infective to man, T. brucei brucei) and ETAR 1 (infective toman, T. brucei rhodesiense) trypanosomes.

Transmission electron microscopy of thin sections

Epimastigotes. In the epimastigote form of the trypanosome the kinetoplast liesanterior to the nucleus and the flagellate multiplies by binary fission while attachedto the salivary gland epithelium. The luminal border of the epithelium is uneven and


covered with tangled microvilli, each approximately 1-1-5 fim long. It is to these thatthe trypanosomes attach.

A detailed description of the ultrastructural features of the epimastigote stage of theAfrican trypanosomes has been given for T. vivax (Vickerman, 1973) and theT. brucet epimastigote differs most strikingly from this in the nature of its attachmentto the vector, as T. vivax epimastigotes attach to the chitinous lining of the tsetse foodcanal. As in T. vivax, the body and flagellar membranes are uncoated and attachmentto the host is by the flagellum; in T. brucet, however, the flagellar membrane andunderlying sheath are greatly expanded to form arborescent outgrowths, whichpenetrate between the supporting host microvilli and embrace them, bringing theparasite into intimate contact with the apical surface of the host epithelial cell (Figs1,2,18). Where microvilli indent the branched flagellar outgrowths, local attachmentplaques approximately 100-130nm in diameter are visible; fibrillar electron-densematerial lines each cup-like plaque only on the flagellar membrane side of the junc-tion ; there is no plaque on the confronting host cell membrane. A gap of approximate-ly 20 nm separates the host and flagellar membranes (Fig. 3). Where the parasitemembrane is in sustained contact with host cell apical membrane, focal plaquespunctuate the flagellar membrane at regular intervals (40-50 nm); there is no con-tinuous attachment 'hemidesmosome' as there is in the junction between the T. vivaxflagellum and the chitinous lining of the tsetse fly proboscis. The body membrane ofthe epimastigote does not appear to attach to host surfaces. The tentacular outgrowthswith their attachment plaques are absent from the recently emerged flagellum andappear to be predominantly a feature of its distal end, though less marked on its 'free'(non-body-attached) terminal portion; they arise largely from the peri-axonemalsheath (Fig. 2), but not infrequently from that half of the flagellar sheath that sur-rounds the paraxial rod; the membrane of the free portion of the flagellum may formjunctional complexes around its entire perimeter.

Figs 1-10, 11 and 12 are of AnTAR 1; Figs 9 and 13-17 of ETAR 1 trypanosomes.Fig. 1. Tangential section through apical region of microvillar border of tsetse salivarygland cell with attached flagella of T. brucei epimastigotes. Three profiles of trypanosomeflagellar shafts (f\,fi,fz) are visible, each showing extensive branched outgrowths (fo) ofthe flagellar sheath. The outgrowths penetrate between the microvilli (BID) and focalattachment plaques (ap) are visible where the microvilli indent the flagellar outgrowths.Electron-dense material lines each cup-like plaque only on the flagellar membrane side ofthe junction. The flagellar membrane of/2 has been traced in black to indicate more clearlythe extent of branching in this section. X42 000.

Fig. 2. Transverse section of epimastigote showing flagellum with outgrowth (fo) andseveral attachment plaque junctions (ap) with adjacent host microvilli (mv). Both flagellarand body surface membranes of the epimastigote lack a surface coat, though a thin unevenprecipitate of salivary protein covers all visible surface membranes. The trypanosomemitochondrion (m) shows the tubular cristae characteristic of the epimastigote stage.X48800.

Fig. 3. Detail of junctions between epimastigote flagellum (/) and host cell (he). Note thefibrillar plaque regions (ap) on the flagellar membrane where it is indented by host cellmicrovilH (mv). The gap between confronting parasite and host membranes is consider-ably wider between plaques (large arrowheads) than in the plaque region (smallarrowheads). X150000.

Differentiation in Trypanosoma brucei

Figs 1-3

L. Tetley and K, Vickerman


Other ultrastructural features of the epimastigote include the branched mitochon-drion (usually two strands are visible in the pre-kinetoplastic portion of the body andat least two in the post-kinetoplastic portion alongside the nucleus) with its crowdedtubular cristae (Fig. 2), and the dense bacilliform glycosomes (Fig. 4).

Premetacyclic trypomastigotes. Scattered among the attached epimastigotes areflagellates, which, because they have a postnuclear kinetoplast and flagellar origin canbe identified as trypomastigotes, but they differ from the mature metacyclictrypomastigotes and resemble epimastigotes in that they are uncoated, patentlycapable of division (Fig. 6), and are attached. Flagellar outgrowths are again present,but noticeably shorter and not as well developed as in the epimastigote. Attachmentplaques are abundant but mainly along the membrane of the flagellar shaft (Fig. 3).The mitochondrion is still branched in the pre-kinetoplastic part of the body and theglycosomes are bacilliform.

Nascent metacyclics. Profiles of coated trypomastigotes whose flagella lacktentacular outgrowths but retain the attachment-plaque junctional complexes withthe microvillar membrane are frequently encountered in sections (Fig. 7). In theircytoplasmic organization these flagellates resemble mature metacyclics in that the pre-kinetoplastic mitochondrion is a single strand with sparser ampulliform cristae andthe glycosomes are spherical (Fig. 5), less electron-dense and not bacilliform. Divid-ing stages of these coated attached trypomastigotes have not been observed. In all buttheir attached flagella then, these flagellates are metacyclic trypanosomes and are hereinterpreted as 'nascent metacyclics', i.e. trypanosomes that have recently acquired thecoat but have not yet been released.

A notable feature of these nascent metacyclics is the persistence of an extension ofthe flagellum beyond the anterior tip of the body - the so called 'free flagellum', whichis reputedly absent or greatly truncated in the mature metacyclic (Hoare, 1972). Inthe nascent metacyclic this flagellar tip either lies attached by focal junctions amongthe host microvilli or is inserted into the apical cytoplasm of the host cell (Fig. 8).Whether embedding of the flagellar tip occurs before or after coating is unknown butinserted uncoated flagella have not been observed.

Mature metacyclics. These lie free in the lumen of the salivary gland. They have neverbeen observed in division. Their flagella bear no traces of junctional complexes otherthan the desmosome-like junctions between trypanosome body and flagellum. Themitochondrion and glycosomes are essentially similar to those of the nascent metacyclic.

Fig. 4. Bacilliform glycosomes (g) of epimastigote stage showing dense homogeneouscontents. X78000.

Fig. 5. Part of section of metacyclic trypanosome showing spherical glycosome (g) withless-dense contents characteristic of trypomastigote stages that bear a surface coat (sc).m,mitochondrion. X78 000.

Fig. 6. Longitudinal section of uncoated dividing trypomastigote (pre-metacyclic). Noteelongate nucleus (n) with spindle microtubules, passing from pole to pole (poles arrowed),two kinetoplasts (k) and profiles of branched mitochondrion (m). The morphology of theglycosomes (g) suggest that these organelles resemble those of the uncoated epimastigoterather than those of the coated (metacyclic) trypomastigotes./, flagellum. X26250.

WlsL. Tetley and K. Vickerman

•** '


Fig. 9. Scanning electron micrograph of carpet of trypanosomes attached to salivary glandepithelium. Epimastigotes (e) have anterior insertion of the flagella (ei) and long drawn-out posterior extremities. Premetacyclic trypomastigotes show posteriorly inserted flagellaand retain the pointed extremity, which may be short (pm\) or long (pmz), depending onthe stage in development. Nascent metacyclic trypanosomes (nra) have a blunt posteriorend. X6700.

Scanning electron microscope observations

Owing to dense crowding of the flagellates in the carpet of parasites lining thesalivary gland, and too-deep penetration of the microvillar layer by flagella, details ofattachment (including the arborization of the epimastigote flagellum) cannot bevisualized with the SEM. The different stages in development of the metacyclic,however, can be distinguished by their posterior ends (Fig. 9). During the transition

Fig. 7. Transverse section of anterior extremity of attached coated trypomastigote(nascent metacyclic). Attachment plaques (ap) are still present where host cell microvilli(mi;) indent the flagellar membrane but outgrowths are not present on the metacyclicflagellum ( / ) . sc, surface coat; m, mitochondrion. X92000.

Fig. 8. Oblique longitudinal section through 'free flagellum' (jf) and part of body ofnascent metacyclic trypanosome. The flagellum is inserted beyond the microvillar (mv)border into the cytoplasm of a host epithelial cell (he). Evenly spaced attachment plaques(ap) occur throughout the region of parasite-host cell membrane contact (compare withattachment plaques {ap') of adjacent epimastigote flagellar outgrowths), m, mitochon-drion; pmt, pellicular microtubules of trypanosome body. X66000.


from epimastigote to metacyclic, progressive shortening of the distance from flagellarbase to posterior end occurs and the acute angle formed by the posterior extremity inprofile increases. In the nascent and mature metacyclic the posterior extremity isblunt and not acute.

Freeze-fracture observations

Survey views of replicas with the transmission electron microscope at low magnifi-cation confirm the branched nature of epimastigote flagellar outgrowths and showthem penetrating between irregularly shaped host cell microvilli (Figs 10,13).Attachment-plaque sites with interposed plaque-free areas are seen as conspicuousconcavities (PF) and convexities (EF) in cleaved flagellar membranes and occur ingroups along the length of attached flagella. Trypanosome body membranes and theflagellar membranes of luminal flagellates lack these plaques.

Fracture faces of the surface membranes of attached epimastigotes differ strikinglyin their intramembranous particle (IMP) distribution from those of maturemetacyclic trypanosomes found free in the lumen of the gland. During the trans-formation from epimastigote to metacyclic trypomastigote the density of IMPs in thePF becomes reduced, while there is a slight reduction in IMP density in the EF (Figs11,12). The cleaved flagellar membranes of both forms show a reduced IMPdistribution compared with their respective trypanosome body membranes (Figs13,14). Trypanosome attachment plaques are of uniform size (100 nm diameter) asseen in replicas of cleaved flagellar membrane and largely devoid of IMPs on bothfracture faces.

The cleaved host cell membrane, however, shows patches with regimented lineararrays of IMPs on the PF (Figs 14,15); these arrays correspond in size to the con-cavities/convexities of the flagellar membrane attachment plaques and in somereplicas (Fig. 14) can be seen to be apposed to them. PFs of the basal region of thehost cell microvillar border may show several IMP arrays, confirming that indentationof the flagellar membrane by microvilli is not necessary for the generation of attach-ment plaques. Such PFs show that the regular IMP arrays are composed of particlesof a particular size class (lOnm) and clearly distinct from larger (16 nm) unaggregatedparticles also present in the host membrane PF (Fig. 15). Particle aggregates are not

Figs 10-16 are of freeze-fracture replicas. The direction of shadowing is from bottom totop of each micrograph.Fig. 10. Survey view of microvillar border of infected tsetse salivary gland as seen infreeze-fracture replica preparation. Fracture faces of numerous flagella and host cellmicrovilli (mv), pointing in different directions, are visible. Attachment plaques areevident as circular concavities (at arrowheads) in the flagellar membrane PF (PFf) and asconvexities (arrowed) in the EF (EFf). he, cytoplasm of host cell; sgl, salivary glandlumen. X30000.

Figs 11, 12. Fracture faces of surface membranes of bodies of mature metacyclic (coatedtrypomastigote; Fig. 11) and epimastigote (uncoated; Fig. 12) to show differences inintramembranous particle distribution, x 100 000. Particle counts (with S.D.). CoatediiF,1096 ± 150 fim-2 ( n=10) ; PF, 4872 ± 397 ̂ m"2 (w=10). Uncoated: EF, 1697 ±159/un"2 (n = 14); PF, 7814 ± 873 /im"2 (n = 7).


sgl

\ t

Figs 10-12


Figs 13-14. For legend see p. 14.


Figs 15-17. For legend see p. 14.


found in the freeze-fractured microvillar membrane of uninfected salivary glands.Complementary replicas of the flagellar membrane attachment plaques (Fig. 17)confirm that the particle arrays are entirely absent from the cleaved parasite mem-brane. Filipin treatment of attached trypanosomes results in characteristic lesions ofthe flagellar membrane (Fig. 16) except at sites of flagellar attachment plaques,suggesting that cholesterol is largely excluded from these regions.

DISCUSSION

Eukaryotic flagella and cilia are primarily contractile organelles involved in celllocomotion or in the propulsion of fluid over cells, but in protists the flagellar or ciliary-membrane may play a part in recognition processes, especially in mating reactions asis now well established for Chlamydomonas spp. (Goodenough & Thorner, 1983) andthe ciliate Paramecium (Miyake, 1981). The all-parasitic trypanosomatid flagellatesappear to be unique in that they have evolved the ability to modify the flagellum asan attachment organ and, in at least one phase of the life cycle, they multiply whileattached to a host surface. This surface may be the inert chitinous lining of the hindgutor proboscis of the arthropod host or the living microvillar border of its midgut,Malpighian tubule or salivary gland epithelium. Although the host in question isusually an arthropod, Trypanosoma congolense has the ability to attach to capillary

Fig. 13. Fractured junction of epimastigote flagellum and microvillar border. The frac-ture plane exposes the EF of part of the host cell membrane (EFHc) and the PF of partof the apposed flagellar shaft (PFf) and some of its outgrowths (Jo) between host microvilli(mv). The indentations (arrowed) that mark the sites of junctional complexes are relativelyfree of intramembranous particles. X56 000.

Fig. 14. Fractured body (tb) and (unbranched) flagellum (f\) of nascent metacyclic; theflagellum shows indented (attachment plaque) and non-indented regions. The line- ofextracellular particles (at arrows) is believed to represent the surface coat. Note differencein IMP density between PFs of body (PFtb) and flagellum {PFf) (suggesting differencesin composition although both bear the glycoprotein coat) and relative paucity of IMPs onmetacyclic flagellar PF compared with that of epimastigote (Fig. 13). In lower right-handcorner note array of IMPs (arrowhead) on host microvillus PF at site of indentation ofadjacent flagellum (fi) (attachment plaque), mv, host cell microvillus. X75 000.

Fig. 15. Fractured basal region of microvillar border of host epithelial cell to whichflagellates are attached. The exposed PF of the intermicrovillar host membrane (PFhc)shows clustered regular aggregates of intramembranous particles (arrowed) at sites ofhost-parasite attachment plaques. A residual fragment of adhering parasite membrane(large arrowhead) shows no corresponding aggregates in its EF. Stumps of microvilli arevisible (mv). X72000.Fig. 16. Fractured flagellum of metacyclic trypanosome after filipin treatment. Charac-teristic filipin-induced lesions (arrowed) indicating jS-hydroxysterol-rich areas of theflagellar membrane are visible on the PF. Such lesions are lacking on the EF convexitiesthat correspond to parasite attachment plaques, suggesting that the membrane of theplaque region is less rigid than that of the rest of the flagellum. X37 000.

Fig. 17. Complementary replicas of the PF and EF of part of a trypanosome flagellumattached to salivary gland microvilli (mv). Note the absence of particle arrays (such as arefound in host cell membrane PF) at sites of parasite attachment (arrowed) in complemen-tary PF and EF. X67000.


vessel endothelia in its mammalian host (Banks, 1978). Sometimes these attachmentsoccur between the flagella of adjacent parasites so that attachment to the host surfaceis indirect (Vickerman, 1973). Flagellar beating continues unimpaired in suchattached forms and may help to circulate the surrounding medium about the multiply-ing parasites, or, in some cages drive this medium through the flagellar pocket fromwhich pinocytosis occurs (Vickerman & Preston, 1976).

It is uncertain how specific these trypanosomatid flagellar attachments are. ThusCrithidia fasciculata will adhere to the chitinous intima of the mosquito hindgut(Brooker, 1971a) in vivo, but in vitro it will attach to Millipore filters (Brooker,19716). The epimastigote stage of T. congolense, which multiplies while clinging tothe chitinous wall of the food canal in the tsetse proboscis will attach to the wall ofplastic culture flasks in vitro (Gray et al. 1981). On the other hand, the specificityshown by attachment of the flagella of certain species of Leptomonas for the water andsalt-transporting rectal ampullae as opposed to other chitinized regions of the hindgut.of their insect hosts is quite remarkable (Lauge & Nishioka, 1977). Those flagellatesthat attach to living host cells, however, have not been cultivated on artificial sub-strates and it is possible that either growth of the attached phase in the life cycle ormaturation of the free-swimming phase that develops from it is dependent uponexchange between parasite and host cell.

The ultrastructure of the hemidesmosome-like attachments formed where theflagellar membrane contacts an inert substratum has been described for severaltrypanosomatid species by numerous investigators (reviewed by Vickerman &Preston, 1976; Molyneux, 1977), but attachments to living host microvilli have beenpoorly characterized. No hemidesmosomes were discerned with microvilli for Leish-mania mexicana in Lutzomya longipalpis (Killick-Kendrick, Molyneux & Ashford,1974) or Trypanosoma melophagium in Melophagus ovinus (Molyneux, 1975).Steiger (1973) briefly described the punctate attachment plaques of T. brucei epi-mastigote flagella with their insect host's microvilli, but not the remarkable dendroidflagellar outgrowths that also play a part in attachment. He envisaged coating takingplace after release of the trypanosome from its mooring.

We report here that the acquisition of the variable antigen-containing surface coatoccurs while the trypanosome is still attached and that this non-dividing nascentmetacyclic stage is preceded by a dividing uncoated trypomastigote (the premeta-cyclic), which arises from the main multiplicative stage in the salivary gland (theepimastigote). The recognition of these different stages in metacyclic differentiation(Fig. 18) is crucial to the problem of the generation of trypanosome variable-antigendiversity in the vector's salivary glands and to the interpretation of data on thispopulation heterogeneity (Le Ray et al. 1978).

In our experience, mature (coated) metacyclics only are discharged from the fly'ssalivary gland; it is possible, however, that the practice in some laboratories of squeez-ing the abdomen of the fly to force it to discharge more trypanosomes will result indislodgements of attached uncoated trypanosomes. This practice should therefore beavoided as it unnecessarily complicates the picture of metacyclic variable-antigen type(VAT) heterogeneity as studied using the immunofluorescence reaction. The ability


•Attached to microvillar honk-r of salivary gland epithelium

Flagellaroutgrowths withattachmentplaques

Free in gland lumen—|

Variableantigencoat

NASCENT-METACYCLIC-I EPIMASTIGOTE 11- PREMETACYCLICHI METACYCLIC 11— METACYCLIC—I

I—Prenuclear kinetoplast—11 Postnuclcar kinetoplast 1

-Uncoated, dividing- -Coated, non-dividing- H

f- -Branched mitochondrion-

—Bacilliform glycosomes—

-Unbranched mitochondrion-

Spherical glycosomes

Fig. 18. Schematic diagrams of stages in development of mature metacyclic fromepimastigote via premetacyclic and nascent metacyclic stages, showing principal mor-phological changes taking place during differentiation.

to recognize nascent metacyclics in sections of the vector salivary gland will be usefulin answering the question, 'is VAT heterogeneity present ab initio or do all nascentmetacyclics initially acquire a coat of the same antigenic type so that heterogeneityarises as a consequence of antigenic change in the mature metacyclic population?'Electron immunocytochemical studies on nascent metacyclics in situ are in progresstowards this end.

The host-parasite junctional complex

Junctional complexes described to date between cells of the same organism may beclassified as adhering (desmosome, hemidesmosome), impermeable (tight, septate)and communicating (gap, synapse) junctions. The punctate junctions described hereare most probably of the adhering type, performing an anchoring function anddistributing any shearing forces through the salivary gland epithelium. Each time thetsetse fly feeds, muscular contraction of the wall of the salivary glands to expel saliva


will create a shearing force along the microvillar border, threatening to dislodge thetrypanosome.

The flagellar attachment plaques differ from hemidesmosomes in that there is littleevidence for a tono6bril-like filament system connecting the plaques to one anotheror to the main cytoskeletal elements, in this case the axoneme-paraxial rod complex.This absence may reflect lack of preservation. In T. vivax (Vickerman, 1973) andT. congolense (Evans, Ellis & Stamford, 1979; Thevenaz & Hecker, 1980) the belt-like hemidesmosome that attached the flagellum to the chitinous lining of the tsetseproboscis does appear to show such connections to particular doublets of the axoneme(6,7,8,9 and 1). Similarly, the spot desmosome-like junctions that bind the flagellumto the body in all trypanosomes show a filamentous 'Y' connection between theflagellar plaque and axonemal doublet 7 and the paraxial rod (Vickerman, 1969).Comparable connectives between the axoneme or paraxial rod and surface mas-tigonemes of the flagella of euglenid flagellates have been demonstrated (Melkonian,Robenek & Rassat, 1982).

In 71. brucei the efficiency of anchoring is increased by the great expansion ofthe flagellar membrane, so that many more attachment plaques can be accom-modated on the dendroid outgrowths and attachment can be effected to a largenumber of microvilli in the plane of shear. Although limited flowing of the flagellarcortex into branches can be observed in T. vivax and T. congolense attached toinert substrata (cf.Thevenaz & Hecker, 1980; Gray et al. 1981), the extraordinaryscale on which branching occurs in T. brucei has no known parallel among othertrypanosomatids. In the related biflagellate kinetoplastid family, Bodonidae, how-ever, comparable branching of the anterior flagellum for the purpose of attachmenthas been described for Cryptobia helicis in the spermatheca of pulmonate snails,though no differentiated junctional complexes were seen in these attachments(Current, 1980).

It is interesting to note that these junctional complexes persist in the trypanosomeflagellum during the acquisition of the surface coat, so that the forces that bind hostand parasite membranes together must endure, at least initially, the intervention ofthe parasite glycoprotein coat. Barry (1978) has presented evidence that the variableantigen glycoprotein is freely diffusible in the plane of the surface membrane of thetrypanosome and that during coat acquisition the density of packing of the glyco-protein molecules increases over the entire surface of the flagellate. We postulate herethat the achievement of maximum glycoprotein packing disrupts the bond betweenhost and parasite. It is equally possible, however, that metabolic changes taking placein the trypanosome during metacyclic maturation results in products that disrupt theunusual junctional complex, liberating the mature metacyclic into the lumen of thegland.

We thank Dr A. M. Jordan, Tsetse Research Laboratory, Bristol, for supplying tsetse fly pupae,Miss C. R. Cameron and Dr S. L. Hajduk for maintaining infected flies, and Mr P. Tindall for helpin fly microdissection.

This work was supported by grants from the U.K. Overseas Development Administration andthe Medical Research Council to Professor K. Vickerman.


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{Received 30 July 1984 -Accepted 6 September 1984)

differentiation in trypanosoma brucei: host-parasite … · ture of antar 1 (non-infective to man,...

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