Transformation of monomorphic Trypanosoma brucei bloodstream form trypomastigotes into procyclic forms at 37°C by removing glucose from the culture medium

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Abstract

African trypanosomes have been shown previously to undergo efficient transformation from bloodstream forms to procyclic (insect dwelling) forms in vitro by adding citrate and/or cis-aconitate to the culture medium and lowering incubation temperature to 27°C. In this paper, it is shown that strain 427 monomorphic bloodstream forms of Trypanosoma brucei grown in axenic culture at 37°C can be transformed to procyclic forms by simply replacing the glucose carbon source in the culture medium with glycerol. The removal of glucose from the medium results in the loss of the variant surface glycoprotein, the acquisition of cell surface procyclic acidic repetitive protein, the synthesis of procyclic-specific glycosylphosphatidylinositol precursors and the acquisition of substantial resistance to salicyl hydroxamic acid and glycerol within 72 h. A procyclic-specific cytoskeletal protein, known to be a marker of the late stage of transformation, is fully expressed by 96 h but full trans-sialidase activity appears only after 18–30 days. The transformation process described here is slower and less efficient than that previously described for monomorphic trypanosomes, using citrate and/or cis-aconitate and temperature shift as triggers. However, the separation of the transformation process from these stimuli is significant and the effects of glucose deprivation described here may reflect some of the events that occur in vivo in the tsetse fly midgut, where glucose levels are known to be very low.

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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