Differentiation of Trypanosoma brucei bloodstream trypomastigotes from long slender to short stumpy-like forms in axenic culture

doi:10.1016/0166-6851(90)90075-W

Molecular and Biochemical Parasitology

Volume 40, Issue 1, April 1990, Pages 13-22

https://doi.org/10.1016/0166-6851(90)90075-W Get rights and content

Abstract

An axenic cultivation system [10] was used to study the differentiation of Trypanosoma brucei bloodstream forms from long slender to short stumpy-like forms. Trypanosomes in the logarithmic phase are similar to long slender bloodstream forms freshly isolated from infected mice, differing only in the rate of oxygen uptake. In contrast, trypanosomes in the stationary phase show a decreased level of glucose oxidation, express pyrroline-5-carboxylate reductase (proline oxidase), are inhibited in oxygen uptake to about 44% by KCN, undergo considerable morphological changes on the cellular and subcellular level, and have a significantly smaller cell volume. These results are comparable to those observed during the differentiation of long slender to short stumpy forms in infected animals, suggesting that the differentiation process towards insect procyclic forms can be initiated in culture at 37°C. As judged from immunofluorescence and electron microscopy analysis, the surface coat remains intact.

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Cited by (69)

Protocol to analyze the transmigration efficiency of T. brucei using an in vitro model of the blood-cerebrospinal fluid barrier
2022, STAR Protocols
Citation Excerpt :
Always keep cell density between 1e5 / mL and 1e6 / mL. Do NOT use trypanosomes if cell density was higher than 1e6 / mL, as the parasites might have differentiated into a cell-cycle arrested “short stumpy” form (Hamm et al., 1990). Incubate diluted cells at 37°C in a water-saturated atmosphere and 5% (v/v) CO2.
At present, the only approach to investigate the transmigration of Trypanosoma brucei, the causative agent of human African trypanosomiasis, from blood to cerebrospinal fluid is through animal experiments. This protocol details how to analyze the transmigration efficiency using an in vitro model of the blood-cerebrospinal fluid (blood-CSF) barrier. We describe how to grow human choroid plexus epithelial cells on cell culture filter inserts to form the barrier, followed by isolating and quantifying genomic DNA of transmigrated parasites by qPCR.
For complete details on the use and execution of this protocol, please refer to Speidel et al. (2022).
Transmigration of Trypanosoma brucei across an in vitro blood-cerebrospinal fluid barrier
2022, iScience
Citation Excerpt :
To prevent the host’s premature death from uncontrolled parasitic growth, the parasite secretes a stumpy induction factor (SIF) which induces differentiation of proliferating slender parasites into the cell cycle arrested “short stumpy” form (Hamm et al., 1990; Reuner et al., 1997; Rojas et al., 2019; Seed and Black, 1997). On the one hand, this can be interpreted as an altruistic cell density control, since stumpy trypanosomes secrete prostaglandin D2, an autocrine inductor of apoptosis in the stumpy parasite itself (Duszenko et al., 2006; Hamm et al., 1990). On the other hand, the stumpy form is preadapted to change hosts, as it will transform into procyclic trypanosomes as described above.
Trypanosoma brucei is the causative agent of human African trypanosomiasis. The parasite transmigrates from blood vessels across the choroid plexus epithelium to enter the central nervous system, a process that leads to the manifestation of second stage sleeping sickness. Using an in vitro model of the blood-cerebrospinal fluid barrier, we investigated the mechanism of the transmigration process. For this, a monolayer of human choroid plexus papilloma cells was cultivated on a permeable membrane that mimics the basal lamina underlying the choroid plexus epithelial cells. Plexus cells polarize and interconnect forming tight junctions. Deploying different T. brucei brucei strains, we observed that geometry and motility are important for tissue invasion. Using fluorescent microscopy, the parasite’s moving was visualized between plexus epithelial cells. The presented model provides a simple tool to screen trypanosome libraries for their ability to infect cerebrospinal fluid or to test the impact of chemical substances on transmigration.
The conserved hypothetical protein Tb427.10.13790 is required for cytokinesis in Trypanosoma brucei
2018, Acta Tropica
Trypanosoma brucei, a flagellated protozoan causing the deadly tropical disease Human African Trypanosomiasis (HAT), affects people in sub-Saharan Africa. HAT therapy relies upon drugs which use is limited by toxicity and rigorous treatment regimes, while development of vaccines remains elusive, due to the effectiveness of the parasite´s antigenic variation. Here, we evaluate a hypothetical protein Tb427.10.13790, as a potential drug target. This protein is conserved among all kinetoplastids, but lacks homologs in all other pro- and eukaryotes. Knockdown of Tb427.10.13790 resulted in appearance of monster cells containing multiple nuclei and multiple flagella, a considerable enlargement of the flagellar pocket and eventually a lethal phenotype. Furthermore, analysis of kinetoplast and nucleus division in the knockdown cell line revealed a partial cell cycle arrest and failure to initiate cytokinesis. Likewise, overexpression of the respective protein fused with enhanced green fluorescent protein was also lethal for T. brucei. In these cells, the labelled protein appeared as a single dot near kinetoplast and flagellar pocket. Our results reveal that Tb427.10.13790 is essential for the parasite´s viability and may be a suitable new anti-trypanosomatid drug target candidate. Furthermore, we suggest that it might be worthwhile to investigate also other of the many so far just annotated trypanosome genes as a considerable number of them to lack human homologs but may be of critical importance for the kinetoplastid parasites.
Trypanosoma brucei aquaglyceroporins mediate the transport of metabolic end-products: Methylglyoxal, D-lactate, L-lactate and acetate
2018, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
p < 0.05, p < 0.01, p < 0.001 was considered as statistically significant and were represented with one, two or three asterisks, respectively. Trypanosoma brucei bloodstream forms were cultivated in a modified minimum essential medium containing 10% fetal bovine serum and incubated at 37 °C in a 5% CO2 atmosphere [24]. For the IC50 cytotoxicity assay, the concentration necessary to inhibit cell growth by 50% was calculated.
Bloodstream forms of Trypanosoma (T.) brucei, the causative agent of African sleeping sickness, possess a highly active glycolysis, which generates as main end-products: pyruvate under aerobic conditions, and pyruvate and glycerol under anaerobic conditions. To secrete them into the extracellular milieu, the parasites have at least two main specific membrane proteins, the pyruvate transporter and the aquaglyceroporins However, there are several other minor products from the glycolysis that must be excreted by the parasites and whose exit pathway until now remained elusive. As aquaglyceroporins from T. brucei (TbAQP1, 2, and 3) show a wide permeability profile for small solutes, we decided to evaluate if these proteins allow the passage of methylglyoxal, L-lactate, D-lactate and acetate molecules. We expressed heterologously TbAQP1, 2, and 3 in aquaglyceroporin-null yeast cells or in Xenopus laevis oocytes and demonstrated that these channels are permeable for methylglyoxal, L-lactate, D-lactate and acetate. We further demonstrate that methylglyoxal is highly toxic for bloodstream forms of T. brucei, while L-lactate and D-lactate appear almost harmless. Additionally, we discuss all our findings in the light of the novel metabolic discoveries, putting in context the participation of TbAQP1, 2, 3, and other proteins in the excretion of unwanted metabolic end-products.
The lane to the brain: How African trypanosomes invade the CNS
2014, Trends in Parasitology
African trypanosomes induce sleeping sickness. Although it is clear that this parasite moves from the blood to the central nervous system (CNS) to induce the second stage of the disease, little is known about the molecular details of this process. Considering new findings of the trypanosome localization, this opinion paper will summarize the current knowledge about CNS infection, propose a different perception of the invasion process, and discuss possible consequences for drug development.
The Trypanosoma brucei zinc finger protein ZC3H18 is involved in differentiation
2011, Molecular and Biochemical Parasitology
Citation Excerpt :
The trypanosome life cycle involves proliferative as well as cell-cycle-arrested stages in both hosts [8]. Upon attaining a threshold cell density, the long slender bloodstream form (the form that grows in the mammalian blood and tissue fluids) is subject to a quorum-sensing-like mechanism, which results in cell cycle arrest and formation of stumpy forms [9,10]. These are pre-adapted to differentiation into the procyclic form, which multiplies in the Tsetse fly midgut.
In mammalian cells, the degradation of mRNAs that have AU-rich elements in their 3′-untranslated regions is accelerated by the binding of proteins that contain two CCCH-zinc-finger-domains. Three CCCH zinc-finger proteins, TbZFP1, TbZFP2, and TbZFP3, have been shown to have roles in trypanosome differentiation. We here studied another protein, ZC3H18, which has two CCCH zinc finger domains. The ZC3H18 gene is not essential in bloodstream forms, but in an in vitro model of differentiation, depletion of ZC3H18 delayed the transformation of bloodstream-form trypanosomes to the procyclic form that grows in the Tsetse fly.

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Differentiation of Trypanosoma brucei bloodstream trypomastigotes from long slender to short stumpy-like forms in axenic culture

Abstract

Int. J. Parasitol.

Mol. Biochem. Parasitol.

J. Biol. Chem.

Cell

Mol. Biochem. Parasitol.

J. Biol. Chem.

J. Biol. Chem.

Analyt. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Mol. Biochem. Parasitol.

Developmental cycles and biology of pathogenic trypanosomes

Br. Med. Bull.

Trypanosoma brucei: biochemical and morphological changes during in vitro transformation of bloodstream to procyclic trypomastigotes

Exp. Parasitol.

Repression of glycoprotein synthesis and release of surface coat during transformation of Trypanosoma brucei

EMBO J.

Trypanosoma brucei: differentiation of in vitro-grown bloodstream trypomastigotes into procyclic forms

J. Protozool.