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Mild acid stress as a differentiation trigger in Trypanosoma brucei
Molecular and Biochemical Parasitology 93 (1998) 251 – 262
Mild acid stress as a differentiation trigger in Trypanosoma brucei Sylvie Rolin, Jacqueline Hanocq-Quertier, Franc¸oise Paturiaux-Hanocq, Derek P. Nolan, Etienne Pays * Laboratory of Molecular Parasitology, Department of Molecular Biology, Free Uni6ersity of Brussels, 67 Rue des Che6aux, B-1640, Rhode St Gene`se, Belgium Received 4 December 1997; received in revised form 11 March 1998; accepted 11 March 1998
Abstract In vitro differentiation of Trypanosoma brucei from the bloodstream to the procyclic form is efficiently induced by the combination of cold shock from 37 to 27°C and the addition of citrate/cis-aconitate (CCA) to the incubation medium. Here it is reported that exposure of pleomorphic bloodstream trypanosomes to mild acidic conditions (pH 5.5 for 2 h at 37°C) not only accelerated the process of morphological transformation from long slender and intermediate to short stumpy bloodstream forms but also allowed their subsequent differentiation into procyclic forms even in the absence of CCA. This process appeared to involve the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), since null GPI-PLC mutants (PLC − ) appeared to be largely refractory to acid stress-induced differentiation. However, an effective response was restored upon reintegration of the GPI-PLC gene in the genome (PLC + ) © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Trypanosoma; Cellular differentiation; pH stress; Phospholipase C; Cell signalling
1. Introduction The initial event in the differentiation of Trypanosoma brucei from bloodstream to procyclic Abbre6iations: CCA, citrate/cis-aconitate; GPI-PLC, glycosylphosphatidylinositol specific phospholipase C; VSG, variant surface glycoprotein. * Corresponding author. Tel.: + 32 2 6509621; fax: +32 2 6509656.
forms occurs in the bloodstream of the mammalian host where all forms of the parasite are covered with a major antigen termed the variant surface glycoprotein (VSG). As the parasitaemia reaches high levels, the rapidly dividing slender forms progressively differentiate into short stumpy forms incapable of further division. These forms contain expanded mitochondrial cristae and some mitochondrial enzymes, probably as a
0166-6851/98/$19.00 © 1998 Published by Elsevier Science B.V. All rights reserved. PII S0166-6851(98)00046-2
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preadaptation to a drastic switch in energy metabolism from glycolysis in glycosomes in bloodstream forms to oxidative phosphorylation in mitochondria in procyclic cells [1,2]. After ingestion of a bloodmeal by the tsetse fly, the parasites further transform into dividing procyclic trypomastigotes and colonize the insect midgut. Among many morphological and biochemical transformations, insect procyclic forms are characterized by some distinctive features. The kinetoplast is not subterminal, the undulating membrane is poorly developed and the flagellum is either absent or very short [3]. There is a metabolic switch from glucose-based to amino acid-based respiration which relies on the activity of a fully developed mitochondrion [1 – 4]. The synthesis of the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is rapidly repressed and the enzyme activity declines to undetectable level during subsequent growth of procyclic forms [5]. The VSG is no longer expressed and is replaced by a completely different major surface glycoprotein termed procyclin [6 – 8] and, last but not least, the procyclic forms are no longer infective for mammals. In the insect vector it has been observed that the majority of the ingested bloodstream forms, including the stumpy forms which are believed to be preadapted for differentiation [2], are eliminated. The successful infection of the insect midgut appears to be initiated by a very small fraction of the cells actually ingested by the fly (less than 1%) differentiating into proliferative procyclics (J. Van Den Abbeele, Y. Claes, D. Le Ray and M. Coosemans, personal communication). Although nothing is known about the physiological signals involved in the transformation events occurring within the fly, the process of differentiation from bloodstream forms to procyclic forms can be readily and very efficiently performed in vitro. Several experimental conditions have been devised which allow a more or less synchronous transformation of either monomorphic or pleomorphic populations. For example if monomorphic bloodstream trypanosomes are placed in procyclic culture medium at 27°C, most cells die but a small number begin to grow as procyclics after about 20 days or more
[3,5,9]. The addition of Krebs cycle intermediates, citrate/cis-aconitate (CCA) to the culture medium speeds up the rate of differentiation, so that monomorphic population starts to proliferate as procyclic after 24 h [10–13]. Differentiation of the cell population occurs even more rapidly, and synchronously, if a predominantly intermediate and stumpy population of bloodstream trypomastigotes is used. Under these experimental conditions, typical changes such as release of the VSG coat, entry in S-phase and complete re-programming of protein synthesis, occur only 2–12 h after the trigger of differentiation [5,7,8,12–19]. The physiological basis of the experimental stimuli used for differentiation in vitro is unclear. While temperature naturally decreases during the transfer of trypanosomes from the mammalian blood into the insect vector, the role of CCA, although critically important in stimulating in vitro trypanosome differentiation [20], is less obvious in vivo. In a search for other possible transformation-promoting factors, the effect of exposure to low pH on a pleomorphic population of T. brucei was studied. This experiment was prompted by our observation [21] that this treatment is representative of several environmental stresses which do not kill bloodstream trypomastigotes but induce the release of VSG and activation of adenylate cyclase, both of which occur during the transformation of bloodstream to procyclic forms [18]. Moreover, acidic stress has been reported to trigger differentiation of the flagellated extracellular forms of T. cruzi or Leishmania into the aflagellar intracellular forms [22– 25].
2. Material and methods
2.1. Trypanosome strains and transformation protocol The pleomorphic T. brucei variant clone AnTat 1.1 and a GPI-PLC null mutant (PLC − ) [26] were grown in mice that had been immunosuppressed by X-irradiation (600 rads), and harvested 7 days after infection as previously described [18]. The transformation experiments were initiated by a
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preincubation for 2 h at 37°C in a phosphate buffer (1×106 cells ml − 1 in 125 mM sodium phosphate containing 1% glucose, 20 mg ml − 1 leupeptin and 15% heat-inactivated fetal bovine serum [27]), either at pH 7.5 (control cells) or at pH 5.5 (acid-treated cells). The cells were then transferred to modified DTM medium containing 15% heat inactivated fetal bovine serum and incubated in the presence and absence of 3 mM citrate/cis-aconitate (CCA) at 27°C [13,18]. It is important to stress that the majority of cells (both AnTat 1.1 and PLC − ) exposed to either neutral or acidic pH remained motile and intact during the preincubation and entire incubation period preceding cellular proliferation, as judged by phase microscopy.
2.2. Light microscopy The course of transformation from bloodstream to procyclic forms was followed by determining the percentage of bloodstream forms (slender, intermediate and stumpy), transforming forms and transformed procyclic cells, according to the criteria of Vickerman [2] and Ghiotto et al. [3]. Nine stages were examined. The first stage (t = − 2 h) represents the bloodstream population immediately after isolation from the blood, the second stage (t=0 h) represents the bloodstream population after the 2 h-preincubation period at either pH 7.5 or 5.5, and the following stages represent the different forms found in the population incubated at 27°C in DTM medium in the presence ( + CCA) or absence (− CCA) of citrate/cisaconitate at different time intervals after the beginning of transformation. At each stage, samples of cells were fixed overnight in 2% (v:v) formaldehyde, 0.05% (v:v) glutaraldehyde in PBS, and then Giemsa stained. At least 100 trypanosomes were classified and the percentage of each group was determined. The time course of differentiation was also followed by counting the number of motile cells with a hemocytometer. Another different criterion applied to distinguish the long slender forms from the other forms, was by cytochemical staining of NAD
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diaphorase activity [1,28]. Air dry smears were fixed for 5 min at 4°C in 2.5% glutaraldehyde/1M cacodylate buffer (pH 7.2), then processed as described by Vickerman [1]. Sites of NADH oxidation were registered as blue-black formazan deposits derived from reduction of nitro-blue tetrazolium. Photomicrographs were taken with light or contrast phase microscopy at 1000× magnification.
2.3. Flow cytometry and Western blotting Flow cytometry analysis was performed using a fluorescence activated cell sorter (FACscan, Becton Dickinson), on cells fixed and treated for 1 h at 0°C with primary and secondary antibodies. The following antibodies were used: rabbit antiAnTat 1.1 VSG (1:400), mouse monoclonal antiprocyclin antibody (Cedarlane, 1:500), FITClabeled donkey anti-rabbit antibody (Amersham, 1:500) and FITC-labeled sheep anti-mouse antibody (Amersham, 1:200). Trypanosome extracts were electrophoresed in SDS-10% polyacrylamide gels and analyzed by Western blotting as described previously [29,30]. The primary antibody was the hyperimmune antiAnTat 1.1 antibody (dilution 1:100000) and the secondary antibody was anti-rabbit IgG alkalinephosphatase conjugate from Promega. In the case of the PLC − cells, the VSG was detected following incubation of the trypanosome extracts with exogenous bacterial GPI-PLC (0.07 U per sample), which generated the cross-reacting determinant (CRD) recognized by the polyclonal anti-AnTat 1.1 hyperimmune antiserum.
3. Results The differentiation of bloodstream forms into procyclic forms was analysed by four different criteria: (i) morphological transformations (Figs. 1 and 6) and NADH diaphorase activity (Fig. 3); (ii) cell growth (Fig. 2); (iii) switching of the major surface antigen (Figs. 4 and 5); and (iv) loss of infectivity.
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Fig. 1. Effect of low pH and CCA on morphological changes that occur during a representative transformation experiment with the wild type pleomorphic AnTat 1.1 variant. Bloodstream forms were preincubated at 37°C into either pH 7.5 (A, B) or pH 5.5 (C, D) buffer, then transferred to DTM medium in the presence (A, C) or absence (B, D) of CCA and cultivated at 27°C for the period indicated. The first point (t = −2 h) represents the parasite population immediately after isolation from the blood.
3.1. Effect of mild acid treatment on in 6itro differentiation of a wild type pleomorphic AnTat 1.1 6ariant The AnTat 1.1 bloodstream population used to initiate the typical experiment illustrated in Fig. 1 consisted of 33% slender, 47% intermediate and 20% stumpy forms (Fig. 1; t = − 2 h). Under previously described conditions (27° +CCA), the cells acquired the procyclic morphology (Fig. 1A, thick line) and started to divide (Fig. 2A) after about 12 h. As expected from previous reports [10 – 13] and shown in Fig. 1B (thick line) and 2A, the addition of CCA was absolutely required for successful transformation when cells were first preincubated at neutral pH. Preincubation at pH 5.5 did not affect this process and led to transfor-
mation into procyclic forms at 27°C. Significantly, this pretreatment allowed trypanosomes to transform even in the absence of CCA, albeit with differences in the kinetics of transformation and cellular growth compared to that observed in the presence of CCA (Fig. 1C and D thick line). In the former case stumpy forms remained predominant for at least 24 h and the first procyclic-like forms were detected after more than 10 h and then increased to only 10% of the population after 22 h (Fig. 1D). These cells did not divide for a further 24–48 h and thereafter, they grew slowly and almost linearly (Fig. 2A). In fact the only obvious difference was the rapid loss of the slender forms, which decreased from 10 to 0% within 2 h after transfer to 27°C, and an increase in short stumpy forms, from 10 to \ 90% within 4 h (Fig.
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Fig. 2. Effect of low pH and CCA on cell growth and transformation of the wild type (A), PLC − (B) and PLC + (C) trypanosomes. Bloodstream forms were preincubated at 37°C in either neutral buffer (open symbols) or acidic buffer (black symbols) and then cultivated at 27°C in the presence or absence of CCA, as indicated. Times refer to the transformation protocol in Fig. 1. Average values obtained from several independent experiments 9 S.E. are shown.
1C and D). This transformation was not a consequence of artefactual cell swelling due to a deleterious effect of the preincubation medium, but reflected cellular differentiation into true stumpy forms, as revealed by the acquisition of NADH diaphorase activity, a marker of mitochondrial activation (Fig. 3A%, B% and C%). In order to relate the morphological changes with the time course of VSG release and procyclin expression, whole cells or trypanosome extracts were probed with antibodies against these proteins, using flow cytometry and Western blotting, respectively. In the presence of CCA, transformation of acid-treated bloodstream forms was associated with the shedding of VSG (Fig. 4A, panels b,c) and the simultaneous expression of procyclin (Fig. 5A, panels b,c). These processes also occurred after preincubation for 2 h at neutral pH (Fig. 4A, panel f; Fig. 5A, panel g). In the absence of CCA, no change in the nature of surface antigen was observed in cells preincubated for 2 h at neutral pH (Fig. 4A, panel g, and Fig. 5A, panel h). In contrast, under the same conditions, cells preincubated for 2 h at pH 5.5 lost their VSG (Fig. 4A, panels d, e) and expressed procyclin (Fig. 5A, panels d – f). In agreement
with the morphological observations described above, there were differences in the kinetics and synchrony between these processes and those that occurred in cells incubated with CCA (compare panels d,e and b,c in Fig. 4A; d,e,f and b,c in Fig. 5A). Fig. 4B illustrates the time course of VSG release assessed by Western blot analysis of acidtreated cells cultivated at 27°C in the presence (upper left panel) and absence (upper right panel) of CCA. The disappearance of VSG in the last lanes clearly demonstrated that acid-treated cells were able to release their VSG even in the absence of CCA, albeit at a slower rate than in the presence of CCA (compare the two top panels in Fig. 4B). It was noted that after pretreatment at pH5.5 the VSG remained associated with the pellet of surviving stumpy cells, whereas under the same conditions monomorphic trypanosomes released VSG through the activation of GPI-PLC [21]. The infectivity of the culture during the pH 5.5 induced-transformation was investigated by injecting equal numbers of trypanosomes (1× 106) into the intraperitoneal cavity and counting blood samples up to day 20 after inoculation. A progressive decrease in infectivity was observed as the time in culture increased, with a complete loss of
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Fig. 3. Light micrographs of wild type (A, B, C) and of PLC − (D, E, F) trypanosomes after exposure to low pH. Bloodstream form trypanosomes were first incubated at 37°C in pH 5.5 buffer for 2 h and then cultivated at 27°C in DTM without CCA for the periods indicated. Panels A to F show morphological changes observed in Giemsa stained wild type (A, B, C) and PLC − (D, E, F) trypanosomes 0 h (A, D), 6 h (B, E) and 24 h (C, F) after triggering cell differentiation (sl, slender; im, intermediary; ss, stumpy; tr, transforming forms). Panels A%,B% and C% show different stages of wild type trypanosomes stained for NAD diaphorase activity. Arrowheads indicate sites of NADH2 oxidation.
infectivity 72 h after transfer to 27°C in the medium with CCA, and 96 h after transfer to 27°C in the medium without CCA. Interestingly, it was also observed that the pleomorphic trypanosomes inoculated into mice immediately after acid treatment also gave rise to infection, but with an increase of the prepatent period compared to control cells (9–10 vs 2 days). This was probably due to the rapid loss of slender forms from the population after preincubation at low pH (Fig. 1C and D ). All the experiments described above have been repeated several times with the same results. Altogether, the data showed that a pretreatment under mild acidic conditions can circumvent the need for CCA in the transformation of bloodstream into procyclic forms in vitro, although the efficiency of this process was lower.
3.2. Effect of mild acid treatment on in 6itro differentiation of a GPI-PLC null mutant In order to evaluate whether the induction of bloodstream transformation by low pH depended on the activation of the GPI-PLC, the effect of acid pretreatment on the differentiation of trypanosomes deprived of GPI-PLC (PLC − ) targeted homologous recombination [26] was studied. The parasite population used to initiate the experiment illustrated in Fig. 6 consisted of 29% slender, 61% intermediate and 10% stumpy forms (Fig. 6; t= −2 h). In the presence of CCA, the transformation of PLC − occurred as in the wild type in both control and acidic stress conditions (Fig. 6, panels A, C and Fig. 2B). As might be expected from these results, the release of VSG (Fig. 4B, lower left panel) and appearance of procyclin (Fig. 5B, panels b, c, g) followed the
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Fig. 4. Effect of low pH and CCA on VSG release. Panel A shows FACS analysis of AnTat 1.1 bloodstream forms preincubated for 2 h at either acid or neutral pH before cultivation at 27°C in the presence or absence of CCA. At the times indicated (0, 24 and 72 h) samples were removed, fixed, treated with primary and secondary antibodies (rabbit anti-AnTat 1.1 VSG antibody and FITC-labeled donkey anti-rabbit antibody) and analysed by flow cytometry. Panel B shows the time course of VSG release as measured by Western blot analysis of pellets of wild type (upper panels) and null GPI-PLC mutant (PLC − , lower panels) trypanosomes preincubated for 2 h at acid or neutral pH and then grown at 27°C in the presence (left) or absence (right) of CCA. The cell pellets were subjected to SDS-PAGE and probed with a polyclonal hyperimmune anti-AnTat 1.1 VSG antibody. In the case of the null mutant, the extracts were incubated with bacterial GPI-PLC to generate an epitope (CRD) recognized by the anti-AnTat 1.1 antibody.
same kinetics as in the wild type. In contrast, when cultivated in the absence of CCA the PLC − trypanosomes appeared largely unable to differentiate into procyclic forms, whether preincubated under neutral or acidic conditions (Fig. 6B and D, thick line). As judged by their morphology, these cells did not transform into stumpy forms but retained their phenotype of bloodstream intermediate forms (Fig. 6B and D and Fig. 3D, E and F) until they died after about 5 days. However, it must be noted that the acid pretreatment appeared to retard the rate of cell death (Fig. 2B) and to allow some transformation compared to cells preincubated at neutral pH. After 22 h a few
stumpy forms (20%) succeeded in differentiating into transforming forms, and upon prolonged incubation (] 1 week) a small fraction of cells transformed successfully into procyclic-like cells and then divided (data not shown). The results from direct analysis of VSG release (Fig. 4B) and procyclin expression (Fig. 5B) supported these morphological observations and demonstrated that when acid-pretreated PLC − cells were cultivated at 27°C in the absence of CCA, the majority of those cells retained their VSG for at least 24 h (Fig. 4B, lower right panel) and failed to express procyclin, although a moderate increase in procyclin expression was observed after 72 h in about
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Fig. 5. Effect of low pH and CCA on expression of procyclin. Wild type (AnTat 1.1, panel A), PLC − (panel B) and PLC + (panel C) bloodstream trypanosomes were preincubated at acid or neutral pH before cultivation at 27°C in the presence or absence of CCA. At the times indicated (t), samples were removed, fixed, treated with primary and secondary antibodies (mouse monoclonal anti-procyclin antibody and FITC-labeled goat anti-mouse antibody) and analysed by flow cytometry.
20% of the acid-treated parasites (Fig. 5B, panel f). To determine whether the inability of PLC − cells to differentiate in the absence of CCA was indeed due to the absence of the GPI-PLC gene, the effect of the pretreatment at low pH was investigated after reintroduction of the GPI-PLC gene in the mutant. These cells (PLC + ) express 5 – 10% of the GPI-PLC level of the wild type [26]. The PLC + trypanosomes responded to acidic stress by differentiating to procyclic forms at a rate intermediate between wild type and PLC − cells. They did not divide for about 48 h, then slowly began to proliferate (Fig. 2(C)). Procyclin was almost fully expressed after 72 h (Fig. 5(C), panel f).
The continuous loss of infectivity observed when acid-pretreated PLC − and PLC + trypanosomes were incubated at 27°C in the presence or absence of CCA was essentially similar to that observed with wild type trypanosomes, with a loss of infectivity 48–72 h after transfer to medium without CCA. Even though the PLC − cells were no longer infective after 48–72 h incubation, they failed to express procyclin and retained their bloodstream morphology. Moreover PLC − and PLC + trypanosomes inoculated into mice immediately after acid treatment were still infective but only in a few mice and with a prolonged prepatent period (7–11 vs 3–4 days for control cells), indicating that only few trypanosomes were still infective.
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Fig. 6. Effect of low pH and CCA on morphological changes that occur during a representative transformation experiment with a null mutant for GPI-PLC. Same legend as for Fig. 1, except that PLC − trypanosomes were analyzed.
4. Discussion When T. brucei intermediate and stumpy forms are submitted to a cold shock (from 37 to 27°C) in the presence of CCA, most cells undergo a rapid, complete and synchronous differentiation into proliferative procyclics [13,18]. This process is absolutely dependent on the combination of these two stimuli. However, in vitro systems are necessarily artificial, and the situation could be quite different in vivo where a natural counterpart to CCA might not exist. In this study, an alternative to CCA as inducer of differentiation in vitro has been identified. Preincubation of a population of predominantly intermediate and stumpy forms at pH 5.5 for 2 h at 37°C was found to induce their differentiation to procyclic forms at 27°C, even in the absence of CCA. It should be stressed that this acid pretreatment had no obvious effects on cellular viability
and motility of the majority of the cells present in this pleomorphic population, as demonstrated by direct morphological observation and by monitoring the total number of cells which remained relatively constant throughout the period preceding cell division. Our data are consistent with the view that the primary effect of this acid stress was to change the relative abundance of slender, intermediate and stumpy forms present in the initial cell population. The loss of slender forms was almost certainly due to the death of these forms since it has been observed that monomorphic long slender bloodstream forms incubated under the same conditions quickly died (survival rate 30 and 0%, respectively, 1 and 2 h after the preincubation at pH 5.5; data not shown). The fact that there was no significant drop in the total number of cells present in the population suggests that the intermediate forms had switched to stumpy forms. This switch allowed the differentiation of those
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forms into procyclic forms independently of CCA, whereas at neutral pH CCA is absolutely required for this process. However, it is clear that acid stress was less efficient than CCA in triggering differentiation, as determined by all the relevant criteria (morphological transformation, expression of stage specific antigens, cell proliferation and loss of infectivity). Actually the lower efficiency of transformation observed under the conditions described in this study may be more closely aligned to the low rate of transformation efficiency in the fly, and in any case it cannot be concluded that this process is physiologically less relevant than the previously described CCA-supported differentiation. Whether an acid stress actually occurs during ingestion of a bloodmeal by the fly is unknown. Although the pH of the alimentary tract of the tsetse fly appears to be alkaline (J. Van Den Abbeele, personal communication), this observation may not be relevant to the processes under consideration here since the parasites living in this compartment have already transformed into procyclics. Therefore, it remains possible that acidic extracellular conditions may play a role in the initiation of differentiation in vivo, for instance during the transfer from the carefully buffered milieu of the mammalian bloodstream to the insect vector where large changes in [H + ] may occur. In this respect, it is interesting to note that in other closely related kinetoplastids, T. cruzi and Leishmania, the transformation of extracellular flagellated promastigotes into intracellular amastigotes is also influenced by low extracellular pH, both in vivo and in vitro [22–25,31]. Finally, whatever the situation in vivo, the fact remains that mild acid treatment belongs to a family of extracellular stress conditions (osmotic shock, Ca2 + , local anaesthetics, pH 5.5, trypsin, PKC inhibitors) that all efficiently stimulate simultaneously adenylate cyclase and VSG release which are both part of the cellular differentiation program from VSG coated bloodstream forms to uncoated procyclics [21,32–36]. Interestingly, it is noted that in contrast to slender forms [21], intermediate and stumpy forms remained largely covered with their VSG even after a 2 h incubation at 27°C in pH 5.5 buffer. Studies are currently under way to
characterize this interesting result and to analyze the GPI-PLC activity during the transition from slender to stumpy forms. The results obtained with trypanosomes lacking GPI-PLC were consistent with the hypothesis that GPI-PLC activity may be involved in the acid stress-induced differentiation observed in wild type cells. Indeed, PLC − trypanosomes preincubated at pH 5.5 were largely incapable of differentiating into stumpy and procyclic forms unless CCA was present. Our data also show that the reintroduction of the GPI-PLC gene allowed these cells to recover their ability to transform into procyclics in the absence of CCA. The reversion was incomplete, which was consistent with the fact that only low amounts of GPI-PLC (5–10%) were measured in these revertants [26]. In T. brucei, the role of the GPI-PLC is still unclear, especially since it is not absolutely required at any stage of the parasite life-cycle [26]. Significantly, this enzyme is not necessary for VSG shedding during the differentiation of VSG-coated bloodstream forms into procyclic forms since this process appears to be due to a proteolytic cleavage in the C-terminal region of the VSG [17]. At present the role of the GPI-PLC in acid stress-induced differentiation remains to be established. In conclusion, it has been shown that in wild type cells a pretreatment at pH 5.5 has the effect not only of speeding up the transition from intermediate to stumpy forms but also of removing the requirement for the presence of CCA for subsequent differentiation to procyclics at 27°C. It is then proposed that at least two distinct pathways can lead to in vitro differentiation from the bloodstream to the procyclic form. A very efficient GPI-PLC-independent process is specifically triggered by CCA at 27°C, whereas a more general pathway involving GPI-PLC is triggered at 37°C by environmental stress.
Acknowledgements We thank G. Van Santen for help in the FACS analysis, P. Viart for invaluable help in the preparation of the figures and D. Franckx for photography. This work was supported by the Fonds de
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la Recherche Scientifique (FRSM and Cre´dit aux Chercheurs), by the International Brachet Stiftung, by a research contract with the Communaute´ Franc¸aise de Belgique (ARC) and by the Interuniversity Poles of Attraction ProgrammeBelgian State, Prime Minister’s Office-Federal Office for Scientific, Technical and Cultural Affairs. J.H.Q. is fellow of the FNRS.
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[13] Ziegelbauer K, Quinten M, Schwarz H, Pearson TW, Overath P. Synchronous differentiation of Trypanosoma brucei from bloodstream to procyclic forms in vitro. Eur J Biochem 1990;192:373 – 8. [14] Ehlers B, Czichos J, Overath P. Repression and reactivation of the variant surface glycoprotein gene in Trypanosoma brucei. FEBS Lett 1987;225:53 – 8. [15] Bu¨llow R, Nonnenga¨sser C, Overath P. Release of the variant surface glycoprotein during differentiation of bloodstream to procyclic forms of Trypanosoma brucei. Mol Biochem Parasitol 1989;32:85 – 92. [16] Pays E, Hanocq-Quertier J, Hanocq F, Van Assel S, Nolan D, Rolin S. Abrupt RNA change precede the first cell division during the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro. Mol Biochem Parasitol 1993;61:107 – 14. [17] Ziegelbauer K, Stahl B, Karas M, Stierhof YD, Overath P. Proteolytic release of cell surface proteins during differentiation of Trypanosoma brucei. Biochemistry 1993;32:3737 – 42. [18] Rolin S, Paindavoine P, Hanocq-Quertier J. Transient adenylate cyclase activation accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms. Mol Biochem Parasitol 1993;61:115 – 26. [19] Matthews KR, Gull K. Evidence for an interplay between cell cycle progression and the initiation of differentiation between life cycle forms of African trypanosomes. J Cell Biol 1994;125:1147 – 56. [20] Matthews KR, Gul K. Commitment to differentiation and cell cycle re-entry are coincident but separable events in the transformation of African Trypanosomes from their bloodstream to their insect form. J Cell Sci 1997;110:2609– 18. [21] Rolin S, Hanocq-Quertier J, Paturiaux-Hanocq F. Simultaneous but independent activation of adenylate cyclase and glycosylphosphatidylinositol-phospholipase C under stress conditions in Trypanosoma brucei. J Biol Chem 1996;271:10844– 52. [22] Kanbara H, Uemura H, Nakazawa S, Fukama T. Effect of low pH on transformation of Trypanozoma cruzi trypomastigotes to amastigote. Jpn J Parasitol 1990;39:226 – 8. [23] Zilberstein D, Blumenfeld N, Liveanu V, Gepstein A, Jaffe CL. Growth at acidic pH induces an amastigote stage-specific protein in Leishmania promastigotes. Mol Biochem Parasitol 1991;45:175 – 8. [24] Bates PA, Robertson CD, Tetley L, Coombs GH. Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms. Parasitology 1992;105:193 – 202. [25] Tomlinson S, Vandekerckove F, Frevert U, Nussenzweig V. The induction of Trypanosoma cruzi trypomastigote to amastigote transformation by low pH. Parasitology 1995;110:547 – 54. [26] Webb H, Carnall N, Vanhamme L. The GPI-specific phospholipase C of Trypanosoma brucei is non essential but influences parasitaemia. J Cell Biol 1997;139:103 – 14.
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[27] Baltz T, Baltz D, Pautrizel R. Affinite´ de la concanavaline A pour Trypanosoma equiperdum. Applications a` l’isolement de la fraction glycoproteique spe´cifique du type antige´nique. Ann Immunol Pasteur 1976;127C:761–74. [28] Steinert M. Le chondriome de Trypanosoma mega. J Cell Biol 1964;20:192 – 7. [29] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350–4. [30] Paindavoine P, Rolin S, Van Assel S. A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclase located on the flagellum of Trypanosoma brucei. Mol Cell Biol 1992;12:1218 – 25. [31] Zilberstein D, Shapira M. The role of pH and temperature in the development of Leishmania parasites. Annu Rev Microbiol 1994;48:449–70.
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[32] Voorheis H, Martin B. Characteristics of the calcium mediated mechanism activating adenylate cyclase in Trypanosoma brucei. Eur J Biochem 1981;116:471 – 7. [33] Bowles J, Voorheis H. Release of the surface coat from the plasma membrane of intact bloodstream forms of Trypanosoma brucei. FEBS Lett 1982;139:17 – 21. [34] Voorheis H, Bowles J, Smith G. Characteristics of the release of the surface coat proteins from bloodstream forms of Trypanosoma brucei. J Biol Chem 1982;257:2300– 4. [35] Voorheis H, Martin B. Local anaesthetics including benzyl alcohol activate adenylate cyclase in Trypanosoma brucei by a calcium-dependent mechanism. Eur J Biochem 1982;123:371 – 6. [36] Yabu Y, Takayanagi T. Trypsin stimulated transformation of Trypanosoma brucei gambiense bloodstream forms to procyclic forms in vitro. Parasitol Res 1988;74:501 – 6.
Mild acid stress as a differentiation trigger in Trypanosoma brucei Sylvie Rolin, Jacqueline Hanocq-Quertier, Franc¸oise Paturiaux-Hanocq, Derek P. Nolan, Etienne Pays * Laboratory of Molecular Parasitology, Department of Molecular Biology, Free Uni6ersity of Brussels, 67 Rue des Che6aux, B-1640, Rhode St Gene`se, Belgium Received 4 December 1997; received in revised form 11 March 1998; accepted 11 March 1998
Abstract In vitro differentiation of Trypanosoma brucei from the bloodstream to the procyclic form is efficiently induced by the combination of cold shock from 37 to 27°C and the addition of citrate/cis-aconitate (CCA) to the incubation medium. Here it is reported that exposure of pleomorphic bloodstream trypanosomes to mild acidic conditions (pH 5.5 for 2 h at 37°C) not only accelerated the process of morphological transformation from long slender and intermediate to short stumpy bloodstream forms but also allowed their subsequent differentiation into procyclic forms even in the absence of CCA. This process appeared to involve the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), since null GPI-PLC mutants (PLC − ) appeared to be largely refractory to acid stress-induced differentiation. However, an effective response was restored upon reintegration of the GPI-PLC gene in the genome (PLC + ) © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Trypanosoma; Cellular differentiation; pH stress; Phospholipase C; Cell signalling
1. Introduction The initial event in the differentiation of Trypanosoma brucei from bloodstream to procyclic Abbre6iations: CCA, citrate/cis-aconitate; GPI-PLC, glycosylphosphatidylinositol specific phospholipase C; VSG, variant surface glycoprotein. * Corresponding author. Tel.: + 32 2 6509621; fax: +32 2 6509656.
forms occurs in the bloodstream of the mammalian host where all forms of the parasite are covered with a major antigen termed the variant surface glycoprotein (VSG). As the parasitaemia reaches high levels, the rapidly dividing slender forms progressively differentiate into short stumpy forms incapable of further division. These forms contain expanded mitochondrial cristae and some mitochondrial enzymes, probably as a
0166-6851/98/$19.00 © 1998 Published by Elsevier Science B.V. All rights reserved. PII S0166-6851(98)00046-2
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preadaptation to a drastic switch in energy metabolism from glycolysis in glycosomes in bloodstream forms to oxidative phosphorylation in mitochondria in procyclic cells [1,2]. After ingestion of a bloodmeal by the tsetse fly, the parasites further transform into dividing procyclic trypomastigotes and colonize the insect midgut. Among many morphological and biochemical transformations, insect procyclic forms are characterized by some distinctive features. The kinetoplast is not subterminal, the undulating membrane is poorly developed and the flagellum is either absent or very short [3]. There is a metabolic switch from glucose-based to amino acid-based respiration which relies on the activity of a fully developed mitochondrion [1 – 4]. The synthesis of the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is rapidly repressed and the enzyme activity declines to undetectable level during subsequent growth of procyclic forms [5]. The VSG is no longer expressed and is replaced by a completely different major surface glycoprotein termed procyclin [6 – 8] and, last but not least, the procyclic forms are no longer infective for mammals. In the insect vector it has been observed that the majority of the ingested bloodstream forms, including the stumpy forms which are believed to be preadapted for differentiation [2], are eliminated. The successful infection of the insect midgut appears to be initiated by a very small fraction of the cells actually ingested by the fly (less than 1%) differentiating into proliferative procyclics (J. Van Den Abbeele, Y. Claes, D. Le Ray and M. Coosemans, personal communication). Although nothing is known about the physiological signals involved in the transformation events occurring within the fly, the process of differentiation from bloodstream forms to procyclic forms can be readily and very efficiently performed in vitro. Several experimental conditions have been devised which allow a more or less synchronous transformation of either monomorphic or pleomorphic populations. For example if monomorphic bloodstream trypanosomes are placed in procyclic culture medium at 27°C, most cells die but a small number begin to grow as procyclics after about 20 days or more
[3,5,9]. The addition of Krebs cycle intermediates, citrate/cis-aconitate (CCA) to the culture medium speeds up the rate of differentiation, so that monomorphic population starts to proliferate as procyclic after 24 h [10–13]. Differentiation of the cell population occurs even more rapidly, and synchronously, if a predominantly intermediate and stumpy population of bloodstream trypomastigotes is used. Under these experimental conditions, typical changes such as release of the VSG coat, entry in S-phase and complete re-programming of protein synthesis, occur only 2–12 h after the trigger of differentiation [5,7,8,12–19]. The physiological basis of the experimental stimuli used for differentiation in vitro is unclear. While temperature naturally decreases during the transfer of trypanosomes from the mammalian blood into the insect vector, the role of CCA, although critically important in stimulating in vitro trypanosome differentiation [20], is less obvious in vivo. In a search for other possible transformation-promoting factors, the effect of exposure to low pH on a pleomorphic population of T. brucei was studied. This experiment was prompted by our observation [21] that this treatment is representative of several environmental stresses which do not kill bloodstream trypomastigotes but induce the release of VSG and activation of adenylate cyclase, both of which occur during the transformation of bloodstream to procyclic forms [18]. Moreover, acidic stress has been reported to trigger differentiation of the flagellated extracellular forms of T. cruzi or Leishmania into the aflagellar intracellular forms [22– 25].
2. Material and methods
2.1. Trypanosome strains and transformation protocol The pleomorphic T. brucei variant clone AnTat 1.1 and a GPI-PLC null mutant (PLC − ) [26] were grown in mice that had been immunosuppressed by X-irradiation (600 rads), and harvested 7 days after infection as previously described [18]. The transformation experiments were initiated by a
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preincubation for 2 h at 37°C in a phosphate buffer (1×106 cells ml − 1 in 125 mM sodium phosphate containing 1% glucose, 20 mg ml − 1 leupeptin and 15% heat-inactivated fetal bovine serum [27]), either at pH 7.5 (control cells) or at pH 5.5 (acid-treated cells). The cells were then transferred to modified DTM medium containing 15% heat inactivated fetal bovine serum and incubated in the presence and absence of 3 mM citrate/cis-aconitate (CCA) at 27°C [13,18]. It is important to stress that the majority of cells (both AnTat 1.1 and PLC − ) exposed to either neutral or acidic pH remained motile and intact during the preincubation and entire incubation period preceding cellular proliferation, as judged by phase microscopy.
2.2. Light microscopy The course of transformation from bloodstream to procyclic forms was followed by determining the percentage of bloodstream forms (slender, intermediate and stumpy), transforming forms and transformed procyclic cells, according to the criteria of Vickerman [2] and Ghiotto et al. [3]. Nine stages were examined. The first stage (t = − 2 h) represents the bloodstream population immediately after isolation from the blood, the second stage (t=0 h) represents the bloodstream population after the 2 h-preincubation period at either pH 7.5 or 5.5, and the following stages represent the different forms found in the population incubated at 27°C in DTM medium in the presence ( + CCA) or absence (− CCA) of citrate/cisaconitate at different time intervals after the beginning of transformation. At each stage, samples of cells were fixed overnight in 2% (v:v) formaldehyde, 0.05% (v:v) glutaraldehyde in PBS, and then Giemsa stained. At least 100 trypanosomes were classified and the percentage of each group was determined. The time course of differentiation was also followed by counting the number of motile cells with a hemocytometer. Another different criterion applied to distinguish the long slender forms from the other forms, was by cytochemical staining of NAD
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diaphorase activity [1,28]. Air dry smears were fixed for 5 min at 4°C in 2.5% glutaraldehyde/1M cacodylate buffer (pH 7.2), then processed as described by Vickerman [1]. Sites of NADH oxidation were registered as blue-black formazan deposits derived from reduction of nitro-blue tetrazolium. Photomicrographs were taken with light or contrast phase microscopy at 1000× magnification.
2.3. Flow cytometry and Western blotting Flow cytometry analysis was performed using a fluorescence activated cell sorter (FACscan, Becton Dickinson), on cells fixed and treated for 1 h at 0°C with primary and secondary antibodies. The following antibodies were used: rabbit antiAnTat 1.1 VSG (1:400), mouse monoclonal antiprocyclin antibody (Cedarlane, 1:500), FITClabeled donkey anti-rabbit antibody (Amersham, 1:500) and FITC-labeled sheep anti-mouse antibody (Amersham, 1:200). Trypanosome extracts were electrophoresed in SDS-10% polyacrylamide gels and analyzed by Western blotting as described previously [29,30]. The primary antibody was the hyperimmune antiAnTat 1.1 antibody (dilution 1:100000) and the secondary antibody was anti-rabbit IgG alkalinephosphatase conjugate from Promega. In the case of the PLC − cells, the VSG was detected following incubation of the trypanosome extracts with exogenous bacterial GPI-PLC (0.07 U per sample), which generated the cross-reacting determinant (CRD) recognized by the polyclonal anti-AnTat 1.1 hyperimmune antiserum.
3. Results The differentiation of bloodstream forms into procyclic forms was analysed by four different criteria: (i) morphological transformations (Figs. 1 and 6) and NADH diaphorase activity (Fig. 3); (ii) cell growth (Fig. 2); (iii) switching of the major surface antigen (Figs. 4 and 5); and (iv) loss of infectivity.
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Fig. 1. Effect of low pH and CCA on morphological changes that occur during a representative transformation experiment with the wild type pleomorphic AnTat 1.1 variant. Bloodstream forms were preincubated at 37°C into either pH 7.5 (A, B) or pH 5.5 (C, D) buffer, then transferred to DTM medium in the presence (A, C) or absence (B, D) of CCA and cultivated at 27°C for the period indicated. The first point (t = −2 h) represents the parasite population immediately after isolation from the blood.
3.1. Effect of mild acid treatment on in 6itro differentiation of a wild type pleomorphic AnTat 1.1 6ariant The AnTat 1.1 bloodstream population used to initiate the typical experiment illustrated in Fig. 1 consisted of 33% slender, 47% intermediate and 20% stumpy forms (Fig. 1; t = − 2 h). Under previously described conditions (27° +CCA), the cells acquired the procyclic morphology (Fig. 1A, thick line) and started to divide (Fig. 2A) after about 12 h. As expected from previous reports [10 – 13] and shown in Fig. 1B (thick line) and 2A, the addition of CCA was absolutely required for successful transformation when cells were first preincubated at neutral pH. Preincubation at pH 5.5 did not affect this process and led to transfor-
mation into procyclic forms at 27°C. Significantly, this pretreatment allowed trypanosomes to transform even in the absence of CCA, albeit with differences in the kinetics of transformation and cellular growth compared to that observed in the presence of CCA (Fig. 1C and D thick line). In the former case stumpy forms remained predominant for at least 24 h and the first procyclic-like forms were detected after more than 10 h and then increased to only 10% of the population after 22 h (Fig. 1D). These cells did not divide for a further 24–48 h and thereafter, they grew slowly and almost linearly (Fig. 2A). In fact the only obvious difference was the rapid loss of the slender forms, which decreased from 10 to 0% within 2 h after transfer to 27°C, and an increase in short stumpy forms, from 10 to \ 90% within 4 h (Fig.
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Fig. 2. Effect of low pH and CCA on cell growth and transformation of the wild type (A), PLC − (B) and PLC + (C) trypanosomes. Bloodstream forms were preincubated at 37°C in either neutral buffer (open symbols) or acidic buffer (black symbols) and then cultivated at 27°C in the presence or absence of CCA, as indicated. Times refer to the transformation protocol in Fig. 1. Average values obtained from several independent experiments 9 S.E. are shown.
1C and D). This transformation was not a consequence of artefactual cell swelling due to a deleterious effect of the preincubation medium, but reflected cellular differentiation into true stumpy forms, as revealed by the acquisition of NADH diaphorase activity, a marker of mitochondrial activation (Fig. 3A%, B% and C%). In order to relate the morphological changes with the time course of VSG release and procyclin expression, whole cells or trypanosome extracts were probed with antibodies against these proteins, using flow cytometry and Western blotting, respectively. In the presence of CCA, transformation of acid-treated bloodstream forms was associated with the shedding of VSG (Fig. 4A, panels b,c) and the simultaneous expression of procyclin (Fig. 5A, panels b,c). These processes also occurred after preincubation for 2 h at neutral pH (Fig. 4A, panel f; Fig. 5A, panel g). In the absence of CCA, no change in the nature of surface antigen was observed in cells preincubated for 2 h at neutral pH (Fig. 4A, panel g, and Fig. 5A, panel h). In contrast, under the same conditions, cells preincubated for 2 h at pH 5.5 lost their VSG (Fig. 4A, panels d, e) and expressed procyclin (Fig. 5A, panels d – f). In agreement
with the morphological observations described above, there were differences in the kinetics and synchrony between these processes and those that occurred in cells incubated with CCA (compare panels d,e and b,c in Fig. 4A; d,e,f and b,c in Fig. 5A). Fig. 4B illustrates the time course of VSG release assessed by Western blot analysis of acidtreated cells cultivated at 27°C in the presence (upper left panel) and absence (upper right panel) of CCA. The disappearance of VSG in the last lanes clearly demonstrated that acid-treated cells were able to release their VSG even in the absence of CCA, albeit at a slower rate than in the presence of CCA (compare the two top panels in Fig. 4B). It was noted that after pretreatment at pH5.5 the VSG remained associated with the pellet of surviving stumpy cells, whereas under the same conditions monomorphic trypanosomes released VSG through the activation of GPI-PLC [21]. The infectivity of the culture during the pH 5.5 induced-transformation was investigated by injecting equal numbers of trypanosomes (1× 106) into the intraperitoneal cavity and counting blood samples up to day 20 after inoculation. A progressive decrease in infectivity was observed as the time in culture increased, with a complete loss of
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Fig. 3. Light micrographs of wild type (A, B, C) and of PLC − (D, E, F) trypanosomes after exposure to low pH. Bloodstream form trypanosomes were first incubated at 37°C in pH 5.5 buffer for 2 h and then cultivated at 27°C in DTM without CCA for the periods indicated. Panels A to F show morphological changes observed in Giemsa stained wild type (A, B, C) and PLC − (D, E, F) trypanosomes 0 h (A, D), 6 h (B, E) and 24 h (C, F) after triggering cell differentiation (sl, slender; im, intermediary; ss, stumpy; tr, transforming forms). Panels A%,B% and C% show different stages of wild type trypanosomes stained for NAD diaphorase activity. Arrowheads indicate sites of NADH2 oxidation.
infectivity 72 h after transfer to 27°C in the medium with CCA, and 96 h after transfer to 27°C in the medium without CCA. Interestingly, it was also observed that the pleomorphic trypanosomes inoculated into mice immediately after acid treatment also gave rise to infection, but with an increase of the prepatent period compared to control cells (9–10 vs 2 days). This was probably due to the rapid loss of slender forms from the population after preincubation at low pH (Fig. 1C and D ). All the experiments described above have been repeated several times with the same results. Altogether, the data showed that a pretreatment under mild acidic conditions can circumvent the need for CCA in the transformation of bloodstream into procyclic forms in vitro, although the efficiency of this process was lower.
3.2. Effect of mild acid treatment on in 6itro differentiation of a GPI-PLC null mutant In order to evaluate whether the induction of bloodstream transformation by low pH depended on the activation of the GPI-PLC, the effect of acid pretreatment on the differentiation of trypanosomes deprived of GPI-PLC (PLC − ) targeted homologous recombination [26] was studied. The parasite population used to initiate the experiment illustrated in Fig. 6 consisted of 29% slender, 61% intermediate and 10% stumpy forms (Fig. 6; t= −2 h). In the presence of CCA, the transformation of PLC − occurred as in the wild type in both control and acidic stress conditions (Fig. 6, panels A, C and Fig. 2B). As might be expected from these results, the release of VSG (Fig. 4B, lower left panel) and appearance of procyclin (Fig. 5B, panels b, c, g) followed the
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Fig. 4. Effect of low pH and CCA on VSG release. Panel A shows FACS analysis of AnTat 1.1 bloodstream forms preincubated for 2 h at either acid or neutral pH before cultivation at 27°C in the presence or absence of CCA. At the times indicated (0, 24 and 72 h) samples were removed, fixed, treated with primary and secondary antibodies (rabbit anti-AnTat 1.1 VSG antibody and FITC-labeled donkey anti-rabbit antibody) and analysed by flow cytometry. Panel B shows the time course of VSG release as measured by Western blot analysis of pellets of wild type (upper panels) and null GPI-PLC mutant (PLC − , lower panels) trypanosomes preincubated for 2 h at acid or neutral pH and then grown at 27°C in the presence (left) or absence (right) of CCA. The cell pellets were subjected to SDS-PAGE and probed with a polyclonal hyperimmune anti-AnTat 1.1 VSG antibody. In the case of the null mutant, the extracts were incubated with bacterial GPI-PLC to generate an epitope (CRD) recognized by the anti-AnTat 1.1 antibody.
same kinetics as in the wild type. In contrast, when cultivated in the absence of CCA the PLC − trypanosomes appeared largely unable to differentiate into procyclic forms, whether preincubated under neutral or acidic conditions (Fig. 6B and D, thick line). As judged by their morphology, these cells did not transform into stumpy forms but retained their phenotype of bloodstream intermediate forms (Fig. 6B and D and Fig. 3D, E and F) until they died after about 5 days. However, it must be noted that the acid pretreatment appeared to retard the rate of cell death (Fig. 2B) and to allow some transformation compared to cells preincubated at neutral pH. After 22 h a few
stumpy forms (20%) succeeded in differentiating into transforming forms, and upon prolonged incubation (] 1 week) a small fraction of cells transformed successfully into procyclic-like cells and then divided (data not shown). The results from direct analysis of VSG release (Fig. 4B) and procyclin expression (Fig. 5B) supported these morphological observations and demonstrated that when acid-pretreated PLC − cells were cultivated at 27°C in the absence of CCA, the majority of those cells retained their VSG for at least 24 h (Fig. 4B, lower right panel) and failed to express procyclin, although a moderate increase in procyclin expression was observed after 72 h in about
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Fig. 5. Effect of low pH and CCA on expression of procyclin. Wild type (AnTat 1.1, panel A), PLC − (panel B) and PLC + (panel C) bloodstream trypanosomes were preincubated at acid or neutral pH before cultivation at 27°C in the presence or absence of CCA. At the times indicated (t), samples were removed, fixed, treated with primary and secondary antibodies (mouse monoclonal anti-procyclin antibody and FITC-labeled goat anti-mouse antibody) and analysed by flow cytometry.
20% of the acid-treated parasites (Fig. 5B, panel f). To determine whether the inability of PLC − cells to differentiate in the absence of CCA was indeed due to the absence of the GPI-PLC gene, the effect of the pretreatment at low pH was investigated after reintroduction of the GPI-PLC gene in the mutant. These cells (PLC + ) express 5 – 10% of the GPI-PLC level of the wild type [26]. The PLC + trypanosomes responded to acidic stress by differentiating to procyclic forms at a rate intermediate between wild type and PLC − cells. They did not divide for about 48 h, then slowly began to proliferate (Fig. 2(C)). Procyclin was almost fully expressed after 72 h (Fig. 5(C), panel f).
The continuous loss of infectivity observed when acid-pretreated PLC − and PLC + trypanosomes were incubated at 27°C in the presence or absence of CCA was essentially similar to that observed with wild type trypanosomes, with a loss of infectivity 48–72 h after transfer to medium without CCA. Even though the PLC − cells were no longer infective after 48–72 h incubation, they failed to express procyclin and retained their bloodstream morphology. Moreover PLC − and PLC + trypanosomes inoculated into mice immediately after acid treatment were still infective but only in a few mice and with a prolonged prepatent period (7–11 vs 3–4 days for control cells), indicating that only few trypanosomes were still infective.
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Fig. 6. Effect of low pH and CCA on morphological changes that occur during a representative transformation experiment with a null mutant for GPI-PLC. Same legend as for Fig. 1, except that PLC − trypanosomes were analyzed.
4. Discussion When T. brucei intermediate and stumpy forms are submitted to a cold shock (from 37 to 27°C) in the presence of CCA, most cells undergo a rapid, complete and synchronous differentiation into proliferative procyclics [13,18]. This process is absolutely dependent on the combination of these two stimuli. However, in vitro systems are necessarily artificial, and the situation could be quite different in vivo where a natural counterpart to CCA might not exist. In this study, an alternative to CCA as inducer of differentiation in vitro has been identified. Preincubation of a population of predominantly intermediate and stumpy forms at pH 5.5 for 2 h at 37°C was found to induce their differentiation to procyclic forms at 27°C, even in the absence of CCA. It should be stressed that this acid pretreatment had no obvious effects on cellular viability
and motility of the majority of the cells present in this pleomorphic population, as demonstrated by direct morphological observation and by monitoring the total number of cells which remained relatively constant throughout the period preceding cell division. Our data are consistent with the view that the primary effect of this acid stress was to change the relative abundance of slender, intermediate and stumpy forms present in the initial cell population. The loss of slender forms was almost certainly due to the death of these forms since it has been observed that monomorphic long slender bloodstream forms incubated under the same conditions quickly died (survival rate 30 and 0%, respectively, 1 and 2 h after the preincubation at pH 5.5; data not shown). The fact that there was no significant drop in the total number of cells present in the population suggests that the intermediate forms had switched to stumpy forms. This switch allowed the differentiation of those
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forms into procyclic forms independently of CCA, whereas at neutral pH CCA is absolutely required for this process. However, it is clear that acid stress was less efficient than CCA in triggering differentiation, as determined by all the relevant criteria (morphological transformation, expression of stage specific antigens, cell proliferation and loss of infectivity). Actually the lower efficiency of transformation observed under the conditions described in this study may be more closely aligned to the low rate of transformation efficiency in the fly, and in any case it cannot be concluded that this process is physiologically less relevant than the previously described CCA-supported differentiation. Whether an acid stress actually occurs during ingestion of a bloodmeal by the fly is unknown. Although the pH of the alimentary tract of the tsetse fly appears to be alkaline (J. Van Den Abbeele, personal communication), this observation may not be relevant to the processes under consideration here since the parasites living in this compartment have already transformed into procyclics. Therefore, it remains possible that acidic extracellular conditions may play a role in the initiation of differentiation in vivo, for instance during the transfer from the carefully buffered milieu of the mammalian bloodstream to the insect vector where large changes in [H + ] may occur. In this respect, it is interesting to note that in other closely related kinetoplastids, T. cruzi and Leishmania, the transformation of extracellular flagellated promastigotes into intracellular amastigotes is also influenced by low extracellular pH, both in vivo and in vitro [22–25,31]. Finally, whatever the situation in vivo, the fact remains that mild acid treatment belongs to a family of extracellular stress conditions (osmotic shock, Ca2 + , local anaesthetics, pH 5.5, trypsin, PKC inhibitors) that all efficiently stimulate simultaneously adenylate cyclase and VSG release which are both part of the cellular differentiation program from VSG coated bloodstream forms to uncoated procyclics [21,32–36]. Interestingly, it is noted that in contrast to slender forms [21], intermediate and stumpy forms remained largely covered with their VSG even after a 2 h incubation at 27°C in pH 5.5 buffer. Studies are currently under way to
characterize this interesting result and to analyze the GPI-PLC activity during the transition from slender to stumpy forms. The results obtained with trypanosomes lacking GPI-PLC were consistent with the hypothesis that GPI-PLC activity may be involved in the acid stress-induced differentiation observed in wild type cells. Indeed, PLC − trypanosomes preincubated at pH 5.5 were largely incapable of differentiating into stumpy and procyclic forms unless CCA was present. Our data also show that the reintroduction of the GPI-PLC gene allowed these cells to recover their ability to transform into procyclics in the absence of CCA. The reversion was incomplete, which was consistent with the fact that only low amounts of GPI-PLC (5–10%) were measured in these revertants [26]. In T. brucei, the role of the GPI-PLC is still unclear, especially since it is not absolutely required at any stage of the parasite life-cycle [26]. Significantly, this enzyme is not necessary for VSG shedding during the differentiation of VSG-coated bloodstream forms into procyclic forms since this process appears to be due to a proteolytic cleavage in the C-terminal region of the VSG [17]. At present the role of the GPI-PLC in acid stress-induced differentiation remains to be established. In conclusion, it has been shown that in wild type cells a pretreatment at pH 5.5 has the effect not only of speeding up the transition from intermediate to stumpy forms but also of removing the requirement for the presence of CCA for subsequent differentiation to procyclics at 27°C. It is then proposed that at least two distinct pathways can lead to in vitro differentiation from the bloodstream to the procyclic form. A very efficient GPI-PLC-independent process is specifically triggered by CCA at 27°C, whereas a more general pathway involving GPI-PLC is triggered at 37°C by environmental stress.
Acknowledgements We thank G. Van Santen for help in the FACS analysis, P. Viart for invaluable help in the preparation of the figures and D. Franckx for photography. This work was supported by the Fonds de
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la Recherche Scientifique (FRSM and Cre´dit aux Chercheurs), by the International Brachet Stiftung, by a research contract with the Communaute´ Franc¸aise de Belgique (ARC) and by the Interuniversity Poles of Attraction ProgrammeBelgian State, Prime Minister’s Office-Federal Office for Scientific, Technical and Cultural Affairs. J.H.Q. is fellow of the FNRS.
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