Pleomorphic Trypanosoma Brucei Research Articles

The Occurrence of Malignancy in Trypanosoma brucei brucei by Rapid Passage in Mice.

Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals.

Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro.

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo, we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms. BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro, procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.

Culturing and Transfection of Pleomorphic Trypanosoma brucei.

Cultivation of pleomorphic Trypanosoma brucei strains was introduced in 1996 when matrix dependence of growth of natural isolates was recognized. Semisolid agarose or liquid methylcellulose are currently used and here we provide optimized protocols for these culture methods and for transfection of pleomorphic strains. Although more laborious than standard liquid culture, culture of native pleomorphic strains is important for a number of research questions including differentiation, virulence, tissue tropism, and regulated metabolism. Some subclones of pleomorphic strains have acquired matrix independence upon passage in culture but maintained a pleomorphic phenotype. It appears that matrix dependence and pleomorphism are not tightly linked traits, yet phenotypes have to be verified before choosing one of these subclones for given experiments. Based on direct comparisons, we give recommendations for pleomorphic strain selection and culture conditions that guarantee truly pleomorphic and differentiation competent Trypanosoma brucei.

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. Index Descriptors and Abbreviations: Trypanosoma brucei; culture-adapted bloodstream forms; differentiation; cell cycle arrest; cAMP, adenosine 3 ′,5 ′-cyclic monophosphate; pCPTcAMP, 8-(4-chlorophenylthio)-cAMP; pCPTcGMP, 8-(4-chlorophenylthio)-guanosine 3 ′,5 ′-cyclic monophosphate; 4 ′,6-diamidino-2-phenylindole, DAPI; PBS, phosphate-buffered saline; HBSS, Hank’s balanced salt solution.

High molecular mass agarose matrix supports growth of bloodstream forms of pleomorphic Trypanosoma brucei strains in axenic culture

Primary axenic culture of Trypanosoma brucei bloodstream forms almost invariably requires a period of culture adaptation with cell death and clonal selection. This has been particularly difficult and in many cases unsuccessful for natural pleomorphic strains, which are characterized by their ability to differentiate from replicating long slender bloodstream forms into short stumpy forms. Here we show that a representative set of pleomorphic T. brucei strains can be cultured in vitro on agarose plates without any previous adaptation period and selection. The slender morphology was retained and the growth rate was identical to the growth rate in vivo. Long term in vitro culture for 3 months with this method did not affect the ability of the AnTat 1.1 strain to give rise to pleomorphic infections in mice. Stumpy populations emanating from these infections transformed rapidly and synchronously into dividing procyclic forms when triggered with cis-aconitate and a temperature shift. The growth supporting activity of agarose plates could be traced to high molecular mass polymeric agarose; β-agarase digestion destroyed the activity. Membrane chamber experiments show that direct physical contact of trypanosomes with the agarose matrix is essential. In the absence of high molecular mass agarose, the cell division process is grossly impaired. We suggest that agarose mimics an interaction of trypanosomes with the host's extracellular matrix. Applications of the culture method are discussed.

The in vitro differentiation of pleomorphic Trypanosoma brucei from bloodstream into procyclic form requires neither intermediary nor short-stumpy stage

Our studies on the in vitro differentiation of a pleomorphic Trypanosoma brucei strain TREU667 indicate that the parasite differentiates directly from long-slender into procyclic form when incubated in Cunningham's medium at 26°C. The intermediary and the short-stumpy bloodstream forms harvested from infected mice can also differentiate directly into procyclic form in vitro with time courses similar to that for the long-slender form. Thus, none of the three bloodstream forms appear to be significantly better preadapted for differentiation. Tricarboxylic acid (TCA) cycle intermediates cis-aconitate and l-citrate can shorten the initial lag phase of the differentiation, but an essential trigger appears to be the temperature shift from 37°C to 26°C, when other TCA cycle intermediates such as l-proline, l-malate, α-ketoglutarate, fumarate and succinate are present in Cunningham's medium. The ornithine decarboxylase (ODC) activity in T. brucei showed a gradual increase and the ODC mRNA level remained constant during the differentiation. dl-α-Difluoromethylornithine (DFMO), putrescine, dibutyryl cAMP and theophylline all exerted no discernible effect on the in vitro process, which suggests that neither cAMP increase nor polyamine depletion could be counted among the triggers of T. brucei differentiation. A monomorphic T. brucei strain EATRO110 was tested in the same medium at 26°C but was unable to differentiate.

Active suppression of interleukin 2 secretion in mice infected with Trypanosoma brucei AnTat 1.1.E.

Impairment of T cell proliferation in mice infected with the pleomorphic Trypanosoma brucei AnTat 1.1 E clone was found not to be related to a depletion of T cells or to an absence of functional accessory cells, but rather to an active suppression of interleukin 2 (IL-2) production. Lymph node cells derived from infected mice failed to produce IL-2 following Con A stimulation, whereas an exogenous supply of recombinant IL-2 could restore the impairment of the mitogen (Con A)-induced proliferative responses. Furthermore, lymph node cells derived from infected mice suppressed both secondary T-cell proliferative responses and IL-2 secretion, indicating that the trypanosome-induced suppression is mediated by a suppressive cell which interferes at the level of IL-2 secretion.

Analysis of B cell and T cell proliferative responses induced by monomorphic and pleomorphic Trypanosoma brucei parasites in mice.

Cells collected from C57B1/6 mice infected with monomorphic and pleomorphic clones of Trypanosoma brucei parasites (ILTat 1.4 and GUTat 3.1) were analysed for the incorporation of 125I-Iododeoxyuridine into DNA of total splenic lymphocytes and of B and T lymphocytes isolated on a fluorescence activated cell sorter. The monomorphic T. brucei ILTat 1.4 parasites triggered delayed and low splenic DNA synthetic responses in comparison to those arising in mice infected with the pleomorphic T. brucei GUTat 3.1 organisms. Mice infected with both parasite clones mounted splenic DNA synthetic responses similar to those arising in animals infected with the pleomorphic organisms alone and similar responses were induced by lethally irradiated T. bruceiGUTat 3.1 and T. brucei ILTat 1.4 parasites. In mice infected with the pleomorphic parasites, DNA synthesis was first detected in the T cell population and B cell DNA synthetic responses were detected between 1 and 2 days later. In contrast only T cell DNA synthetic responses were detected after infection with the monomorphic T. brucei ILTat 1.4 parasites. It is suggested that the previously reported failure of monomorphic T. brucei parasites to trigger antibody production in infected mice is a result of their inability to stimulate B lymphocytes.

Trypanosoma brucei variable surface antigen is released by degenerating parasites but not by actively dividing parasites.

Surface antigen biosynthesis and fate in monomorphic and pleomorphic Trypanosoma brucei was examined to assess how slender and stumpy form T. brucei parasites present their variant specific glycoprotein (VSG) to the host immune system. Monomorphic and pleomorphic T. brucei did not release recently synthesized VSG in vitro. Slender form T. brucei, either from monomorphic or pleomorphic populations, did not release VSG in vivo. Detection of free VSG in plasma from irradiated mice infected with pleomorphic parasites correlated with the appearance of stumpy form parasites and possibly arose as a result of degeneration of those parasites. The in vivo released VSG was found to react well with some but not all antibodies directed against VSG determinants. Monoclonal and monospecific antibodies which react with VSG on living trypanosomes did not react with the released VSG whereas VSG-specific monoclonal antibodies which do not react with the surface of living T. brucei did react with the released VSG. It was unclear whether released VSG had lost a conformational determinant expressed on trypanosome-attached VSG or whether antibodies which react strongly with VSG on living trypanosomes are of such low avidity that they fail to bind released VSG. The results suggest that trypanosome-attached VSG is more important for stimulation of protective humoral responses than released VSG. The requirements for stimulation of protective anti-VSG responses are reported elsewhere (Sendashonga & Black 1982).

Relapse of monomorphic and pleomorphic Trypanosoma brucei infections in the mouse after chemotherapy.

Infections in mice were initiated with trypomastigotes from two lines of Trypanosoma brucei derived from the same primary isolated. Infections with one line were initiated by inoculation of metacyclic trypomastigotes from infected tsetse flies and the resulting infections were pleomorphic. The other line had been passaged 32 times in rodents and inoculation of bloodstream trypomastigotes gave rise to monomorphic infections. In both infections there were high levels of parasitaemia until death up to 4 weeks later if the infection was untreated. It was shown that after chemotherapy with 40 mg/kg diminazene aceturate (Berenil) relapses occurred in both types of infection after an aparasitaemic period of 2--3 weeks. Further, it was shown that 3 days after chemotherapy, brain tissue but neither spleen, liver nor blood was capable of transferring infection to normal recipient mice. There were two major differences in the response of the two infections to chemotherapy. First, treatment of the pleomorphic infection as soon as day 6 after infection resulted in a subsequent relapse while the monomorphic infection had to be at least 12 days old at the time of treatment before occurred. Second, following treatment of the pleomorphic, but not of the monomorphic infection there was an early transient recrudescence of low numbers of trypanosomes which were found to be non-infective to recipient mice. The early transient relapse was followed by a further aparasitaemic period and then the late continuous relapse characterised by large numbers of infective trypanosomes.

Infectivity of Monomorphic and Pleomorphic Trypanosoma brucei Stocks Cultivated at 28 C with Various Tsetse Fly Tissues

Noninfective procyclic forms of Trypanosoma brucei stocks derived from the pleomorphic EVE 10 were cultivated at 28 C in Cunningham's liquid medium in the presence of head-salivary gland, alimentary tract, and abdominal body wall explants of Glossina morsitans morsitans. After 8 to 10 days of cultivation some of the procyclic forms transformed into metacyclic stages infective for mice. Infectivity persisted for varying periods up to 66 days, when the experiments were terminated. Only 10 explants of alimentary tract or abdominal body wall tissues were required in the flasks to render the culture infective for most of the mice inoculated. Similar trypanosome suspensions grown with 10 head-salivary gland explants produced an infection on only one occasion. Cultures of procyclic organisms derived from the monomorphic stock 427 grown inthe presence of all three types of tsetse fly explants produced only sporadic infections in mice. Metacyclic forms failed to develop in trypanosome populations of stock EVE 10 cultivated at 28 C in the liquid medium alone or supplemented with mouse embryo tissues.

Synthesis and content of polyamines in bloodstream Trypanosma brucei.

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.

New culture medium for maintenance of tsetse tissues and growth of trypanosomatids.

SYNOPSIS. A new culture medium (SM), based on the amino‐acid composition of tsetse hemolymph and containing fetal bovine serum, was designed for the maintenance of tsetse organs and the cultivation of various trypanosomatids. For optimum growth 20% (v/v) serum was required. The medium supported prolonged peristalsis of the alimentary tract and salivary glands of pre‐emerged Glossina morsitans morsitans. In established cultures, derived from bloodstream forms of pleomorphic Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense strains, inocula of ∼ 106 procyclics/ml yielded 4–5 × 107 organisms/ml after 4 or 5 days of incubation at 28 C. Bloodstream forms of a cloned monomorphic T. b. brucei strain were also able to transform into procyclics, which, however, multiplied at a lower rate, with maximum yields of ∼ 2 × 107 after 5 days. Cultures of Trypanosoma congolense and of a nearly monomorphic Trypanosoma brucei gambiense strains could be established in SM medium only in the presence of tsetse alimentary tract. The procyclic trypomastigotes of these species, adapted to SM medium and able to grow in it without Glossina organs, gave maximum populations of ∼ 4.5 × 107 cells/ml.Promastigotes of Leishmania donovani, cultivated routinely in a diphasic Table's medium, multiplied actively upon being transferred into SM medium, producing yields of ∼ 4 × 107 cells/ml.