A single-point mutation in the RNA-binding protein 6 generates Trypanosoma brucei metacyclics that are able to progress to bloodstream forms in vitro.

We previously established an in vitro differentiation system based on the inducible expression of the RNA binding protein 6 (RBP6), which initiated differentiation of Trypanosoma brucei non-infectious procyclics to infectious metacyclics (MFs). However, further differentiation to bloodstream forms (BFs) required infection of mice. Here we report the serendipitous isolation of a single point mutation in RBP6 (Q109K), whose expression not only generated MFs, but purified MFs continued the developmental cycle in vitro to BFs expressing variant surface glycoprotein-2 (VSG-2), formerly known as VSG 221. This transition occurred over a period of 11 days and by RNA-Seq, VSG-2 was first measureable on day 1, whereas metacyclic VSGs were detected up to 8 days. We further showed that inducible expression of mutant RBP6 appeared to skip the intermediate epimastigote stage and we highlight the potential involvement of RBP33 in the establishment of metacyclics and in particular in the generation of metacyclics uncharacteristically arrested at the G2/M checkpoint.

Trypanosoma cruzi strains in the Calomys callosus: parasitemia and reaction of intracellular forms with stage-specific antibodies in the acute and chronic phase of infection and after immunosuppression.

An experimental model for chronic Chagas disease was developed to investigate whether reactivation is influenced by the genetic origin of Trypanosoma cruzi isolates. In addition, we examined whether the distribution of T. cruzi stage-specific epitopes, as defined by monoclonal antibodies (Mab), raised against mammalian-stage parasite forms, exhibited comparable distribution patterns in Calomys callosus myocardium during the acute phase and after reactivation of the infection. Animals were infected with parasites of the G (T. cruzi I), Y (T.cruzi II) or CL strains (T. cruzi VI). Heart sections were labelled with the Mabs 2C2, 1D9, 2B7, 3B9 and 4B9, which react with carbohydrate epitopes on Ssp-4, a major amastigote surface glycoprotein. Mab 1D9 and 2B7 showed polymorphic distributions over amastigotes among animals infected with the G, Y or CL strains. Mab 3B2, which recognises a non-carbohydrate epitope in flagellated forms, showed an active state of parasite dissemination in the myocardium of C. callosus that were infected with Y or CL strains and then immunosuppressed after 6 or 12 months. C. callosus infected with the G strain (T. cruzi I) displayed absence of amastigote nests in the heart after immunosuppression. Our results permit us to suggest that parasites of the G strain may be more sensitive to the immune response, since we could not find either evidence of parasitemia or amastigote nests. Conversely, parasites from the Y and CL strains appeared able to escape the immune response, as evidenced by an inflammatory infiltrate and disseminated infection after immunosuppression.

Involvement of Ssp-4-related carbohydrate epitopes in mammalian cell invasion by Trypanosoma cruzi amastigotes.

We examined whether the expression of Ssp-4-related carbohydrate epitopes defined by monoclonal antibodies 1D9 and 2B7 was related to cell invasion by Trypanosoma cruzi amastigotes from different isolates and whether the highest expression of the epitope defined by MAb 1D9 would confer greater infectivity. Confocal microscopy showed that both epitopes localize to the membrane of amastigotes from 569, 588, 573, 587 and SC2005 isolates, similar to the G isolate, whereas the CL isolate showed a punctate and diffuse staining. Flow cytometry revealed inter- and intra-isolate variable expression of these epitopes. Apart from the lower expression of MAb 2B7 epitope by intracellular amastigotes of the SC2005 isolate, amastigotes from chagasic patient isolates expressed both epitopes similar to the G isolate, in contrast to CL isolate, that showed lower expression of both epitopes. MAb 1D9 did not react with CL isolate on immunoblots and reacted poorly with 588 and 587 parasites. MAb 2B7 preferentially reacted with an epitope on an 84 kDa component in G and 573 isolates. Invasion assays revealed that despite the fact that amastigotes from chagasic patient isolates displayed high levels of the epitope defined by MAb 1D9, only isolate 588 invaded host cells in levels comparable to that of isolate G. Both MAbs specifically inhibited cell invasion by G and 588, but not CL. These results suggested that the highest expression of MAb 1D9 epitope was not sufficient to confer higher infectivity on the isolate, and besides the two epitopes, other factors may modulate the invasiveness of extracellular amastigotes from the different isolates.

Comparison of the glycosyl-phosphatidylinositol cleavage/attachment site between mammalian cells and parasitic protozoa.

It was previously hypothesised that the requirements for glycosyl-phosphatidylinositol (GPI) anchoring in mammalian cells and parasitic protozoa are similar but not identical. We have investigated this by converting the GPI cleavage/attachment site in porcine membrane dipeptidase to that found in the trypanosomal variant surface glycoprotein 117 and expressing the resulting mutants in COS-1 cells. Changing the entire (omega), (omega)+1 and (omega)+2 triplet in membrane dipeptidase from Ser-Ala-Ala to Asp-Ser-Ser resulted in efficient GPI anchoring of the mutant proteins, as assessed by cell-surface activity assays and susceptibility to release by phosphatidylinositol-specific phospholipase C. Immunoelectrophoretic blot analysis with antibodies recognising epitopes either side of the native (omega) residue in porcine membrane dipeptidase, and expression of a mutant in which potential alternative cleavage/attachment sites were disrupted, indicated that alternative GPI cleavage/attachment sites had not been used. These results indicate that the requirements for GPI anchoring between mammalian and protozoal cells are not as different as previously suggested, and that rules for predicting the probability of a sequence acting as a GPI cleavage/attachment site need to be applied with caution.

Cell lysis induces redistribution of the GPI-anchored variant surface glycoprotein on both faces of the plasma membrane of Trypanosoma brucei.

African trypanosomes are coated by 10 million copies of a single variant specific glycoprotein (VSG) which are anchored in the plasma membrane by glycosylphosphatidylinositol (GPI). A GPI-specific phospholipase C (GPI-PLC) triggers fast VSG release upon cell lysis but in vivo it is safely controlled and topologically concealed from its substrate by being intracellular. One enigmatic aspect of GPI-PLC action therefore consists of how it could gain access to the VSG in the exoplasmic leaflet of the membrane. The data presented herewith disclose an unexpected possible solution for this puzzle: upon cell rupture the VSG invades the cytoplasmic face of the plasma membrane which thus becomes double coated. This unusual VSG rearrangement was stable in ruptured plasma membrane from GPI-PLC null mutant trypanosomes but transiently preceded VSG release in wild-type parasites. The formation of double coat membrane (DCM) was independent of the presence or activation of GPI-PLC, occurred both at 4 degrees C and 30 degrees C and was unaffected by the classical inhibitor of VSG release, p-choromercuryphenylsulfonic acid (PCM). DCMs conserved the same coat thickness and association with subpellicular microtubules as in intact cells and were prone to form vesicles following gradual detachment of the latter. Our data also demonstrate that: (i) GPI-PLC expressed by one trypanosome only targets its own plasma membrane, being unable to release VSG of another parasite; (ii) DCMs concomitantly formed from trypanosomes expressing different VSGs do not intermix, an indication that DCM might be refractory to membrane fusion.

Mechanisms mediating antigenic variation in Trypanosoma brucei.

Antigenic variation in Trypanosoma brucei is a highly sophisticated survival strategy involving switching between the transcription of one of an estimated thousand variant surface glycoprotein (VSG) genes. Switching involves either transcriptional control, resulting in switching between different VSG expression sites; or DNA rearrangement events slotting previously inactive VSG genes into an active VSG expression site. In recent years, considerable progress has been made in techniques allowing us to genetically modify infective bloodstream form trypanosomes. This is allowing us to reengineer VSG expression sites, and look at the effect on the mechanisms subsequently used for antigenic variation. We can now begin a dissection of a highly complicated survival strategy mediated by many different mechanisms operating simultaneously.

Mild acid stress as a differentiation trigger in Trypanosoma brucei.

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 degrees 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 degrees 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+).

Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei.

The paradigm of antigenic variation in parasites is the variant surface glycoprotein (VSG) of African trypanosomes. Only one VSG is expressed at any time, except for short periods during switching. The reasons for this pattern of expression and the consequences of expressing more than one VSG are unknown. Trypanosoma brucei was genetically manipulated to generate cell lines that expressed two VSGs simultaneously. These VSGs were produced in equal amounts and were homogeneously distributed on the trypanosome surface. The double-expressor cells had similar population doubling times and were as infective as wild-type cells. Thus, the simultaneous expression of two VSGs is not intrinsically harmful.

Effects of phenothiazine neuroleptic drugs on the microtubular-membrane complex in bloodstream forms of Trypanosoma brucei.

African trypanosomes are parasitic protozoa causing sleeping sickness in humans and related diseases in domestic animals against which no entirely satisfactory forms of chemotherapy are yet available. It was previously shown that related species of trypanosomes, as well as procyclic (insect) forms of Trypanosoma brucei are extremely sensitive to the action of phenothiazine neuroleptic drugs in vitro. In this work, we have carried out a more detailed investigation of the effects of thioridazine, one of the most potent neuroleptic phenothiazine drugs known, on the morphology of the infective bloodstream forms of T. brucei, with particular reference to the parasite's prominent pellicular membrane complex. Our data show that this drug induces rapid changes in cell shape that appear to involve some reorganization of the microtubular membrane skeleton, but does not affect the structural integrity of the microtubular complex. Another early consequence of drug action involved damage to nuclear and cytoplasmic membranes and the appearance of tubular arrays of coated membrane within the flagellar pocket. It was also revealed that the drug induces a rapid release of the variant-specific glycoprotein (VSG) which makes up the surface coat protecting bloodstream forms of the parasite against the host immune system. Our evidence suggests that this release of VSG involves cleavage of the protein's glycosyl-phosphatidylinositol (GPI) membrane anchor by endogenous GPI-specific phospholipase C, probably as a consequence of minor damage to the parasite plasma membrane induced by the drug.

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 lytic factor (TLF). T.b. brucei rapidly develops resistance to TLF when incubated at 26 degrees 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 degrees C, and precedes the loss of variant surface glycoprotein. In 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.

The induction of Trypanosoma cruzi trypomastigote to amastigote transformation by low pH.

Following cell invasion, Trypanosoma cruzi trypomastigotes transform into amastigotes, which are the mammalian replicative forms of the parasite. Although amastigotes represent a critical stage in the life-cycle of T. cruzi, little is known of the factors controlling trypomastigote to amastigote transformation. Kanbera et al. (1990) observed that exposure of trypomastigotes to acidic pH induced their transformation into rounded forms resembling amastigotes. We confirm their observation and, using two strains of T. cruzi, establish that these transformants are ultrastructurally and biochemically indistinguishable from natural amastigotes. Incubation of trypomastigotes in medium at pH 5.0 for 2 h was sufficient to trigger their transformation into forms resembling amastigotes. Electron microscopical analysis confirmed that the kinetoplast structure, and general morphological features of the acid-induced, extracellular amastigotes were indistinguishable from those of intracellular-derived amastigotes. The extracellular transformation was accompanied by the acquisition of the stage-specific surface antigen of the naturally transformed amastigotes (Ssp-4), and loss of a stage-specific trypomastigote antigen (Ssp-3). Trypomastigotes incubated at neutral pH did not transform into amastigotes, and did not acquire the Ssp-4 epitope or lose the Ssp-3 epitope. Finally, acid-induced amastigotes subsequently incorporated [3H]thymidine into their DNA, indicating that the important replicative property of intracellular amastigotes is also exhibited by these in vitro transformants. This effect of low pH appears to be of physiological relevance, and acid-induced extracellular transformation appears to represent a valid experimental technique for studies of the molecular mechanisms involved in the differentiation process.

Characterization of concanavalin A-binding glycoproteins from procyclic culture forms of Trypanosoma congolense, T. simiae and T. brucei brucei.

Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) of Trypanosoma congolense, T. simiae, and T. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCF T. congolense K45/1 identified surface-accessible glycoproteins of 81.5, 59, and 38-42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high-mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies.

A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei.

In T. brucei, a transferrin-binding protein has been found to share sequence homology with pESAG-7 and -6, the products of two related genes present in the VSG gene polycistronic transcription unit. When expressed in Xenopus oocytes, they appear as N-glycosylated proteins secreted in the medium (pESAG-7) and GPI anchored to the membrane (pESAG-6). These proteins are able to homo- or heterodimerize, probably through association in the same orientation. Only heterodimers can bind Tf, possibly two molecules per dimer. A comparison of Tf binding to pESAG-7/6-expressing oocytes and trypanosomes suggests that pESAG-7/6 is the Tf receptor of the parasite. In trypanosomes, the majority of pESAG-7/6 is released from the membrane and associates, together with Tf, with a glycosylated matrix present in the lumen of the flagellar pocket. Both pESAG-7/6 and Tf are internalized via coated pits and vesicles. These observations suggest a novel mode of Tf binding and uptake in trypanosomes.

Hydrophilic-interaction chromatography of complex carbohydrates.

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

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.

Immunoelectron microscopic studies on the specific adhesion of Trypanosoma congolense to cultured vascular endothelial cells.

Bloodstream forms of Trypanosoma congolense were cocultivated in vitro with vascular endothelial cells. The trypanosomes adhere specifically to the endothelial surfaces of the anterior part of their flagella, as shown by scanning and transmission electron microscopy. The interaction between parasite and host cell is very tight, and frequently the accumulation of endocytotic vesicles near the contact site is observed. Immunoelectron microscopy revealed a compound distributed over the total surface of the trypanosomes and reacting with antibodies against the beta 1 integrin chain, but no reaction was found with anti-alpha 1 or anti-alpha 2 antibodies. Integrins are typical adhesion molecules and are now shown to be present at the surface of T. congolense by electron microscopy and by immunofluorescence. A direct participation of this substance in the specific adhesion to endothelium, however, could not be proven.

A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-phosphatidylinositol membrane anchor.

The procyclic acidic repetitive protein is the major cell-surface glycoprotein of the insect-dwelling procyclic forms of the Trypanosoma brucei species of African trypanosomes. The glycoprotein contains an acidic Glu-Pro repeat domain, a glycosyl-phosphatidylinositol membrane anchor and a putative asparagine glycosylation site. In this paper we describe a rapid purification scheme for this glycoprotein, using solvent extraction and hydrophobic interaction chromatography, and a partial characterization of the glycosylphosphatidylinositol membrane anchor. The carbohydrate composition of the anchor is extremely unusual; it contains on average nine GlcNAc, nine Gal, and five sialic acid residues. This is the first description of such a heavily substituted and negatively charged anchor. A comparison between the trypanosome procyclic surface and the Leishmania promastigote surface is also presented.

An integral membrane glycoprotein associated with an endocytic compartment of Trypanosoma vivax: identification and partial characterization.

A 65-kD glycoprotein (gp65) of Trypanosoma (Duttonella) vivax was identified using a murine monoclonal antibody (mAb 4E1) that had been raised against formalin-fixed, in vitro-propagated, uncoated forms. Intracellular localization studies utilizing the mAb in immunofluorescence on fixed, permeabilized T. vivax bloodstream forms and immunoelectron microscopy on thin sections of Lowicryl K4M-embedded cells revealed labeling of vesicles and tubules in the posterior portion of the parasite. Some mAb-labeled vesicles contained endocytosed 10 nm BSA-gold after incubation of the parasites with the marker for 5-30 min at 37 degrees C, and the greatest degree of colocalization was observed after 5 min. Double labeling experiments using the mAb and a polyclonal anti-variant surface glycoprotein (VSG) antibody to simultaneously localize both gp65 and VSG demonstrated that there was little overlap in the distribution of these antigens. Thus, gp65 is associated with tubules and vesicles that are involved in endocytosis but which appear to be distinct from VSG processing pathways within the cell. Using the mAb for immunoblot analyses, gp65 was shown to be enriched in a fraction of solubilized membrane proteins eluted from either immobilized Con A or Ricinus communis agglutinin and was found to possess carbohydrate linkages cleaved by both endoglycosidase H and O-glycosidase, suggesting the presence of N- and O-linked glycans. Protease protection and crosslinking experiments suggest that gp65 is a transmembrane protein with trypsin cleavage and NH2-crosslinking sites on the lumenal face of the vesicles.

Identification of the surface components of Trypanosoma evansi.

125I-labelling was used to characterise the surface components of five stocks of Trypanosoma evansi. Two components of 67 and 60.5 kD were labelled in two of the stocks, a single 60.5 kD component in two other stocks and no components in the remaining stock. These differences are probably related to the labelling method and biochemical differences between the stocks.

Evidence for the participation of the Ssp-3 antigen in the invasion of nonphagocytic mammalian cells by Trypanosoma cruzi.

Trypomastigotes of Trypanosoma cruzi have to invade mammalian cells in order to multiply. They bear on their plasma membrane a sialic acid-containing epitope (Ssp-3) defined by a series of monoclonal antibodies (mAbs). Previous investigations have shown that Fab fragments of these mAbs inhibit the attachment of trypomastigotes to 3T3 fibroblasts. To further define the role of Ssp-3 in invasion, here we use, as targets for infection, L cells and CHO cells stably transfected with cDNA coding for the mouse Fc receptors genes. When the trypomastigotes are incubated with small, nonagglutinating amounts of antibodies to Ssp-3, their attachment to the transfected cells is greatly enhanced, without a parallel increase in invasion. The enhancement in attachment is Fc mediated, since it is abolished by treatment of the transfected cells with mAbs to Fc receptors. In contrast, both attachment to, and invasion of, the transfected cells are increased if the parasites are incubated with polyclonal or monoclonal antibodies against T. cruzi surface membrane antigens other than Ssp-3. If, however, antibodies to Ssp-3 are added to the incubation mixtures containing any of the other anti-T. cruzi antibodies, the enhancement of invasion (but not of attachment) is reversed. These results suggest that Ssp-3-bearing molecules participate in the process of parasite internalization.