Transformation of monomorphic Trypanosoma brucei bloodstream form trypomastigotes into procyclic forms at 37°C by removing glucose from the culture medium
Introduction
Trypanosoma brucei is responsible for African sleeping sickness in man and Nagana in cattle. The parasite exists in the mammalian host as the bloodstream trypomastigote form and in the tsetse fly midgut as the procyclic form. The bloodstream form is characterised by its morphology and ultrastructure [1], the presence of a dense cell surface coat of variant surface glycoprotein (VSG) 2, 3and the lack of a functional citric acid cycle and mitochondrial respiratory chain 4, 5. The bloodstream form avoids the hosts immune system by antigenic variation, i.e. the sequential expression of different VSG variants, and invariant surface molecules, such as ISG65 and ISG75, appear to be masked by the dense packing of the VSG coat [6]. The procyclic form is characterised by its morphology and ultrastructure, a lack of VSG, the presence of a fully functional citric acid cycle and respiratory chain [5]and the expression of cell surface trans-sialidase 7, 8, kinetoplastid membrane protein-11 [9], the CAP5.5 cytoskeletal protein 10, 11, 12and the major cell surface glycoprotein procyclin (also known as procyclic acidic repetitive protein (PARP)) 13, 14, 15. Trypanosomes can express PARP glycoproteins containing -Glu-Pro- repeats and -Gly-Pro-Glu-Glu-Thr- repeats, known as EP-PARP and GPEET-PARP, respectively 16, 17, 18. Both forms of PARP contain identical glycosylphosphatidylinositol (GPI) anchors [16]which are based on the procyclic-specific GPI anchor precursor PP1 EtN-P-Man3GlcN(2-O-acyl)myo-inositol1-HPO4-(sn-1-stearoyl-2-lyso-glycerol) [19].
The transformation of bloodstream form trypomastigotes to procyclic forms in the tsetse fly takes 2–3 days [1]. A similar process can be produced in vitro by placing bloodstream forms into culture medium at 27°C, where some cells can successfully transform and multiply 20, 21, 22. The efficiency of the transformation process can be greatly improved by including citric acid and/or cis-aconitate in the transformation medium 23, 24, 25, 26or by including trypsin or Pronase [27]. Trypanosome strains and clones are referred to as either pleomorphic or monomorphic, according to whether they exist in the bloodstream in multiple forms, ranging from dividing slender forms to non-dividing (stumpy forms), or as slender forms alone. Slender trypanosomes can undergo transformation to procyclic forms in vitro and in vivo 24, 26, 28, 29, 30, although stumpy forms transform more efficiently 10, 11, 31, 32. The parameters that have been followed during in vitro transformation triggered by a shift to 27°C and the addition of citrate and/or cis-aconitate include: VSG and PARP glycoprotein and mRNA levels 29, 32, 33, 34, the rate of VSG synthesis 26, 35, oxidative metabolism [28], adenylate cyclase activity [36], cyclin-1 expression [37], RNA levels and turnover 35, 38, kinetoplast repositioning, cell cycle events and the expression of a cytoskeleton associated protein called CAP5.5 10, 11, 12. A recent study, exploiting the synchronous transformation of stumpy forms, has shown that cis-aconitate is the pre-eminent trigger for transformation and that the temperature shift to 27°C is required for the efficient re-entry of the transformed cells into the cell cycle [39].
This paper describes the serendipitous observation that removing glucose from the culture medium of monomorphic bloodstream trypanosomes growing at 37°C produces continuous cultures of procyclic form cells after 4–5 days. During this transformation period the trypanosome population, in approximately the following order, loses the VSG surface coat, expresses PARP, produces procyclic form specific GPI intermediates, becomes resistant to salicyl hydroxamic acid (SHAM) and glycerol, expresses CAP5.5, acquires procyclic morphology, begins to grow in cell number and finally starts to express trans-sialidase. Although this transformation process is probably less efficient than those previously described using citrate and/or cis-aconitate and temperature shift, the independence of this process from these ‘essential’ in vitro triggers may be significant.
Section snippets
Cell cultures
A clone of strain 427 trypanosomes expressing VSG MlTat.1.4 was established in continuous axenic culture from infected mouse blood in a modified minimum essential medium (MEM) with Earle’s salts similar to that described in 40, 41but supplemented with 2 mg l−1 myristic acid/bovine serum albumin (1 mol mol−1), 12 mg l−1 adenosine, 5.64 mg l−1 bathocuprione disulfonate, 1 mg l−1 catalase, 20 mg l−1 myo-inositol, 6 mg l−1 monothioglycerol, 10 mg l−1 ornithine and 110 mg l−1 sodium pyruvate. The
Growth characteristics of trypanosomes grown in glucose-free medium
In the course of trying to adapt axenic bloodstream form trypomastigote cultures to a medium containing glycerol in place of glucose for sugar-labelling studies, a dramatic change was noticed in the expression of cell surface VSG. In order to follow this more closely, bloodstream form trypomastigotes were first adapted to culture medium containing 30 mM glucose 40, 41for several days and then logphase cells were transferred into glucose-free medium supplemented with 30 mM glycerol at 37°C. The
Discussion
The transformation protocol reported here, using axenic monomorphic bloodstream form cultures of T. brucei strain 427, variant MlTat.1.4, can be compared with that described in 24, 26, 28, 29, 32for the same trypanosome variant using citrate, cis-aconitate and temperature shift to 27°C (herein referred to as the conventional transformation protocol). In those studies, VSG coat loss started after 6–30 h and was essentially complete by 48 h 24, 26, 29. PARP mRNA and glycoprotein were expressed to
Acknowledgements
We thank Alison Sparks for help with the FACScan flow cytometer. We also thank Keith Matthews and Keith Gull for useful discussions and for communicating unpublished observations and Alan Fairlamb and Manu Tetaud for helpful advice. We are grateful to Isabel Roditi for GPEET-PARP specific antibody, Terry Pearson for EP-PARP specific antibody, Keith Gull for CAP5.5 specific antibody and Angela Mehlert for VSG specific antisera. This work was supported by the Wellcome Trust, KGM is a Beit
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