Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point

Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.     

Differential protein synthesis during the life cycle of the protozoan parasite Trypanosoma brucei

Two-dimensional polyacrylamide gel electrophoresis has been used to analyze changes in protein content and protein synthesis in three stages of the life cycle of the protozoan parasite Trypanosoma brucei. The stages examined were slender and stumpy mammalian bloodstream forms and procyclic forms, which are analogous to the tsetse fly midgut stage. Two-dimensional gels of 35S-methionine-labeled proteins were examined by autoradiography to analyze newly synthesized protein, and gels were stained with ammoniacal silver to analyze proteins present. Several stage-specific molecules were noted. The most obvious was the variant surface glycoprotein, which was only present in bloodstream forms. Some other proteins were also bloodstream form specific; they had molecular weights of 120,000 and 38,000. Proteins of 52,000, 46,000, 25-30,000, and 16,000 daltons were present both in stumpy forms and procyclics but not in slender-form trypanosomes. Several proteins (molecular weights of 50-70,000, 43,000, 40,000, 26-24,000, 20-25,000, and 15,000) were present only in one of the three stages. One protein, a molecule of about 18,000 daltons present in both slender and stumpy parasites, did not appear to be synthesized in the stumpy stage. In vitro translation products of mRNA purified from the three stages were also examined. The abundance of mRNA encoding a protein of about 40,000 daltons appeared to be greater in slender than in stumpy parasites although the stumpy forms contained more of the protein and synthesized it at a higher rate.     

Differential Protein Synthesis During the Life Cycle of the Protozoan Parasite Trypanosoma brucei1

Two-dimensional polyacrylamide gel electrophoresis has been used to analyze changes in protein content and protein synthesis in three stages of the life cycle of the protozoan parasite Trypanosoma brucei. The stages examined were slender and stumpy mammalian bloodstream forms and procyclic forms, which are analogous to the tsetse fly midgut stage. Two-dimensional gels of ³⁵S-methionine-labeled proteins were examined by autoradiography to analyze newly synthesized protein, and gels were stained with ammoniacal silver to analyze proteins present. Several stage-specific molecules were noted. The most obvious was the variant surface glycoprotein, which was only present in bloodstream forms. Some other proteins were also bloodstream form specific; they had molecular weights of 120,000 and 38,000. Proteins of 52,000, 46,000, 25–30,000, and 16,000 daltons were present both in stumpy forms and procyclics but not in slender-form trypanosomes. Several proteins (molecular weights of 50–70,000, 43,000, 40,000, 26–24,000, 20–25,000, and 15,000) were present only in one of the three stages. One protein, a molecule of about 18,000 daltons present in both slender and stumpy parasites, did not appear to be synthesized in the stumpy stage. In vitro translation products of mRNA purified from the three stages were also examined. The abundance of mRNA encoding a protein of about 40,000 daltons appeared to be greater in slender than in stumpy parasites although the stumpy forms contained more of the protein and synthesized it at a higher rate.     

Protein tyrosine phosphatase TbPTP1: A molecular switch controlling life cycle differentiation in trypanosomes

Differentiation in African trypanosomes (Trypanosoma brucei) entails passage between a mammalian host, where parasites exist as a proliferative slender form or a G0-arrested stumpy form, and the tsetse fly. Stumpy forms arise at the peak of each parasitaemia and are committed to differentiation to procyclic forms that inhabit the tsetse midgut. We have identified a protein tyrosine phosphatase (TbPTP1) that inhibits trypanosome differentiation. Consistent with a tyrosine phosphatase, recombinant TbPTP1 exhibits the anticipated substrate and inhibitor profile, and its activity is impaired by reversible oxidation. TbPTP1 inactivation in monomorphic bloodstream trypanosomes by RNA interference or pharmacological inhibition triggers spontaneous differentiation to procyclic forms in a subset of committed cells. Consistent with this observation, homogeneous populations of stumpy forms synchronously differentiate to procyclic forms when tyrosine phosphatase activity is inhibited. Our data invoke a new model for trypanosome development in which differentiation to procyclic forms is prevented in the bloodstream by tyrosine dephosphorylation. It may be possible to use PTP1B inhibitors to block trypanosomatid transmission.     

Trypanosoma brucei: differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (delta psi) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial delta psi was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.     

Trypanosoma brucei brucei and T. b. gambiense: Stumpy Bloodstream Forms Express More CB1 Epitope in Endosomes and Lysosomes than Slender Forms

CB1-glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1-glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1-glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1-glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle.     

Activation of endocytosis as an adaptation to the mammalian host by trypanosomes

Immune evasion in African trypanosomes is principally mediated by antigenic variation, but rapid internalization of surface-bound immune factors may contribute to survival. Endocytosis is upregulated approximately 10-fold in bloodstream compared to procyclic forms, and surface coat remodeling accompanies transition between these life stages. Here we examined expression of endocytosis markers in tsetse fly stages in vivo and monitored modulation during transition from bloodstream to procyclic forms in vitro. Among bloodstream stages nonproliferative stumpy forms have endocytic activity similar to that seen with rapidly dividing slender forms, while differentiation of stumpy forms to procyclic forms is accompanied by rapid down-regulation of Rab11 and clathrin, suggesting that modulation of endocytic and recycling systems accompanies this differentiation event. Significantly, rapid down-regulation of endocytic markers occurs upon entering the insect midgut and expression of Rab11 and clathrin remains low throughout subsequent development, which suggests that high endocytic activity is not required for remodeling the parasite surface or for survival within the fly. However, salivary gland metacyclic forms dramatically increase expression of clathrin and Rab11, indicating that emergence of mammalian infective forms is coupled to reacquisition of a high-activity endocytic-recycling system. These data suggest that high-level endocytosis in Trypanosoma brucei is an adaptation required for viability in the mammalian host.     

Evaluation of Rhodamine 123 As A Probe For Monitoring Mitochondrial Function In Trypanosoma Brucei Spp.

ABSTRACT. Rhodamine 123, a membrane potential-specific dye, has been evaluated as a probe to monitor the function of the mitochondrion in long slender bloodstream and procyclic trypomastigotes of several Trypanosoma brucei spp. By epifluorescence microscopy, mitochondrial development has been followed in long slender bloodstream and procyclic organisms stained with rhodamine 123. to photograph stained long slender bloodstream forms, it was necessary to develop a method to completely immobilize viable organisms. In both parasite forms, as the cell cycle progressed, the mitochondrion developed from a thread-like structure to a highly branched organelle. A dramatic reorganization occurred preceding cytokinesis to produce two progeny thread-like structures which were partitioned into newly formed daughter cells. the organelle within the long slender trypomastigote was found to stain optimally at 0.3 μ/ml of rhodamine 123, while the procyclic form required 3.0 μ/ml. the results suggest that the plasma membrane potential is higher in the long slender parasite than in the procyclic form. the effects of inhibitors that disrupt mitochondrial function were examined in long slender and procyclic parasites, and some of these agents were shown to affect rhodamine 123 accumulation and retention. In long slender trypomastigotes the trypanosome alternative oxidase does not appear to be coupled to proton pumping, whereas in procyclic organisms the effects of inhibitors indicate that this oxidase may be coupled to a pathway that is branched preceding an antimycin A₁-sensitive site.     

Trypanosoma brucei: in vitro slender-to-stumpy differentiation of culture-adapted,monomorphic bloodstream forms

Pleomorphic Trypanosoma brucei strains are characterized by their ability to differentiate from replicating long slender forms into non-dividing short stumpy forms in the mammalian host. The differentiation process can be efficiently induced in vitro by treatment with the membrane-permeable cAMP derivative 8-(4-chlorophenylthio)-cAMP (pCPTcAMP). In contrast, monomorphic T. brucei strains do not differentiate to stumpy forms in the host. Here, we show that exposure of monomorphic, culture-adapted T. brucei bloodstream forms to pCPTcAMP allowed their subsequent differentiation into short stumpy forms. The stumpy nature of pCPTcAMP-treated parasites was confirmed by (1) morphological change, (2) inhibition of growth and DNA synthesis, (3) cell cycle arrest in the G(1)/G(0) phase, (4) expression of NADH diaphorase activity and dihydrolipoamide dehydrogenase, (5) disappearance of the small subunit of ribonucleotide reductase, (6) up-regulation of the major lysosomal membrane protein, and (7) efficient transformation into replicating procyclic insect forms after induction with citrate/cis-aconitate. Our results indicate that the inability of monomorphic T. brucei bloodstream forms to differentiate into short stumpy forms in the host may be due to a failure in the signalling pathway rather than in the differentiation process itself. Treatment of monomorphic bloodstream trypanosomes with pCPTcAMP could be a useful method for identifying the genes involved in the slender-to-stumpy differentiation process.     

Deletion of a novel protein kinase with PX and FYVE-related domains increases the rate of differentiation of Trypanosoma brucei

Growth control of African trypanosomes in the mammalian host is coupled to differentiation of a non-dividing life cycle stage, the stumpy bloodstream form. We show that a protein kinase with novel domain architecture is important for growth regulation. Zinc finger kinase (ZFK) has a kinase domain related to RAC and S6 kinases flanked by a FYVE-related zinc finger and a phox (PX) homology domain. To investigate the function of the kinase during cyclical development, a stable transformation procedure for bloodstream forms of differentiation-competent (pleomorphic) Trypanosoma brucei strains was established. Deletion of both allelic copies of ZFK by homologous recombination resulted in reduced growth of bloodstream-form parasites in culture, which was correlated with an increased rate of differentiation to the non-dividing stumpy form. Growth and differentiation rates were returned to wild-type level by ectopic ZFK expression. The phenotype is stage-specific, as growth of procyclic (insect form) trypanosomes was unaffected, and Deltazfk/Deltazfk clones were able to undergo full cyclical development in the tsetse fly vector. Deletion of ZFK in a differentiation-defective (monomorphic) strain of T. brucei did not change its growth rate in the bloodstream stage. This suggests a function of ZFK associated with the trypanosomes' decision between either cell cycle progression, as slender bloodstream form, or differentiation to the non-dividing stumpy form.