A modification to the life cycle of the parasite Trypanosoma brucei

Sarah Schuster, Ines Subota, Jaime Lisack, Henriette Zimmermann, Christian Reuter, Brooke Morriswood, Markus Engstler

Posted on: 13 August 2019

Preprint posted on 29 July 2019

Article now published in eLife at

A parasite voyage re-visited: Trypanosoma brucei transmission to the tsetse fly is successful upon infection with slender forms of the parasite.

Selected by Mariana De Niz


African trypanosomes are the causative parasites of sleeping sickness in humans and nagana in cattle. They are transmitted by tsetse flies upon biting mammalian hosts, given their hematophagous behaviour. In the mammalian host’s circulation, tissues, and interstitium, it is conventionally established that at least two main life cycle stages of the parasite Trypanosoma brucei exist. These stages are known as slender and stumpy, which receive their name from their characteristic morphology.  Slender forms undergo a transition into stumpy forms in response to quorum-sensing factors, or stumpy-inducing factors (SIF). This transition is believed to play two important roles: a) to regulate parasite load in the host, preventing hyperparasitemia and early death of the host which would diminish the chance of transmission; and b) to generate a stage that is pre-adapted for survival in the tsetse fly midgut following transmission. Until recently, current knowledge suggested that only stumpy forms were able to infect tsetse flies. The present work by Schuster and Subota et al (1) challenges this paradigm with novel findings that have important epidemiological implications for our understanding of T. brucei transmission from mammalian hosts to tsetse flies.


Key findings and developments

Overall key findings

  • Although conventionally thought that only stumpy forms are able to infect tsetse flies, Schuster and Subota et al propose a revision to this concept by showing that slender forms are equally capable of infecting flies.
  • While an unanswered epidemiological question is how transmission to tsetse flies can exist when brucei are present in very low parasite densities in mammalian hosts, Schuster and Subota et al show that a single parasite (slender or stumpy) is sufficient to produce an effective infection in the fly.
  • Once in the fly, slender parasites can activate expression of the stumpy marker protein associated with differentiation 1 (PAD1) and directly differentiate into procyclic (insect form) stages, without transitioning to the stumpy form and undergoing cell-cycle arrest.
Figure 1. Revised life cycle of T. brucei. While it is known that stumpy trypanosomes can establish a fly infection when taken up in the blood meal, this work shows that slender trypanosomes are equally effective for tsetse transmission.

Key tools

  • Expression of PAD1 is a proxy for transition into brucei stumpy stages. For most of their work, the authors used a parasite line whereby parasites have GFP positive nuclei when the PAD1 gene is active. This line was used to distinguish slender cells (GFP negative) from stumpy cells (GFP-positive). This was used in combination with nuclear dyes to infer the cell cycle stage of parasites during replication as T. brucei replication follows a sequential process that can be visualized through the parasite nucleus (N) and the kinetoplast (K). In this sequence, T. brucei parasites possess 1K, 1N, followed by 2K, 1N, and finally 2 K, 2N. Although slender parasites are present in all 3 ratios, stumpy forms only exist as 1K1N (thought of as a cell cycle arrest).
  • A reporter parasite line was used to study the sequence of events occurring in the fly following ingestion of either stumpy or slender parasites. This line, as the above, encodes the GFP-PAD1 marker, but also an EP1-YFP fusion which allows identifying parasites that begin expressing procyclin on their cell surface, and differentiate to the procyclic life stage.


Specific findings

  • The work quantitatively compared completion of the brucei developmental cycle in the tsetse fly, by inducing stumpy production in different ways, namely by a) ectopic expression of a second variant surface glycoprotein (VSG) isoform (i.e. using expression site-attenuated stumpy parasites), and b) parasites produced by incubation with the quorum-sensing stumpy-inducing factor (SIF). Analysis of parasite presence in tsetse flies showed that upon uptake of even a single stumpy form generated by either of the above conditions, led to robust infections in the fly midgut, proventriculus, and salivary glands, in up to 5% of flies.
  • Upon feeding flies with PAD1-negative slender trypanosomes from the aforementioned lines, infection efficiency was equal to that observed by stumpy parasite infections.
  • Infections with 1-2 slender cells had a higher transmission index than infection with equal numbers of stumpy cells, suggesting that slender cells have at least comparable developmental capacities as the stumpy cells. This finding altogether proposes a revision to the traditional view of the brucei life cycle.
  • Infection with slender trypanosomes also begins procyclin expression and differentiates to procyclic forms. Within the fly, by 48-50 h post-infection with slender trypanosomes, the entire population was GFP-PAD1 positive, and a fifth of the population was EP1:YFP positive.
  • The authors observed increasing numbers of GFP-PAD1 slender cells, and that these were capable of replicating. This shows that slender parasites activate the PAD1 pathway without being pushed to cell cycle arrest.
  • In vitro differentiation experiments using cis-aconitate and temperature drop supported the in vivo observations pointed above.

What I like about this paper

I find this paper very interesting and relevant for parasitology. On one hand because it’s a good example of how unexpected findings can be made based on careful observation and experimentation: it’s important to question established ideas in research fields, and to undertake science that proves or rejects those observations. On the other hand, the finding itself suggests a paradigm-shift for an important piece of knowledge on T. brucei transmission from the mammalian host to the insect vector. This piece of knowledge opens a new research window and could have important implications for our understanding of T. brucei interactions with the vector host, and with the mammalian host at the point of transmission. Equally, while in vitro work is key for research, and has been telling in many parasitology fields, we have often found in various parasites that the relevance and behaviour in vivo, might differ. I find it important that the experiments shown here were performed in relevant vectors.

Open questions

  1. You found that slender parasites can travel more efficiently than stumpy forms to the salivary glands of the fly. Does infection with stumpy or slender forms have a different effect on tsetse fly fitness/immune response to infection?
  2. You found that slender forms have a seamless differentiation into procyclic forms without the need to undergo differentiation into stumpy forms. Do procyclic forms arising from slender and from stumpy forms, differ genetically and molecularly?
  3. While your in vivo experiments in flies open a new research window regarding host-to-vector transmission, what implications does this finding have on how we perceive dynamics, differentiation, and reservoir establishment in the mammalian host? If slender forms and stumpy forms can transmit with equal success and establish an infection in the fly, what is the role of a tissue reservoir- like the skin- in the parasite’s life cycle?
  4. In a natural feed on an infected host, it is perhaps likely that both stumpy and slender forms are ingested by the fly. Based on your work, slender forms will most successfully invade the salivary glands, while stumpy forms are more able to establish midgut infections (yet have a more limited lifetime). Would ingestion of mixed parasite forms by the fly provide a transmission advantage?
  5. In your paper, you show that SIF-induced stumpy forms are the fastest in generating procyclic forms, as well as the ones generating procyclics in largest proportions. How do SIF-induced stumpy forms differ from stumpy forms produced in other ways?
  6. You show that even a single parasite (slender or stumpy) is able to establish infection in the fly. From research in the Plasmodium transmission field, it is known that not every mature gametocyte (the final transmission form) is infectious (2). Moreover, your group has characterized in the fly, various intermediate stages of the parasite’s life cycle in the insect vector (3), while the general concept of the bloodstream forms is that two forms exist: slender forms and stumpy forms. Are intermediate forms transmissible? Are all slender or stumpy forms equally capable of infecting the fly? If not, what is required by brucei to be infectious?
  7. Following up from the question above, you mention in you discussion that congolense infects tsetse flies without manifesting a cell cycle-arrested stumpy life cycle stage. Are all slender forms of T. congolense equally infectious, and equally capable of establishing infections in the tsetse fly?
  8. You conclude your pre-print by mentioning that your finding raises the question of what the true biological function of the stumpy life cycle stage actually is. Findings in various parasitology areas show that the production of parasite forms with arrested cycles (like stumpy forms in brucei, or gametocytes in Plasmodium) regulates parasitemia and ensures the survival of the host – as parasite lines which cannot produce such forms kill the host early in infection. In the case of Plasmodium, gametocytes are also stages which are less efficiently recognized by the immune system, establish reservoirs in tissues with less accessibility to drugs (i.e. the bone marrow and the spleen), and are more resistant to many anti-malarial drugs. This capacity of gametocytes ensures Plasmodium transmission despite a) lack of symptoms in the host, and b) clearance of asexual forms of the parasite due to treatment with anti-malarial drugs. Equally, the gametocyte reservoir in the bone marrow ensures a continuous release of mature gametocytes even after parasites in the bloodstream have been cleared. What is known of T. brucei regarding all these aspects, that would make stumpy forms a successful evolutionary adaptation?


  1. Schuster S, Subota I, Lisack J, Zimmermann H, Reuter C, Morriswood B, Engstler M, A modification to the life cycle of the parasite Trypanosoma brucei, bioRxiv, (2019), doi: 10.1101/717975
  2. Aingaran M, Zhang R, Law SK, Peng Z, Undisz A, Meyer E, Diez-Silva M, Burke TA, Spielmann T, Lim CT, Suresh S, Dao M, Marti M, Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum, Cell Microbiol, (2012), doi: 10.1111/j.1462-5822.2012.01786.x
  3. Schuster S, Krüger T, Subota I, Thusek S, Rotureau B, Beilhack A, Engstler M, Developmental adaptations of trypanosome motility to tsetse fly host environments unravel a multifaceted in vivo microswimmer system, Elife, (2017) doi: 10.7554/eLife.27656





Tags: parasitology, trypanosoma


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