Bloodstream Trypanosomes Research Articles

Isolation of bloodstream trypanosomes by sedimentation field‐flow fractionation

Isolation of bloodstream trypanosomes by sedimentation field‐flow fractionation

Isolation of bloodstream trypanosomes by sedimentation field‐flow fractionation

Isolation of bloodstream trypanosomes by sedimentation field‐flow fractionation

Isolation of bloodstream trypanosomes by sedimentation field-flow fractionation

Isolation of bloodstream trypanosomes by sedimentation field-flow fractionation

Polysomal, procyclin mRNAs accumulate in bloodstream forms of monomorphic and pleomorphic trypanosomes treated with protein synthesis inhibitors

The major surface antigen of insect stage (procyclic and epimastigote form) Trypanosoma brucei is termed procyclin or procyclic acidic repetitive protein (PARP). Procyclin/PARP is not expressed in bloodstream form parasites, which are coated instead with the variant surface glycoprotein (VSG). Although procyclin/PARP protein is not present and the mRNA is barely detectable, procyclin/PARP genes are transcribed at this life cycle stage. We examined the mechanism for down-regulation of procyclin/PARP expression in bloodstream trypanosomes by using protein synthesis inhibitors to effect accumulation of procyclin/PARP transcripts. We show that the accumulation is not due to increased transcription of procyclin/PARP genes. Further, transcripts which accumulate under these conditions are of mature size, polyadenylated and polysome-associated indicating that normally, in bloodstream trypanosomes, down-regulation of procyclin/PARP expression is exerted either during transcript processing or at the level of mRNA stability. A comparison of the inhibitor-induced accumulation of procyclin/PARP transcripts in bloodstream forms of monomorphic and pleomorphic cell lines of trypanosome stock EATRO 795 shows that accumulation occurs with similar kinetics in both cell lines.

The effect of host blood in the in vitro transformation of bloodstream trypanosomes by tsetse midgut homogenates.

Midgut homogenates prepared from Glossina morsitans morsitans, that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma brucei into procyclic (midgut) forms in vitro. Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.

Lipid metabolism in Trypanosoma brucei: utilization of myristate and myristoyllysophosphatidylcholine for myristoylation of glycosyl phosphatidylinositols.

Myristate is the exclusive fatty acid species in the glycosyl phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG). [3H]Myristate can be incorporated into T. brucei GPIs by two distinct processes known as fatty acid remodelling and myristate exchange. Myristoyllysophosphatidylcholine (M-LPC) can also serve as a myristate donor for VSG in trypanosomes [Bowes, Samad, Jiang, Weaver and Mellors (1993) J. Biol. Chem. 268, 13885-13892]. We have studied in detail the myristoylation of GPIs using a [3H]M-LPC substrate. Labelling of VSG and free GPIs by [3H]M-LPC in cultured trypanosomes occurred at the same rate as with [3H]myristate. Concurrent with GPI labelling, there was rapid hydrolysis of [3H]M-LPC to generate extracellular [3H]myristate. Experiments in a trypanosomal cell-free system indicated that GPI labelling by fatty acid remodelling and myristate exchange was also equally efficient with [3H]M-LPC and [3H]myristate. Furthermore, both ATP and CoA are required for the myristoylation of GPIs by [3H]M-LPC. These experiments suggest that GPI myristoylation from M-LPC involves hydrolysis of M-LPC to free myristate. To address the physiological importance of myristate and M-LPC in VSG myristoylation, we radiolabelled trypanosomes in vivo with both substrates in medium containing serum, and found that [3H]myristate labelled VSG and GPIs more efficiently. Thus, VSG myristoylation by free myristate may be favoured in bloodstream trypanosome infections.

Differential Expression of the Expression Site-associated Gene I Family in African Trypanosomes

A minimum of 20 different mRNA species encoding related members of the expression site-associated gene I (ESAG-I) family occur in metacyclic variant antigen type 4 bloodstream trypanosomes. None of these ESAG-I mRNAs are derived from the metacyclic variant antigen type 4 variant surface glycoprotein (VSG) gene expression site, and some appear to come from pseudogenes. The ESAG-Is are transcribed in both procyclic and bloodstream trypanosomes, but their mRNAs accumulate to a detectable steady state level only in bloodstream trypanosomes. At least five different groups of 3'-untranslated regions (3'-UTRs) are represented among these ESAG-I mRNAs, suggesting that the 3'-UTR does not contribute to their differential expression. Some ESAG-I mRNAs completely lack a 3'-UTR or have only a single nucleotide as a 3'-UTR. Transcription of the ESAG-Is is sensitive to alpha-amanitin, indicating that they are transcribed by a different RNA polymerase than the VSG genes. These results collectively demonstrate that ESAG-I's are a heterogeneous population that can be expressed independently of VSG genes, but like the VSG genes, their mRNAs are present in the bloodstream stage of the parasite and not in the procyclic stage.

Post-transcriptional control of hsp70 mRNA in Trypanosoma brucei

The control of hsp70 mRNA levels was investigated using transgenic bloodstream and procyclic trypanosomes. Heat shock of procyclic and bloodstream trypanosomes caused no significant change in overall protein synthesis, but led to a 2–3-fold increase in the relative hsp70 mRNA level in bloodstream trypanosomes. Incubation of procyclic trypanosomes at 35°C for up to 18 h increased the level of hsp70 mRNA only marginally. The expression of actin and hsp70 mRNAs was markedly reduced in late log phase procyclic trypanosomes but PARP mRNA levels remained constant. Measurements of phleomycin-binding-protein RNAs bearing 3′- and 5′-untranslated regions from the actin, PARP or hsp70 loci indicated that both the heat-shock and cell-density effects were mediated by the untranslated regions. No significant promoter activity was detected in the different hsp70 locus intergenic regions in transient assays.

An Mr 145000 low-density lipoprotein (LDL)-binding protein is conserved throughout the Kinetoplastida order

In view of the importance of the low-density lipoprotein (LDL)-receptor in Trypanosoma brucei, we have examined whether other bloodstream trypanosomes of medical and veterinary importance ( T.b. rhodesiense, T. equiperdum, T. vivax, T. congolense), but also related parasites developing in mammalian ( Leishmania donovani) and non-mammalian hosts ( Crithidia luciliae and Phytomonas sp. isolated from Euphorbia), would possess an LDL-receptor of their own. (1) All these parasites specifically accumulate human 125I-LDL with a relatively 2.5-fold higher rate for bloodstream trypanosomes. (2) A mixture of monoclonal antibodies raised against T.b. brucei LDL-receptor inhibit binding of LDL to all species but with different efficiency. (3) A single glycoprotein of similar M r (gp145) is isolated by LDL-affinity chromatography from all the above species, as well as from both human serum-resistant and sensitive strain of T.b. rhodesiense, and from the bodonid member of the Kinetoplastida Trypanoplasma borelli. (4) Several control experiments including 35S-metabolic labeling of procyclic T.b. brucei and of C. luciliae followed by LDL-affinity chromatography or immunoprecipitation demonstrate that gp145 is indeed synthesised by the parasites and is not a contaminant of the experimental system. (5) In immunoblots and ELISA, these gp145 cross-react with the polyclonal and monoclonal antibodies raised against the LDL-receptor of T.b. brucei, the highest degree of cross-reactivity being found among the members of the Trypanozoon subgroup. (6) Finally, immunisation of mice with the purified LDL-receptor from one strain of T.b. brucei is not sufficient to confer durable protection against another strain of this parasite.

The human trypanosomiases (sleeping sickness and Chagas disease)

The African and American trypanosomiases remain major public health problems. Parasitological diagnosis is often difficult because of the low number of blood-stream trypanosomes present. A number of advances in diagnostic methods have been made recently, ranging from direct parasite detection to polymerase chain reaction-based methods. Vector control remains the most effective transmission control measure in most ecological situations for both types of disease. Chemotherapy and prophylaxis do not hold much promise, except in African trypanosomiasis where a new but expensive drug is now available, and there is better understanding of the pharmacological properties of the established drugs. Impressive advances in our understanding of human trypanosome biology are being made in many areas of study; whether these advances can be translated into improved control and treatment remains to be seen.

Identification and partial purification of a stage-specific 33 kDa mitochondrial protein as the alternative oxidase of the Trypanosoma brucei brucei bloodstream trypomastigotes.

The glycerophosphate oxidase (GPO), the unique terminal oxidase of bloodstream trypanosome (TAO), appears to be functionally similar to the alternative oxidases of some plants and higher fungi. Immunoblotting of mitochondrial proteins of bloodstream trypomastigotes of Trypanosoma brucei with monoclonal or polyclonal antibodies to Sauromatum guttatum (voodoo lily) and Symplocarpus foetidus (skunk cabbage) alternative oxidases respectively revealed two proteins of about 33 kDa (p33) and 68 kDa (p68). These proteins are not present in procyclic trypomastigotes. Electrophoresis under rigorous denaturing conditions indicated p68 to be the dimer of p33. Indirect immunofluorescent studies of bloodstream and procyclic trypomastigotes with monoclonal antibody to plant alternative oxidase also showed the localization of 33 kDa protein in the mitochondria of the bloodstream trypomastigotes. The functional TAO activity could be solubilized efficiently from the mitochondrial membrane of the bloodstream trypomastigotes by 1% NP-40 or 10 mM lauryl maltoside. When fractionated by Superose 12 gel filtration chromatography, p33 was co-purified with the TAO enzymatic activity. The apparent molecular size of the active enzyme complex was found to be 160 kDa. Gradual disappearance of the 33 kDa protein and the TAO enzymatic activity were well correlated during in vitro differentiation of the bloodstream to procyclic trypomastigotes. This study implies that the net biosynthesis of p33, an essential subunit of TAO, is decreased during differentiation from bloodstream to procyclic trypomastigotes.

Developmentally Regulated Sensitivity of Trypanosoma brucei brucei to the Cytotoxic Effects of Human High-Density Lipoprotein

The bloodstream developmental stages of the protozoan parasite Trypanosoma brucei brucei are lysed by normal human serum. The cytotoxic factor is a minor sub-class of human high-density lipoprotein termed trypanosome lyric factor (TLF). T. b. brucei rapidly develops resistance to TLF when incubated at 26°C under conditions which allow differentiation to the procyclic, insect midgut developmental stage. This in vitro differentiation system allowed us to correlate loss of TLF sensitivity with other parameters of differentiation to the procyclic form. The onset of resistance to TLF occurs within 2 hr after shifting bloodstream forms to differentiation conditions. TLF resistance is correlated with a rapid but transient decrease in protein synthesis by the parasite, is acquired prior to cell division at 26°C, and precedes the loss of variant surface glycoprotein. Tn addition, we found binding and uptake of TLF by established procyclic trypanosomes was reduced approximately fivefold relative to bloodstream trypanosomes and the TLF binding observed in procyclics was nonspecific. No TLF was bound to the procyclic flagellar pocket membrane and the procyclics failed to endocytose any of the surface-bound TLF. In contrast, bloodstream forms bind TLF via a flagellar pocket-localized protein and bound TLF is taken up;by endocytosis. These findings suggest that resistance of procyclic T. b. brucei to TLF-mediated lysis is due to a reduction in the endocytosis of TLF by this developmental stage of the parasite.

Inhibition of Protein Phosphatase 1 and 2A Down-Regulates β-Tubulin Gene Expression in Trypanosoma rhodesiense

We studied the effect of okadaic acid, a specific inhibitor of serine/threonine protein phosphatase types 1 and 2A, on the expression of β-tubulin and procyclin genes in Trypanosoma rhodesiense. Okadaic acid was found to decrease the steady-state level of β-tubulin mRNA about 5-fold in differentiating bloodstream trypanosomes and about 3-fold in established procyclic trypanosomes. No effect was observed on the expression of the procyclin gene. The down-regulation of β-tubulin gene expression by okadaic acid in procyclic trypanosomes occurs at the post-transcriptional level. These results demonstrate the involvement of protein phosphatase 1 and/or 2A activity in maintaining the steady-state level of β-tubulin mRNA in African trypanosomes.

The putative promoter for a metacyclic VSG gene in African trypanosomes

During their metacyclic developmental stage, African trypanosomes are coated with one of 12–15 variant surface glycoproteins (VSGs) that define different metacyclic variant antigen types (MVATs). The MVAT VSG genes are located near telomeres of large chromosomes and are expressed without rearrangement in the metacyclic stage. We have cloned and examined the telomere-linked MVAT5 VSG gene and its upstream expression site associated gene (ESAG I) which are separated by 4.5 kb. Within this 4.5-kb intergenic region is an 87-bp sequence that serves as a strong promoter for a luciferase reporter gene in transient transfection assays. This 87-bp sequence is similar, but not identical, to the promoter for another MVAT VSG gene. UV irradiation experiments were used to detect RNA synthesis from this MVAT5 promoter in bloodstream trypanosomes expressing an unrelated VSG. We propose that this sequence is a specific promoter for the MVAT5 VSG mRNA that occurs in about 10% of the trypanosome population during the metacyclic stage of the parasites' life cycle.

Generation by limited proteolysis of a catalytically active 39-kDa protein from the 115-kDa form of phosphatidylinositol-glycan-specific phospholipase D from bovine serum.

It has been suggested previously that small amounts of the mature 115-kDa form of phosphatidylinositol (PtdIns)-glycan-specific phospholipase D from bovine serum may exist as a 47-kDa form which can also be generated in vitro by treatment with proteases. In this study, we investigated the possible proteolytic processing by trypsin of partially purified PtdIns-glycan- specific phospholipase D from bovine serum and found that tryptic digestion caused an apparent activation of the enzyme when assayed in the presence of 0.1% (mass/vol.) Triton X-100. Trypsin cleaved the 115-kDa form of PtdIns-glycan-specific phospholipase D into three major polypeptides with molecular masses of 33, 39, and 47 kDa. Under non-denaturing conditions, the polypeptides remained tightly but noncovalently associated with each other. However, in the presence of 6 M urea, the polypeptides could be separated by anion-exchange chromatography. After renaturation, PtdIns-glycan-specific phospholipase D activity was found to be associated with a 39-kDa fragment. Based on its size and its amino acid sequence, the active-site-containing fragment consisted of approximately 275 residues of the N-terminal region of PtdIns-glycan-specific phospholipase D. The active 39-kDa fragment hydrolyzed the PtdIns-glycan-anchors of solubilized acetylcholinesterase from bovine erythrocytes and variant surface glycoprotein from blood stream trypanosomes. However, this fragment was inactive on membrane-associated acetylcholinesterase and PtdIns.

A Monocistronic Transcript for a Trypanosome Variant Surface Glycoprotein

Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45- to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.

A monocistronic transcript for a trypanosome variant surface glycoprotein.

Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45- to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.

Reconstitution of a surface transferrin binding complex in insect form Trypanosoma brucei.

In the bloodstream of the mammalian host, Trypanosoma brucei takes up host transferrin by means of a high-affinity uptake system, presumably a transferrin receptor. Transferrin-binding activity is seen in the flagellar pocket and is absent in insect form trypanosomes. By transfection we have reconstituted a transferrin-binding complex in insect form trypanosomes. Formation of this complex requires the products of two genes that are part of a variant surface glycoprotein expression site, expression site-associated gene (ESAG) 6 (encoding a protein with GPI-anchor) and ESAG 7 (encoding a protein without any obvious membrane attachment). This complex can be precipitated by transferrin-Sepharose and by an antibody directed only against the ESAG 6 protein. Transfection of ESAG 6 or 7 alone did not result in transferrin binding. In the transfected trypanosomes, the products of ESAG 6 alone and the combination of ESAG 6 and 7 did not exclusively localize to the flagellar pocket, but were present all over the surface of the trypanosome. The reconstituted transferrin-binding complex also did not result in the uptake of transferrin. Additional proteins present in bloodstream trypanosomes, but not in sufficient amounts in insect form trypanosomes, may therefore be required for the correct routing of the transferrin-binding complex to the flagellar pocket, and for its rapid internalization after ligand binding.

A strand bias occurs in point mutations associated with variant surface glycoprotein gene conversion in Trypanosoma rhodesiense.

We previously described a bloodstream Trypansoma rhodesiense clone, MVAT5-Rx2, whose isolation was based on its cross-reactivity with a monoclonal antibody (MAb) directed against a metacyclic variant surface glycoprotein (VSG). When the duplicated, expressed VSG gene in MVAT5-Rx2 was compared with its donor (basic copy) gene, 11 nucleotide differences were found in the respective 1.5-kb coding regions (Y. Lu, T. Hall, L. S. Gay, and J. E. Donelson, Cell 72:397-406, 1993). Here we describe a characterization of two additional bloodstream trypanosome clones, MVAT5-Rx1 and MVAT5-Rx3, whose VSGs are expressed from duplicated copies of the same donor VSG gene. The three trypanosome clones each react with the MVAT5-specific MAb, but they have different cross-reactivities with a panel of other MAbs, suggesting that their surface epitopes are similar but nonidentical. Each of the three gene duplication events occurs at a different 5' crossover site within a 76-bp repeat and is associated with a different set of point mutations. The 35, 11, and 28 point mutations in the duplicated VSG coding regions of Rx1, Rx2, and Rx3, respectively, exhibit a strand bias. In the sense strand, of the 74 total mutations generated in the three duplications, 54% are A-to-G or G-to-A (A:G) transitions and 7% are C:T transitions, while 26% are C:A transversions and 13% are C:G transversions. No T:G or T:A transversions occurred. Possible models for the generation of these point mutations are discussed.

Identification of a specific epitope on the extracellular domain of the LDL-receptor of Trypanosoma brucei brucei

We have previously shown that an antiserum raised against the 86-kDa fragment of the low-density lipoprotein-receptor (LDL-receptor) of bloodstream Trypanosoma brucei brucei shows extensive cross-reactivity with the mammalian LDL-receptor [7]. Here we report on the production and characterisation of 30 monoclonal antibodies (mAbs) raised against the same 86-kDa fragment of the T. b. brucei LDL-receptor. Of these, only 8 mAbs recognise in an ELISA test the purified (presumably intact) 145-kDa LDL-receptor. Seven of them also recognise the LDL-receptors isolated from rat and rabbit, whereas one mAb (1A9) is specific for the trypanosome LDL-receptor. A pool of several mAbs inhibits by 90% the specific binding of 125I-LDL to trypanosomes at 4°C, but does not interfere with binding of 125I-LDL to rat fibroblasts. 125I-mAb 1A9 is efficiently taken up by T. b. brucei at 30°C and its uptake is inhibited by an excess of unlabelled LDL particles, indicating that mAb 1A9 follows the LDL-receptor pathway. Uptake of 125I-mAb 1A9 by rat fibroblasts is less efficient and is not significantly reduced by an excess of unlabelled LDL. MAb 1A9 as well as other pooled mAbs activate rabbit complement, leading to lysis of trypanosomes in vitro. We conclude that the T. b. brucei LDL-receptor contains at least one specific epitope that is accessible on live cells to antibodies and which can activate the complement system.