Disruption of synchrony between parasite growth and host cell division is
a determinant of differentiation to the merozoite in Theileria annulata
BRIAN SHIELS1*, JANE KINNAIRD1, SUSAN McKELLAR1, JOANNE DICKSON1, LEILA BEN
MILED2, RON MELROSE2, DUNCAN BROWN2 and ANDY TAIT1
1 Wellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, University of Glasgow, Glasgow G61 1QH, Scotland,UK2Centre for Tropical Veterinary Medicine, University of Edinburgh, Roslin, Scotland, UK
•Author for correspondence
Summary
The multinucleated macroschizont stage of the proto-zoon Theileria annulata is an intracellular parasite ofbovine leukocytes. The parasite induces the host cell toproliferate, and divides in synchrony with the immorta-lised host cell. Differentiation to the next stage occurswithin the host cell culminating in the release ofmerozoites and destruction of the leukocyte. In thisstudy clones of Theileria annulata macroschizont-infected cell lines were isolated by (uniting dilution andtested for differentiation to the merozoite stage (mero-gony). Two cloned cell lines underwent differentiationwith enhanced efficiency, while two others were of lowerefficiency. Quantification was carried out using mono-clonal antibodies, which showed that over 90% of thecells in an enhanced cloned cell line could be induced todifferentiate. By carrying out induction at 41°C forlimited periods of time followed by culture at 37°C
evidence was obtained that differentiation to the mero-zoite is a two-step process: a preliminary reversiblephase, followed by a second irreversible phase ofdifferentiation. Analysis of the nuclear number of themacroschizont and the growth rate of the cloned celllines showed that the ability to differentiate wasassociated with an increase in nuclear number (size) ofthe macroschizont, generated by a disruption in thesynchrony between parasite growth and host celldivision. We believe that these results reveal a relation-ship between a reduction in parasite division anddifferentiation, and that there are similarities betweenstage differentiation in parasites and cellular differen-tiation in higher eukaryotes.
Differentiation of a cell frequently occurs after changesin the cellular environment. In higher eukaryotes thesechanges can be caused by an alteration in the level ofpolypeptide growth factors, which regulate the prolifer-ative activity of cellular populations (Cross and Dexter,1991). Differentiation of protozoan parasites from onelife-cycle stage to another can also occur in response toenvironmental changes, particularly during the tran-sition from the invertebrate vector to the vertebratehost. The molecular mechanisms that regulate parasitestage differentiation have not been fully elucidated,although it has been postulated that an increase in heatshock gene expression is involved (Van Der Ploeg etal., 1985).
Theileria annulata is a protozoon parasite of cattleand is the causative agent of tropical theileriosis, adisease of livestock in areas of Europe, North Africaand Asia (Purnell, 1978). The bovine phase of the life
cycle (Fig. 1) is initiated by inoculation of thesporozoite stage with a feeding tick. The sporozoiteinvades mononuclear leukocytes and differentiatesthrough the trophozoite to the macroschizont stage(Jura et al., 1983). Macroschizont-infected host cellsbecome immortalised, and division of parasite and hostcell occurs in synchrony (Hulliger, 1965), the parasiteassociating closely with the microtubules of the host cellspindle to achieve distribution of the schizont to thedaughter leukocytes. Differentiation to the merozoitestage (merogony) takes place within the host cell withthe generation of merozoite nuclei, rhoptry andmicroneme organelles (Melhorn and Schein, 1984).Differentiation culminates in the destruction of theleukocyte and the release of merozoites into theextracellular environment. Free merozoites sub-sequently invade erythrocytes, where they mature intopiroplasms (Conrad et al., 1985). Completion of thecycle occurs with the uptake of infected erythrocytes bythe tick vector.
100 B. Shiels and others
Sporozoites inoculated by feedingtick invade mononuclearleukocytes
Differentiation to multinucleatedmacroschizont stage.Immortalisation of infected hostcell. Parasite and host cell dividein synchrony
Differentiation to merozoitestage. Destruction of host cell,release of merozoites
Merozoites invade erythrocytesand differentiate into piroplasms.Uptake of infected erythrocytesby feeding ticks
Fig. 1. Schematic outline of T. annulata stagedifferentiation in the bovine host.
In a previous study differentiation of the macroschi-zont to the merozoite was induced by raising thetemperature of the culture to 41°C. Reactivity ofmacroschizont-infected cells and merozoites with apanel of monoclonal antibodies demonstrated that thisprocess results in significant changes in the antigenicprofile of the parasite, indicating that merogony is amajor point of differentiation in the mammalian phaseof the parasite life cycle and that both positive andnegative regulation of gene expression occur during thisprocess (Glascodine et al., 1990). These experimentswere carried out with a macroschizont-infected cell linederived by infection of peripheral blood mononuclearcells with sporozoites from a parasite stock. It has beenshown that parasite stocks, and the macroschizont celllines derived from them, consist of more than one typeof parasite, which can be distinguished by monoclonalantibody reactivity and restriction fragment lengthpolymorphisms on Southern blots (Shiels et al., 1986;Conrad et al., 1989; Toye et al., 1991). In the presentstudy we have isolated clones of macroschizont-infectedcell lines by limiting dilution, and demonstrated thatcertain cloned cell lines undergo differentiation with ahigh degree of efficiency. Using these clones we haveobtained evidence that merogony is a two-step process,with a preliminary reversible phase leading to a secondirreversible phase of differentiation. In addition, wepresent data that demonstrate that a disruption in thesynchrony between parasite growth and host celldivision is a determinant of differentiation to themerozoite, and that similarities exist between differen-tiation in parasites and higher eukaryotic cells.
Materials and methods
Cell culture and cloning of the TaA2 macroschizont-infected cell lineThe T. annulata (Ankara) macroschizont-infected cell line(TaA2) was obtained by in vitro infection of peripheral bloodmononuclear cells with sporozoites from a mixed parasitestock. Maintenance of the cell line in culture was carried outat 37°C in RPMI-1640 (Gibco) supplemented with 20% heat-inactivated foetal calf serum, 8 fig ml"1 streptomycin, 8 unitsml"1 penicillin, 0.6 \i% ml"1 amphotericin B and 0.05%NaHCC>3. Cloning of a low passage number of this cell linewas carried out by limiting dilution as previously described(Shiels et al., 1986). To determine whether the cell lines hadisoenzyme patterns characterstic of clones, analysis wasperformed for glucose 6-phosphate isomerase as outlined byMelrose et al. (1984).
Induction of differentiation in vitroInduction of macroschizont-infected cell lines to differentiatewas carried out by increasing the temperature of culture from37°C to 41°C. The cell number of each culture was estimatedby counting, using a haemocytometer, and was adjusted to 1.4xlO5 cells ml"1 by dilution with fresh medium, before andevery second day after transfer to 41°C.
Giemsa staining and indirect immunofluorescenceassayMorphological examination of induced cultures was routinelycarried out by light microscopy of Giemsa-stained cytospinpreparations. A 50 jA sample of culture was spun at 1,500revs min"1 (240 g) for 5 min using a Shandon cytospin 2. Thepreparations were then air dried for 10 min and fixed inmethanol for 30 min. Staining was performed with Gurr'simproved R66 Giemsas stain (BDH) at 4% in distilled water.
Indirect immunofluorescence assay (IFA) on preparationsof differentiating cultures with monoclonal antibodies raisedagainst the piroplasm stage of the parasite (Glascodine et al.,1990) was carried out as described previously (Shiels et al.,1986). Differentiating cultures were centrifuged at 500 g for 5min and washed three times with sterile phosphate-bufferedsaline (PBS). Fixation was carried out on ice in 1.8%formaldehyde (BDH) for 10 min and the cells were thenwashed three times in PBS. Cells were resuspended in PBS(approx. 5xl06 ml"1), spotted onto PTFE Multispot slides(C.A. Hendley, Essex) and air dried. Analysis and photogra-phy of immunofluorescence was carried out with a LeitzOrtholux II fluorescent microscope and an Orthomat-Wcamera attachment.
Southern blottingThe isolation of DNA from the different cell lines, gelelectrophoresis and Southern blotting were performed usingstandard methods (Maniatis et al., 1982). The L16 DNA usedas a probe was isolated from a genomic library of T. annulata(Jedaida 2), cloned in pUC 18. The probe was radiolabelledby the random priming method (Feinberg and Vogelstein,1983). Hybridisation and washing were carried out at 65°C asdescribed by Church and Gilbert (1984).
Counts and statistical analysisTo quantify the number of cells that reacted with themonoclonal antibodies by IFA the numbers of positive andnegative cells were counted in random microscope fields.Between 500 and 1000 cells were counted for each time pointand cell line. Statistical analysis of the values obtained for the
Host cell division and differentiation in Theileria 101
different cell lines was carried out using the chi2 distributionand 2x2 contingency tables. Estimation of the macroschizontnuclei number was carried out by counting the nuclei of over100 infected cells in random fields of Giemsa-stained cytospinpreparations. This was done for both cloned cell lines at eachtime point. The standard error of the mean (S.E.M.) wasestimated, and the difference between means tested, usingstandard statistical formulae (Harper, 1971). In order toanalyse the growth rate of the different cell lines the cellnumber per ml was estimated by counting samples in ahaemocytometer, after the culture had been diluted toapproximately 1.4 x 105 cells ml"1 at the end of a period ofgrowth. After a further 48 h of culture the cell number wasagain estimated, and the increase in cell number wascalculated by dividing the second count by the first. Thisprocedure was carried out at 37°C and at 2-day intervals at41°C, until day 6. Counting was carried out in duplicate forduplicate cultures of each cloned cell line. The S.E.M. foreach time point was estimated, and the difference betweenmeans tested by using the t distribution.
Results
Isolation of cloned macroschizont-infected cell linesFour cell lines were successfully isolated by limitingdilution of the TaA2-infected macroschizont cell lineand showed clonal phenotypes by isoenzyme analysis(data not shown). These cloned cell lines were thentested for their ability to differentiate at 41°C. Two ofthese clones (C9 and D7) exhibited morphologycharacteristic of differentiating cultures after 10 days at41°C (Fig. 2B and C). In approximately 50% of thesecells the number of nuclei was greatly increased and theparasite was enlarged, filling most of the host cellcytoplasm. In just over one third of these parasites thenuclei were bulky and stained densely. The macroschi-zont was so large in some cases that it completely filledthe host cell, which also appeared to be enlarged. Inaddition to the enlarged macroschizonts, parasitescontaining multiple small densely staining particleswere observed within approximately 22% of the hostcells. This morphology is indicative of merozoiteformation, and from previous studies these forms wereidentified as microschizonts. A high level of extracellu-lar particles (between 300 and 400 per microscope field)with small densely staining nuclei were also observed,and these were identified as merozoites (Glascodine etal., 1990).
After 10 days at 41°C the level of differentiation ofthe other two cloned cell lines (E3 and D3) did notappear as extensive (Fig. 2F). Only 8% of the infectedhost cells contained enlarged macroschizonts (volumeof parasite greater than 50% of the host cell cytoplasm),with approximately 1% showing the microschizontmorphology consistent with merozoite formation. Thesmall extracellular densely staining merozoites wereobserved at low levels (20-30 per field). Continuedculture over 14 days at 41°C resulted in the loss of thecultures of cloned cell lines C9 and D7. In contrast,after a period of poor growth (from day 4 to 14) thecloned cell lines D3 and E3 recovered and could be keptat 41°C indefinitely. These cells showed the morphology
of macroschizont-infected cells (see Fig. 2H), with anoccasional infected cell displaying morphology consist-ent with merozoite formation. From these initialobservations we designated the cloned cell lines ashaving an enhanced (C9 and D7) or a diminished (E3and D3) differentiation phenotype.
Reactivity of differentiating parasites with monoclonalantibodiesMonoclonal antibodies have been raised that reactagainst the merozoite and piroplasm, but not againstthe macroschizont of T. annulata (Glascodine et al.,1990). Using IFA, three of these monoclonals (5E1,1D11 and 1C2) were tested against fixed slide prep-arations of the cloned cell lines after 8 days of culture at41°C. Fig. 3A shows that within the represented fieldthe majority (but not all) of cells of the enhanced clonedcell line (C9) reacted with monoclonal antibody 5E1;with most of the fluorescence being located at theperiphery of the infected cell. None of the cells of thediminished cloned lines (D3 or E3), however, showedreactivity with antibody 5E1 (Table 1).
In contrast to 5E1, antibody 1C2 clearly reactedagainst a small proportion of cells of the diminished (D3and E3) cloned cell lines but showed no reactivityagainst cells of the enhanced (D7 or C9) cloned celllines. The staining pattern differed from that ofmonoclonal 5E1 in that the fluorescence obtained wasstippled and distinct (see Fig. 3 B). The thirdmonoclonal (1D11) gave the same staining pattern asmonoclonal 1C2 (Fig. 3 C) but, unlike the other twoantibodies, reacted against cells of all the cloned celllines (Table 1). From these results we concluded that inaddition to reacting with the liberated merozoite thethree antibodies also detected parasites that were in theprocess of merogony, and could therefore be used asmarkers for differentiation.
Genotypic analysis of cloned cell lines by SouthernblottingIn order to determine whether any differences betweenthe enhanced and the diminished cloned cell linesexisted at the genomic level, Southern blot analysis wascarried out. A restriction fragment length polymor-phism was observed when the T. annulata DNA probeL16 (Ben Miled, unpublished data) was hybridized to£coRI-digested cell line DNA. This was most evident
Table 1. Reactivity of monoclonal antibodies againstcloned cell lines cultured at 41 °C for 8 days
Assessment of reactivity was carried out by analysis of randommicroscope fields. A minimum of between 200 and 500 cells wereanalysed for each antibody and cell line.
102 B. Shiels and others
Fig. 2. Giemsa-stained cytocentrifuge preparation of T.annulate (Ankara) cloned cell lines C9 and E3 followingculture at 41°C. (A-D) Cloned cell line C9. (A) At 37°C;(B) day 6 at 41°C; (C) day 10 at 41°C; and (D) day 16 at41°C. (E-H) Cloned cell line E3. (E) At 37°C; (F) day 6 at41°C; (G) day 10 at 41°C; and (H) day 44 at 41°. Host cellnucleus (hn), macroschizont (ms), macroschizont nuclei(msn), enlarged macroschizont (ems) and merozoites (me).Bar, 10 fan.
in the major bands of 7.1 kb and 1.3 kb, which werespecific to either the enhanced (7.1 kb; Fig. 4, tracks 2and 3) or the diminished (1.3 kb; Fig. 4, tracks 4 and 5)cloned cell lines. Both of these bands were present inDNA isolated from the T. annulata (Ankara) cell lineTaA46 (Fig. 4, track 1).
Quantitification of differentiation using monoclonalantibodies 1D11 and 5ElTo compare the ability of enhanced (cloned cell lineC9), diminished (cloned cell line E3) and parental(TaA2) cell lines to differentiate, fixed preparations ofcells after different time points of culture at 41°C weretested by IFA with monoclonal antibodies 1D11 and5E1, and the percentage of positive cells was estimated.The analysis with monoclonal 1D11 (Table 2) showedthat at all time points tested the percentage of cells thatreacted positively to antibody ID 11 was highest withthe enhanced cloned cell line (C9). At day 12 thepercentage of positive cells for this cell line was ninetimes greater than that estimated for the diminishedcloned cell line (E3) and over seven times greater thanthe parental cell line (TaA2). The number of positivecells in the diminished cloned cell line (E3) increased to8% at day 14 (data not shown), but after culture for 44days at 41°C this level was reduced to 0.2%.
Owing to the inability of monoclonal antibody 5E1 todetect differentiating cells of the diminished cloned celllines (see Table 1) the analysis with this antibody wasconfined to the enhanced cloned cell line (C9) and theparental line (TaA2). At day 10 over 80% of the cells ofclone C9 reacted with monoclonal 5E1, and this highlevel increased to 92% at day 12. These valuescompared with a maximum (at day 10) of 18% for theparental cell line (TaA2).
In addition to comparing the abilities of the three celllines to differentiate, the quantitative analysis showed adifference in the number of cells of the enhanced cloned
Host cell division and differentiation in Theileria 103
1 2 3 4 5
«1-3kb
Fig. 4. Southern blotanalysis of EcoRlrestriction-digested DNAisolated from differentcell lines. The blot washybridised with the T.annulata L16 probe asdescribed in Materialsand methods. Track 1,DNA isolated from cellline TaA46; tracks 2 and3, DNA isolated fromcloned cell lines C9 andD7; tracks 4 and 5,DNA isolated fromcloned cell lines E3 andD3.
cell line (C9) that reacted with antibody 5E1 comparedwith antibody 1D11. Thus, after 10 days of culture at41°C three times more cells were positive with antibody5E1 than with 1D11. Interestingly, by day 12 thedifference was reduced to one and a half times.
Limited incubation of cultures at 41°C results in areduction in the level of differentiationTo test whether placing the cultures of macroschizont-infected cells at 41GC resulted in a rapid induction ofdifferentiation, the enhanced cloned cell line (C9) wasplaced in culture at 41°C. Following incubation fordifferent time periods at the increased temperature,cultures were returned to 37°C. The cultures were thenmaintained at 37°C up to the time at which visibledifferentiation, as assessed by Giemsa-stained mor-phology, had occurred in the control culture at 41°C.Fixed slide preparations were then made and the levelof differentiation was assessed by IFA with monoclonal5E1. Table 3 shows that 24 h at 41°C followed by 6 daysat 37°C produced the same result as the negative control(37°C) culture in that no cells were induced todifferentiate by this procedure. A proportion of thecells (14%) were positive for differentiation after 48 h in
TableDays
2. Percentageat 41°C
Monoclonal antibody
of cells positive0
5E1
with
1D11
monoclonal
5E1
5E110
or 1D11
1D11
before and
5E1
after12
induction
1D11
at 4PC44
1D11
82n.d.18*
262it
92n.d
5*
63ff
t
n.d.0.2n.d.
Cell line C9E3TaA2
n.d., test not done, due to non-reactivity of monoclonal antibody 5E1 or the inability of cell line C9 to survive culture at 41°C.•Significant difference with monoclonal antibody 5E1 between cloned cell line C9 and cell line TaA2 (P<0.01).tSignificant difference with monoclonal antibody 1D11 between cloned cell lines C9 and E3, and between C9 and TaA2 (P<0.01).
104 B. Shiels and others
Table 3. Percentage cells positive with monoclonal5E1 after different times of culture at 41°C, followed
by culture at 3TC
Values are the mean obtained from duplicate cultures ± thestandard deviation (S.D.). The duplicate values at each time pointwere compared by x analysis. The only time point to show asignificant difference between the duplicate cultures was day 3 at41 °C (P<0.01).
culture at 41°C followed by 5 days at 37°C. Thisproportion increased the longer the cells were main-tained at the higher temperature, so that after 4 days at41°C followed by 3 days at 37°C 50% of the cells wereestimated to be differentiating. In agreement with theabove observation, it was found that the highest level ofdifferentiation (73%) was achieved by continuousculture at 41°C for 7 days (see Table 3).
Estimation of number of macroschizont nuclei duringculture at 41° CMorphological analysis of macroschizont-infected cellscultured at 41°C has shown an increase in the number ofnuclear particles per macroschizont, which is associatedwith a larger parasite size, during differentiation to themerozoite (Hulliger et al., 1966; Glascodine et al.,1990; see Fig. 2B). To compare the extent of thesechanges in the enhanced (C9) with those in thediminished (E3) cloned cell lines, the mean number ofnuclei per macroschizont was estimated at successive 2-day time points at 41°C, and the data are presented inFig. 5. For the enhanced cloned cell line (C9) at 37°Cthe average number of nuclei per schizont was 20. Thisnumber increased by 40% (8 nuclei) after 2 days ofculture at 41°C, and over the next 48 h between day 2and day 4 the mean number of nuclei rose from 27 to 57,an increase of almost 300% from day 0. An accurateestimation of the number of nuclei was not possible atday 6, because many of the cells had macroschizontswith a very large number of nuclei (300-500) closelypacked together (see Fig. 2 B).
The increase in the mean number of macroschizontnuclei for the diminished cloned cell line E3 over thesame period of time was found to be much smaller.Thus, from day 0 (37°C) to day 4 the mean number ofnuclei increased by only 30%, from 13 to 17, and it tookuntil day 12 for the mean number of nuclei to rise to alevel of 32. Following prolonged (44 days) culture ofthese cells at 41°C, the mean number of nuclei fell to 11,a level not significantly different (P<0.01) from themacroschizont-infected cells cultured at 37°C.
Comparison of infected cell growth at 41°C and 3TCIn order to analyse the gTowth rate of the cloned cell
eo
so
Days at 41°C
012347
Days at 37°C
765430
% Cells5E1 +ve ± S.D.
00
14±1.430±5.650±2.173±2.1
O
SB 4 012 30COCD
I 20
10
12 44
Days at 41°C
Ics
Fig. 5. Mean number of nuclei per macroschizont afterculture of cloned cell lines C9 and E3 at 37°C and atprogressive 2-day time points after induction at 41°C. Filledbar, cloned cell line E3; hatched bar, cloned cell line E3.A dot above the column represents a significant difference(P<0.01) between the means estimated at this and thepreceding time point.
10
2 4ill!6
Days at 41°CI CS M E3
Fig. 6. Increase in cell number of cloned cell line C9 (filledbar) and cloned cell line E3 (hatched bar) over 48 hperiods of culture. Days shown represent the valueestimated at 37°C (day 0) and at 41°C (days 2-6). A dotabove the column represents a significant difference(P<0.01) between the means estimated at this and thepreceding time point.
lines C9 and E3 during the first 6 days of differentiationat 41°C, cell counts were taken every 48 h. Fig. 6 showsthat over the first 2 days of growth at increasedtemperature the amplification in cell number (7.3-fold)of the enhanced cloned cell line (C9) culture wasgreater than that estimated for the culture over 48 h at37°C (4.2-fold). This level of growth at 41°C was notmaintained, however, and as the time course pro-gressed the increase in cell number of the culturesteadily declined to 6-fold from day 2 to 4 and 3.1-foldfrom day 4 to 6. In general, the fluctuation in growthobserved over the time course was similar for theculture of the diminished cloned cell line (E3).However, at each time point the estimated increase incell number for the diminished cloned cell line wasalways greater than for the enhanced (see Fig. 6). For
Host cell division and differentiation in Theileria 105
example, after 48 h at 37°C the increase in cell numberof the diminished cloned cell line (E3) was estimated at6.7-fold, compared to 4.2-fold for the enhanced clonedcell line (C9).
Discussion
We have isolated clones of Theileria macroschizont-infected cell lines that showed an enhanced or dimin-ished ability to differentiate to the next stage in theparasite life cycle, the merozoite. Thus, we haveestablished cell lines in which the majority of theparasites (>90%) differentiate over approximately thesame time period. These cloned cell lines are aconsiderable improvement on cultures that have beenused in previous studies of merozoite differentiation(Hulliger et al., 1966; Danskin and Wilde, 1976; Fritschet al., 1988; Glascodine et al., 1990), and are essentialfor analysis of the molecular events that occur duringmerogony.
The percentage of cells of the enhanced cloned cellline (C9) that reacted with monoclonal ID 11 was foundto be smaller than the number positive with monoclonal5E1. In addition to this quantitative difference, mono-clonal 5E1 produced a halo type pattern of immunoflu-orescence, whereas parasites that reacted with mono-clonal 1D11 had a stippled pattern (see Fig. 3).Immunoelectron microscopic studies have shown thatmonoclonal antibody 5E1 binds to the surface of thedifferentiating macroschizont, whereas monoclonal1D11 reacts with the rhoptry located within the differ-entiating parasite (L. Tetley and B. Shiels, unpublisheddata). We conclude that during the differentiationprocess the molecule detected by monoclonal 5E1 isexpressed on the surface of the differentiating parasitebefore formation of the rhoptry is completed.
The reactivity of two of the monoclonal antibodieswas found to be specific for either the enhanced(monoclonal 5E1) or the diminished (monoclonal 1C2)cloned cell line. Therefore, the epitopes recogised bythese monoclonals are not conserved between theparasites of the different cloned cell lines, and thedifferentiating macroschizonts and merozoites gener-ated from the two types of cloned cell line must beantigenically distinct. From these results it was pre-dicted that the parasite genotypes represented by thedifferent type of cloned cell line would be distinct, andthis was confirmed by the result of the Southern analysiswhich showed a restriction fragment polymorphismbetween the enhanced and the diminished cloned celllines (Fig. 4). Thus, like other stocks of Theileria(Allsop and Allsop, 1988; Conrad et al., 1989) ourparental (TaA2) cell line contained at least twodifferent parasite genotypes. These genotypes corre-lated with the enhanced or diminished differentiationphenotypes of the infected cell lines. This suggests thatparasites of different genotype can have differingabilities to differentiate.
The results of the pulse experiment (see Table 3)showed that differentiation was not an immediate
response to the increased temperature, as the cells hadto be in culture for more than 24 hours at the highertemperature before a proportion were induced todifferentiate. Furthermore, it was evident that return-ing the cultures to 37°C reduced the level of differen-tiating cells from that obtained by continuing culture at41°C for 7 days. From these results it appears that inorder to differentiate the cells must go through apreliminary phase at 41°C, and that this early phase isreversible if the cells are returned to 37°C. However,returning the culture to the lower temperature after 4days at 41°C did not result in a reduction in differen-tiation, when compared with the level observed im-mediately after 4 days of culture at 41°C (data notshown). Thus, we believe that, as in a number of othersystems, including protozoon parasites (Watson et al.,1987; Bruce et al., 1990), the macroschizont oncetriggered becomes committed to an irreversible phaseof differentiation that can proceed to completion at37°C.
A characteristic that has been observed in the earlystages of differentiation to the merozoite is an enlarge-ment of the macroschizont, with a concomitant increasein its nuclear number (Hulliger et al., 1966; Glascodineet al., 1990). Our results show that the number of nucleiper macroschizont and the parasite size increasedgreatly during the first 6 days of culture of the enhancedcloned cell line C9 (see Fig. 5 and Fig. 2B), but with thediminished cloned cell line (E3) the increase wassignificantly smaller and took longer to occur. Further-more, after 44 days of culture of the diminished line at41°C the level of differentiation was very low (Table 2)and the mean number of nuclei per macroschizont wasnot significantly different from the number estimated at37°C (Fig. 5). Thus, there is an association between theincrease in the number of nuclei per macroschizont(and parasite size) and the ability of the cell lines todifferentiate. We propose that this increase in parasitegrowth takes place during the initial reversible phase ofthe differentiation process until a condition is reachedthat triggers the second irreversible phase of differen-tiation to the merozoite.
In the growth analysis of the enhanced cell line (C9)we found, as postulated by Hulliger et al. (1966), thatthe growth of the macroschizont becomes asynchronouswith the division of the host cell at 41°C. For the first 2days the infected cell grew faster at the highertemperature (see Fig. 6). Therefore, in order to accountfor the increase in the number of parasite nuclei andsize observed over this time (see Fig 5; cell line C9), therate of parasite nuclear division must have been evengreater than the increased host cell division. Thisincrease in parasite nuclear division was then coupledwith a decline in host cell division from two to six days,and it is likely that at a certain time point host celldivision was inhibited completely. Owing to theassociation of the parasite with host cell spindle forseparation of the macroschizont, as the rate of host celldivision is reduced the division of parasites alsobecomes slower. Thus, an increase in parasite nucleardivision/growth is accompanied by a decrease in
106 B. Shiels and others
parasite cell division, and consequently the macroschi-zont is enlarged to the point where the host cellcytoplasm is completely filled (see Fig. 2B). Thediminished cell line (E3) divided faster than theenhanced cell line at all time points tested, and thedisruption of synchrony between parasite and host celldivision was less at 41°C. This and the smaller size of themacroschizonts at 37°C probably caused the diminishedphenotype of the E3 cell line, as it would take longer forthe parasite to reach a predetermined size or conditionwhich triggers differentiation.
Studies on the kinetics of replication of T. parva invivo showed that an increase in the nuclear number ofthe macroschizont preceded the appearance of pirop-lasm infected erythrocytes. It was argued that micros-chizont formation (i.e. differentiation to the merozoite)was time-dependent and was preceded by a fixednumber of macroschizont-infected cell multiplications(Jarrett et al., 1969). The hypothesis of a mitotic clockregulating differentiation has been proposed for highereukaryotic cells (Temple and Raff, 1986). Recentstudies have demonstrated that the timing of such aclock is determined by control over proliferation, as inoligodendrocyte progenitor cells differentiation occursprematurely when their proliferation potential isreduced by removal of growth factor (Raff et al., 1988).Moreover, differentiation can be inhibited by continuedstimulation of the cells to proliferate by the addition ofgrowth factors (Bogler et al., 1990). We believe thesituation to be similar for differentiation to themerozoite, and that the timing of the initial reversiblephase is regulated by the rate of increase in parasitesize, with a concomitant decrease in the proliferationpotential of both host cell and parasite.
In addition to studies carried out on higher eukary-otes, a correlation between a reduction in proliferationand differentiation has also been found for otherprotozoon parasites. For example, during its life cycleTrypanasoma brucei alternates between proliferativeand non-proliferative phases (Vickerman, 1985), andrepeated passage of the trypomastigote (bloodstream)stage can result in a loss of differentiation (Hajduk andVickerman, 1981). In Leishmania differentiation fromthe insect promastigote stage to the mammalianinfective stage (metacyclic) involves a change fromlogarithmic- to stationary-phase growth (Sacks andPerkins, 1984, 1985), and the inability of Leishmaniamajor promastigotes to differentiate after transfer of anin vitro culture from 25°C to 35°C has been associatedwith proliferation at the higher temperature (Shapira etal., 1988). It is possible, therefore, that one of theparameters that leads to the triggering of stagedifferentiation in protozoan parasites is a reduction inthe rate of parasite division, and that there arefundamental similarities between the mechanisms thatregulate differentiation in parasites and higher eukary-otic cells.
Thanks to Alan May for help with photomicroscopy andphotographic printing, to Frank Wright for advice on
statistical analysis, and to the Wellcome Trust for continuedsupport.
References
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