1. Microbiology and Infectious Disease
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Unexpected plasticity in the life cycle of Trypanosoma brucei

  1. Sarah Schuster
  2. Jaime Lisack
  3. Ines Subota
  4. Henriette Zimmermann
  5. Christian Reuter
  6. Tobias Mueller
  7. Brooke Morriswood
  8. Markus Engstler  Is a corresponding author
  1. Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum, Julius-Maximilians-Universität, Germany
  2. Lehrstuhl für Bioinformatik, Biozentrum, Julius-Maximilians-Universität, Germany
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Cite this article as: eLife 2021;10:e66028 doi: 10.7554/eLife.66028

Abstract

African trypanosomes cause sleeping sickness in humans and nagana in cattle. These unicellular parasites are transmitted by the bloodsucking tsetse fly. In the mammalian host’s circulation, proliferating slender stage cells differentiate into cell cycle-arrested stumpy stage cells when they reach high population densities. This stage transition is thought to fulfil two main functions: first, it auto-regulates the parasite load in the host; second, the stumpy stage is regarded as the only stage capable of successful vector transmission. Here, we show that proliferating slender stage trypanosomes express the mRNA and protein of a known stumpy stage marker, complete the complex life cycle in the fly as successfully as the stumpy stage, and require only a single parasite for productive infection. These findings suggest a reassessment of the traditional view of the trypanosome life cycle. They may also provide a solution to a long-lasting paradox, namely the successful transmission of parasites in chronic infections, despite low parasitemia.

Introduction

Trypanosomes are among the most successful parasites. These flagellated protists infect all vertebrate classes, from fish to mammals, and can cause devastating diseases. African trypanosomes, which are transmitted by the tsetse fly, are the agents of nagana in livestock and sleeping sickness in humans (Bruce, 1895). The most intensively s tudied African trypanosome subspecies is Trypanosoma brucei brucei, which in the past decades has emerged as a genetic and cell biological model parasite. The life cycle of T. brucei was initially elucidated more than a century ago. As part of their life cycle, the trypanosomes undergo a full developmental program in the tsetse fly in order to become infective to the mammalian host (Koch, 1909). This finding, made by Kleine in 1909, showed that transmission was not a purely mechanical event (Kleine, 1909). Kleine subsequently found that the life cycle in the fly could take up to several weeks to complete, a discovery that was shortly afterwards confirmed by Bruce et al., 1909. More details of the general life cycle of Trypanosoma brucei were then elucidated by Robertson in 1913, with several key observations concerning the transmission event (Robertson and Bradford, 1913). Subsequent work has resulted in a detailed picture of the passage through the fly, beginning with the ingestion of trypanosomes in an infected bloodmeal (Rotureau and Van Den Abbeele, 2013). After entering through the tsetse proboscis, the infected blood is either held for a short time in the crop, which acts as a storage site and allows the tsetse to drink more blood per meal, or is passed directly to the midgut. Upon entering the tsetse midgut, the trypanosomes differentiate into the proliferative procyclic stage. Once established in the midgut, the parasites must pass the peritrophic matrix, a protective sleeve that separates the bloodmeal from midgut tissue. To do this, the parasites are thought to swim up the endotrophic space to the proventriculus, the site of peritrophic matrix synthesis, where they are able to cross to the ectotrophic space (Rose et al., 2020). After having crossed the peritrophic matrix and entered the ectroperitrophic space, procyclic trypanosomes may either further colonise the ectotrophic anterior midgut, becoming the cell-cycle arrested mesocyclic stage, or continue directly to the proventriculus. In the proventriculus, trypanosomes further develop into the long, proliferative epimastigote stage (Rose et al., 2020). The epimastigotes then swim from the proventriculus to the salivary glands, while undergoing an asymmetric division to generate a long and a short daughter cell. Once in the salivary gland, the long daughter cell is thought to die while the small one attaches via its flagellum to the salivary gland epithelium (Vickerman, 1969). The attached epimastigotes are proliferative, producing either more attached epimastigote daughter cells or freely swimming, cell cycle-arrested metacyclic trypanosomes. As early as 1911, it was clear that the metacyclic stage (at that time called metatrypanosomes) is the only mammalian-infective stage (Bruce et al., 1911).

In the mammalian host, trypanosomes have been found in many different organs, including brain, skin, and fat, but are hard to study experimentally (Capewell et al., 2016; Goodwin, 1970; Krüger et al., 2018; Trindade et al., 2016). The two main stages found in the bloodstream, and the best-characterised experimentally, are the proliferating slender bloodstream stage and the cell cycle-arrested stumpy bloodstream stage (Krüger et al., 2018; Matthews et al., 2004; Vickerman, 1985). The stumpy stage is formed in response to quorum sensing of the stumpy induction factor (SIF), a signal produced by slender bloodstream trypanosomes (Vassella et al., 1997). As the stumpy stage only survives for 2–3 days after formation, the generation of stumpy parasites is thought to control the burden the parasites impose on the host (Turner et al., 1995). The SIF pathway that controls the slender-to-stumpy transition has been described down to the molecular level, with the protein associated with differentiation (PAD1) being the first recognised molecular marker for the stumpy stage trypanosomes (Dean et al., 2009; Mony and Matthews, 2015). More recently, it was also shown that the stumpy pathway can be triggered independently of SIF, although the extent to which this occurs in the general population remains unclear (Batram et al., 2014; Zimmermann et al., 2017). Besides its proposed role in controlling parasitaemia in the mammalian host, the stumpy stage has a second essential function in the trypanosome life cycle: it is believed to be the only life cycle stage that can infect the tsetse fly (Rico et al., 2013). Thus, arrest of the cell cycle and differentiation to the stumpy stage are presumed essential for developmental progression to the procyclic insect stage. As early as 1912, Robertson suggested that the short, stumpy bloodstream trypanosomes represent the fly-infective stage (Robertson, 1912). While this assumption was questioned several times throughout the 20th century, the discovery of quorum sensing and SIF in the 1990s made it become generally accepted (Vassella et al., 1997). However, if stumpy trypanosomes are the only stage that can infect the fly, another problem arises. Although trypanosomes might be found at higher densities in the skin (Capewell et al., 2016), chronic trypanosome infections are characterised by low blood parasitemia, meaning that the chance of a tsetse fly ingesting any trypanosomes, let alone short-lived stumpy ones, is also very low (Frezil, 1971; Wombou Toukam et al., 2011). Mathematical models have been developed that aim to explain how the limited number of short-lived stumpy cells in the host blood and interstitial fluids can guarantee the infection of the tsetse fly, which is essential for the survival of the species (Capewell et al., 2019; MacGregor and Matthews, 2008; Seed and Black, 1999). The present study provides surprising new solutions to this problem. First, systematic quantification of infection efficiencies showed that very few trypanosomes are necessary to infect a tsetse fly, and in fact just one is sufficient. Second, and wholly unexpectedly, slender stages proved at least as competent at infecting flies as stumpy stages. These findings suggest greater plasticity in the life cycle than supposed, prompting a reassessment of the current rigid view of the process.

Results

A single trypanosome is sufficient for infection of a tsetse fly

Slender and stumpy bloodstream stage trypanosomes can be distinguished based on cell cycle, morphological, and metabolic criteria. The genome of the single mitochondrion (kinetoplast, K) and the cell nucleus (N) can be readily visualised using DNA stains, and their prescribed sequence of replication (1K1N, 2K1N, 2K2N) allows cell cycle stage to be inferred (Sherwin and Gull, 1989). Slender cells are found in all three K/N ratios, while stumpy cells, which are cell cycle-arrested, are found only as 1K1N cells (Figure 1A). Expression of the protein associated with differentiation 1 (PAD1) is accepted as a marker for development to the stumpy stage (Dean et al., 2009). As the 3’UTR of the PAD1 gene regulates the expression of pad1 (MacGregor and Matthews, 2012), cells expressing an NLS-GFP reporter fused to the 3' UTR of the PAD1 gene (GFP:PAD1UTR) will have GFP-positive nuclei when the PAD1 gene is active. Hence, slender cells are GFP-negative; stumpy cells are GFP-positive (Figure 1A). The validity of the GFP:PAD1UTR reporter as an indicator for the activation of the PAD1 pathway has been reported previously (Batram et al., 2014; Zimmermann et al., 2017), and was further corroborated by co-staining with an antibody against the PAD1 protein (Figure 1—figure supplement 1). We have previously shown that stumpy cells can be formed independently of high cell population density by ectopic expression of a second variant surface glycoprotein (VSG) isoform, a process that mimics one of the pathways involved in trypanosome antigenic variation (Batram et al., 2014; Cross, 1975; Hertz-Fowler et al., 2008; Zimmermann et al., 2017). These so-called expression site (ES)-attenuated stumpy cells can complete the developmental cycle in the tsetse fly (Zimmermann et al., 2017). It remained an open question whether this occurred with the same efficiency as with SIF-produced stumpy cells. Therefore, we quantitatively compared the transmission competence of stumpy populations generated by either SIF treatment or through ES-attenuation. Tsetse flies (Glossina morsitans morsitans) were infected via membrane feeding (Figure 1B; Figure 1—video 1) with defined numbers of pleomorphic stumpy trypanosomes, capable of completing the entire developmental cycle. This cycle includes entrance through the proboscis, passage through the crop, then establishing infections in the midgut, proventriculus, and finally the salivary glands (Figure 1B). Two transgenic trypanosome cell lines, both of which contained the GFP:PAD1UTR reporter construct, were used. One was subjected to tetracycline-induced, ectopic VSG expression to drive ES attenuation (Figure 2A, lines i-iii, StumpyES) (Zimmermann et al., 2017). The other was treated with stumpy induction factor (SIF) (Figure 2A, rows iv-vi, StumpySIF). Both treatments resulted in expression of the GFP:PAD1UTR reporter and rapid differentiation to the stumpy stage. The resulting stumpy populations were fed to tsetse flies at concentrations ranging from 120,000 to 10 cells/ml. A feeding tsetse typically ingests about 20 µl of blood (Gibson and Bailey, 2003), meaning that, on average, between 2400 and 0.2 trypanosomes were ingested per bloodmeal (Figure 2A, rows i-vi, column 2, Total; Figure 2—figure supplement 1). The trypanosomes had previously been scored for expression of the GFP:PAD1UTR reporter to confirm their identity as the stumpy stage (Figure 2A, columns 3–4). To analyse the infections, we carried out microscopic analyses of explanted tsetse digestive tracts (Figure 1C). The dissection of the flies was done 5–6 weeks post-infection. The presence of mammal-infective, metacyclic trypanosomes in explanted tsetse salivary glands indicated the completion of the life cycle inside the tsetse. Remarkably, the uptake, on average, of just two stumpy parasites of either cell line produced robust infections of tsetse midgut (MG), proventriculus (PV), and salivary glands (SG) (Figure 1D; Figure 2A, columns 5–7, Figure 2B, Figure 2—figure supplement 2). Ingestion, on average, of even a single stumpy cell was sufficient to produce salivary gland infections in almost 5% of all tsetse (Figure 2A, row v). When the stumpy parasite number was further reduced to 0.2 cells on average per bloodmeal, meaning only every 5th fly would receive a stumpy cell, 0.9% of flies still acquired salivary gland infections (Figure 2A, row vi). As a measure of the incidence of life cycle completion in the tsetse fly, we calculated the transmission index (TI) for each condition. The TI has been previously defined as the ratio of salivary gland to midgut infections and hence, it is a measure for successful passage through the second part of the trypanosome tsetse cycle, where trypanosomes are again infective to a mammalian host (Figures 1B, 3-5)(Peacock et al., 2012). We found that for flies infected with two trypanosomes on average, the TI was comparable between SIF-induced (TI = 0.29) and ES-induced (TI = 0.31) stumpy trypanosomes (Figure 2A, rows iii-iv; Figure 3). A similar TI of 0.23 was observed in flies ingesting on average one trypanosome (Figure 2A, row v; Figure 3). Thus, our data not only clearly show that SIF- and ES-induced stumpy parasites are equally efficient in completing the weeks-long, multi-step fly cycle, but also that a single stumpy cell is sufficient to produce a mature fly infection (individual replicates can be seen in Figure 3—figure supplement 1 and Figure 3—figure supplement 2). While this may seem comparable with an observation that has been made before for Trypanosoma congolense (Maudlin and Welburn, 1989), the migration through the fly differs between the two species: T. brucei infects the salivary glands, while T. congolense infects the proboscis. The tsetse fly, however, is much more susceptible to infections with T. congolense than with T. brucei, with a nearly 5-fold increase in percent T. congolense proboscis infections as compared to T. brucei salivary gland infections. As the authors of this work used chemicals (L- glutathione and N-acetylglucosamine) to boost T. brucei infection rates, the fivefold difference is actually a lower estimate (Peacock et al., 2012). Our results demonstrate that very low numbers of T. brucei stumpy cells can also successfully establish mature tsetse fly infections.

Figure 1 with 2 supplements see all
Slender trypanosomes can complete the entire life cycle in the tsetse fly vector.

(A) Cell cycle (G1/S/post-mitotic), morphology, and differentiation of bloodstream form (mammalian-infective stage) trypanosomes. Proliferation of slender trypanosomes is detectable by duplication and segregation of the mitochondrial genome (kinetoplast, K) and nuclear DNA (N) over time. Quorum sensing causes cell cycle arrest (G0) and expression of the stumpy marker PAD1. Images are false-coloured, maximum intensity projections of deconvolved 3D stacks. The green colour indicates the nuclear GFP:PAD1UTR fluorescence, the DAPI-stained kinetoplast and nucleus are shown in light blue, and the AMCA-sulfo-NHS-labelled parasite cell surface are shown in grey. Scale bar: 5 µm. (B) Trypanosome infections of tsetse flies were achieved via bloodmeal, which consists typically of 20 µl, through a silicon membrane. To complete infection in a tsetse fly after an infective blood-meal, trypanosomes first travel to the midgut, followed by the proventriculus, and finally must reach the salivary glands. (C) The first panel depicts a dissected tsetse fly for explantation of the alimentary tract. The second panel shows the explanted alimentary tract of the tsetse, with the different subcompartments labelled. Scale bar: 5 mm. (D) Scanning electron micrograph of a typical trypanosome infection of the tsetse midgut, proventriculus, and salivary glands. Parasites are false-coloured yellow. Scale bar: 1 µm. Figure 1—figure supplement 1. Antibody staining against PAD1 in stumpy trypanosomes. Figure 1—video 1. A tsetse fly taking a blood meal through a silicone membrane.

Figure 2 with 2 supplements see all
Slender trypanosomes can complete the entire tsetse infection cycle, and a single parasite is sufficient for tsetse passage.

The flies were infected with different numbers of either stumpy or slender trypanosomes. SlenderES, SlenderSIF, and monomorphic trypanosome cell lines were cultivated with regular dilution and a maximum population density of 5x105 cells/ml, in order to avoid SIF accumulation. Stumpy development was induced by expression site attenuation (ES), or SIF-treatment (SIF). StumpyES induction was performed by ectopic overexpression of VSG121 for 56 hr, in the absence of SIF. SIF-mediated stumpy transition (StumpySIF) was induced by incubating slender trypanosome populations in the presence of SIF-containing conditioned medium for 48 hr. The expression of the stumpy marker PAD1 was checked before fly feeding. (A) Percent of the total fly infections is shown. MG, midgut infection; PV, proventriculus infection; SG, salivary gland infection; TI, transmission index (number of SG infections divided by MG infections); n, number of independent fly infections. (B) Graphical visualisation (beeswarm plot) of the data shown in panel A, colour-coded according to cell population used. MG, midgut; PV, proventriculus; SG, salivary gland; n, number of independent fly infection experiments. Figure 2—figure supplement 1. The calculated number of trypanosomes per bloodmeal based on the concentration of trypanosome cells per millilitre. Figure 2—figure supplement 2. The total number of fly infections depicted in Figure 2. Appendix 1. Information on the number of trypanosomes in human blood samples, based on original observations reported by Robert Koch in 1906/07, showing that very few trypanosomes would be uptaken by tsetse during a bloodmeal.

Figure 3 with 2 supplements see all
Graphical representation of the transmission index TI (number of salivary gland infections divided by the number of midgut infections) of slender (blue) and stumpy (red) trypanosomes at different numbers of trypanosomes per bloodmeal (data reproduced from Figure 2A, column 8).

A high TI indicates successful completion of the life cycle in the tsetse vector. At low infective doses, slender trypanosomes had a higher TI compared to stumpy parasites. There was no difference between stumpy parasites generated by SIF-treatment (SIF) or expression site attenuation (ES). Figure 3—figure supplement 1. Transmission index shown graphically as individual replicates. Figure 3—figure supplement 2. Total number of midgut (MG) and salivary gland (SG) infections from each replicate.

Proliferating slender bloodstream stage trypanosomes infect the insect vector with comparable efficiency to cell cycle-arrested stumpy bloodstream stage parasites

Originally intended as a control experiment with an easily predictable (negative) outcome, we also infected tsetse flies with proliferating PAD1-negative slender trypanosomes from the two pleomorphic cell lines used (Figure 2A, rows vii-xi). Unexpectedly, we found that slender parasites were not only viable in the midgut, but also infected the proventriculus and the salivary glands. (Figure 2A, rows vii-xi; Figure 2B). Even one slender parasite was on average sufficient to establish midgut infections, proving that slender and stumpy parasites are, in principle, equally viable in the tsetse midgut. The infection efficiency, measured using TI, was similar when the flies were fed with either 20 stumpy trypanosomes or 20 pleomorphic slender trypanosomes (Figure 2A, compare TI in column eight for rows ii and vii). When flies were fed with an average of two slender parasites each, the TI was actually higher for slender cells (0.60) than for stumpy cells (0.31) (Figure 3; Figure 3—figure supplement 1; Figure 3—figure supplement 2). This TI of 0.60 was identical for both populations of slender cells (Figure 3). Next, when given, on average, just one PAD1-negative slender cell per bloodmeal, parasite infections were still established in the midgut, proventriculus, and salivary glands with incidences of 4.7%, 4.1%, and 2.0% respectively, at a TI of 0.44 (Figure 2A, row x; Figure 3). In order to be absolutely sure that slender trypanosomes can passage through the tsetse, we repeated the experiment with naïve slender parasites that had been freshly differentiated from insect-derived metacyclic trypanosomes, that is cells that had just restarted the mammalian life cycle stage (Figure 2A, row xii). Infections with, on average, two freshly-differentiated slender trypanosomes per bloodmeal revealed 6.3% midgut and 2.7% salivary gland infections. The transmission index was 0.43 (Figure 2A. row xiii). This important control formally ruled out that cultivated slender cells had undergone any kind of gain-of-function adaptation in culture that made them transmission-competent.

As another control for the slender infection experiments, tsetse infections were carried out using a monomorphic slender trypanosome strain, that is one that had lost the capacity of differentiating to the stumpy stage (Figure 2A, rows xiii - xv). Monomorphic trypanosomes are able to infect the tsetse midgut, but they are incapable of establishing robust infections and completing the developmental cycle in the fly (Herder et al., 2007; Peacock et al., 2008). As expected, no salivary gland infections were seen using these cells, even at high infection numbers. Interestingly, we found that even two monomorphic slender parasites could establish a fly midgut infection (Figure 2A, row xv). Thus, infection of the tsetse midgut is independent of the capacity for developmental progression and the infective dose, and it does not require the stumpy life cycle stage. This finding also challenges the assumption that slender parasites are selectively eliminated from the parasite population and that only stumpy trypanosomes can survive the harsh conditions thought to prevail within the tsetse crop and midgut (Nolan et al., 2000).

The ES-attenuated cells showed similar midgut, proventriculus, and salivary gland infection incidence as either the stumpy or slender stage (Figure 2A, rows ii-iii and vii-viii, Figure 2B). The SIF-induced stumpy cells, however, appeared more effective in establishing midgut infections than their slender counterparts (Figure 2A, rows iv-vi and ix-xi, Figure 2B). This result could be interpreted as stumpy trypanosomes being more successful in the tsetse fly, but this is a conclusion that is clearly not supported by our data. First, the infections with one to two slender cells produced higher TI values than those with the same numbers of stumpy cells (Figure 3; Figure 3—figure supplement 1; Figure 3—figure supplement 2). This suggests that the proliferative slender cells are actually more capable of progressing from a midgut infection to a salivary gland one, and thus have at least comparable overall developmental competence to the stumpy stage. Second, the lack of correlation between infective dose and midgut infections underlines the importance of the TI as a relative measure. What is biologically relevant is not the initiation of infection but the completion of the tsetse passage. In summary, our experiments not only establish that a single T. brucei cell (either slender or stumpy) can infect the tsetse fly, but also indicate that infections established by slender cells can still result in efficient completion of the passage through the tsetse fly.

In the tsetse midgut, dividing slender bloodstream stage parasites activate the PAD1 pathway and differentiate to the procyclic insect stage without arresting the cell cycle

To determine how pleomorphic slender trypanosomes manage to establish infections, we observed the early events following trypanosome ingestion by tsetse flies (Figure 4—video 1). The canonical version of events is that ingested stumpy (i.e. PAD1-positive) cells reactivate the cell cycle, begin to express the EP procyclin protein on their cell surface, and differentiate to the procyclic life cycle stage (Dean et al., 2009; Matthews and Gull, 1994; Mowatt and Clayton, 1987; Richardson et al., 1988; Roditi et al., 1989; Ziegelbauer and Overath, 1990). We infected tsetse flies with pleomorphic trypanosomes which not only contained the stumpy-specific GFP:PAD1UTR marker, but also encoded an EP1:YFP fusion (Figure 4Engstler and Boshart, 2004). In this way, the onset of stumpy development was observable as GFP fluorescence in the nucleus, and further differentiation to the procyclic life cycle stage as YFP fluorescence on the parasite cell surface. In addition, the cell cycle status (K/N counts, see Figure 1A), morphology, and the characteristic motile behavior of the trypanosomes were also assessed as criteria of developmental progress. In total, 114 tsetse flies (57 male and 57 female) were dissected after at least six independent infections with either 12,000 slender or stumpy parasites each. These high initial parasite numbers allowed the microscopic analysis of individual living slender (n = 1845) and stumpy trypanosomes (n = 1237) within the complex microenvironment of midgut explants (Schuster et al., 2017). As early as 2 hr post-infection with slender trypanosomes, a few (0.8%) 2K1N dividing trypanosomes with a nuclear PAD1 signal could be observed (Figure 5A, blue). After 8 hr however, half (38.3+6.8+5.3=50.4%) of all trypanosomes in the explants were PAD1-positive (Figure 5A; Figure 5—figure supplement 1 shows summed cell cycle category values for PAD1-positive cells). After 24 hr, 84.3% (56.3+15.0+13.0) of the parasites expressed PAD1. Of these, 9.8% had already initiated developmental progression to the procyclic insect stage, as evidenced by EP1:YFP fluorescence on their cell surface (Figure 6, blue). At 48 hr post-infection with slender trypanosomes, virtually the entire trypanosome population (91.8%) expressed PAD1, and almost one fifth (19.1%) of cells were EP1-positive (Figure 6). To examine cell cycle progression, we counted the number of 1K1N, 2K1N, and 2K2N cells in the PAD1-positive and PAD1-negative slender cell populations. Remarkably, 15 hr post-infection, the majority of all replicating (i.e. 2K1N + 2K2N) cells were PAD1-positive (Figure 5B; Figure 5—figure supplement 1). No indication of a transient cell cycle arrest or intermittent impairment of cell cycle progression was observed. Over the duration of the experiment, PAD1-negative cells gradually decreased in numbers, while PAD1-positive slender cells were increasingly observed at all cell cycle stages (Figure 5B blue vs. grey; Figure 4—video 1C). After 2 days, more than 90% of dividing trypanosomes were PAD1-positive. Thus, the PAD1 pathway was triggered in replicating slender trypanosomes upon ingestion by the fly, without prior or subsequent cell cycle arrest.

Figure 4 with 1 supplement see all
Exemplary images of procyclic trypanosomes in the explanted tsetse midgut 24 hr post infection with slender cells.

Morphology (DIC panels, left), cell cycle status (DAPI label, middle panels) and expression of fluorescent reporters (right) were scored. Note that the upper panels show a cell with procyclic morphology that is nonetheless EP1:YFP negative, indicating that the EP1 signal underestimates the total numbers of procyclic cells in the population. Scale bar: 5 µm. Figure 4—video 1. After ingestion by the tsetse fly, slender trypanosomes promptly activated the PAD1 pathway, without arresting in the cell cycle.

Figure 5 with 2 supplements see all
Slender trypanosomes activate the PAD1 pathway upon uptake by the tsetse fly.

Tsetse flies were infected with either slender (3.6% PAD1-positive) or stumpy (100% PAD1-positive) trypanosomes. 72 (slender) or 42 (stumpy) flies were dissected (equal sex ratios) at different timepoints after infection (for each time point, one hour was given to either slender or stumpy infected flies for dissection and cell analysis). Experiments were done at least three times. Living trypanosomes (>100 cells per time point) were microscopically analysed in the explanted tsetse midguts and scored for the expression of the fluorescent stumpy reporter GFP:PAD1UTR in the nucleus. Stumpy cells (n=1237) are red, and slender cells (n=1845) are blue. (A) Percentages of PAD1-positive slender and stumpy cells over time after uptake by the tsetse fly. Points indicate the individual experiments for either slender (blue) or stumpy (red). Point sizes correspond to the total number of cells counted per experiment. These data were fed into a point estimate model and are shown as solid lines, indicating the predicted percentage of PAD1-positive cells, based on time vs. cell type. Transparent colours indicate the associated 95% confidence bands. The difference between slender and stumpy cells over time is strongly significant (p<0.001). (B, C) Slender and stumpy trypanosomes scored as PAD1-positive or -negative were also stained with DAPI, and the cell cycle position determined based on the configuration of kinetoplast (K) to nucleus (N) at the timepoints indicated. The dividing slender population (B) and dividing stumpy population (C) are shown. As seen, the percentage of PAD1-positive slender cells steadily increased (B, blue) while the percentage of PAD1-negative cells steadily decreased (B, grey). This shows that slender cells can seamlessly turn on the PAD1 pathway, within a continuously dividing population. Stumpy cells did not show a normal cell cycle profile until 48 hr after tsetse uptake (C, red), as the cells differentiated to the procyclic stage. They did however remain PAD1-positive even as dividing parasites at 72 hr. Data are shown as mean +/- SD. Points without SD were the result of two measurements at those timepoints. Figure 5—figure supplement 1. Percentage of slender and stumpy PAD1-positive and -negative cell populations in each stage of the cell cycle. Figure 5—figure supplement 2. Slender trypanosomes survive as well as stumpy trypanosomes in the tsetse midgut at early timepoints after infection.

Slender trypanosomes show delayed expression of EP compared to stumpy trypanosomes, while directly differentiating to the procyclic life cycle stage in the tsetse fly.

Tsetse flies were infected with either slender (3.6% PAD1-positive) or stumpy (100% PAD1-positive) trypanosomes. 72 (slender) or 42 (stumpy) flies were dissected (equal sex ratios) at different timepoints after infection (for each time point, one hour was given to either slender or stumpy infected flies for dissection and cell analysis). Experiments were done at least three times. Living trypanosomes (>100 cells per time point) were microscopically analysed in explanted tsetse midguts and scored for the procyclic insect stage reporter EP1:YFP on the cell surface. Stumpy cells (n=1237) are shown in red and slender cells (n=1845) in blue. Points indicate the individual cell counts for either slender (blue) or stumpy (red). Point sizes correspond to the total number of cells counted per experiment. These data were fed into a point estimate model (see Materials and methods) and are shown as solid lines, indicating the predicted percentage of EP-positive cells, based on time vs. cell type. Shading above and below the lines indicates the associated 95% confidence bands. The difference between slender and stumpy cells over time was strongly significant (p<0.001).

In order to directly compare the kinetics of slender-to-procyclic differentiation with that of stumpy stage trypanosomes, we fed flies with SIF-induced, PAD1-positive stumpy trypanosomes (Figure 5A, red). These cells remained as 1K1N cells in cell cycle arrest for the first day, then differentiated to procyclic cells and re-entered the cell cycle after 2 days. Four hours after uptake by the tsetse fly, stumpy trypanosomes started expressing EP1:YFP (Figure 6, red). The fluorescent reporter was visible on 16.2% of stumpy cells after 10 hr, showing that EP expression was initiated before release of cell cycle arrest (Figure 5C, red; Figure 6, red). Uncoupling of EP surface expression from the commitment to differentiation has been reported before (Engstler and Boshart, 2004).

EP1:YFP expression in slender parasites lagged 12 hr behind stumpy cells, only becoming widespread after 24 hr (Figure 6). Thus, the onset of EP1 expression was shifted, but the kinetics of differentiation were comparable in slender and stumpy parasites, and activation of the PAD1 pathway also preceded developmental progression in slender cells. This strongly suggests that expression of PAD1 is essential for differentiation to the procyclic stage, while cell cycle arrest is not.

Of note, EP1 expression did not directly correlate with acquisition of procyclic morphology. At 24 hr, 9.8% of slender cells were EP1-positive (Figure 6), but the EP1-negative cells frequently exhibited procyclic morphology (Figure 4, upper panels). GPEET is another procyclic surface protein that is expressed early in the transition from bloodstream stage to procyclic stage cells in the tsetse midgut, before being replaced by EP (Vassella et al., 2000). Whether these early and morphologically procyclic cells expressed GPEET was not checked, and remains a target of future work. An example of a dividing (2K2N), PAD1-positive, EP1-positive cell is also shown (Figure 4, lower panels; Figure 4—video 1). This strongly suggests that a seamless developmental stage transition from the slender bloodstream stage to the procyclic insect stage took place, which was accompanied by the typical re-organisation of the cytoskeleton and the concomitant switch of swimming styles (Rotureau et al., 2011Heddergott et al., 2012; Schuster et al., 2017).

Pleomorphic slender bloodstream stage trypanosomes can seamlessly differentiate to the procyclic insect stage without preceding cell cycle arrest in vitro

The factor(s) or condition(s) that trigger differentiation of bloodstream stage trypanosomes to the procyclic insect stage in the tsetse midgut are still ill-defined. In the laboratory, differentiation to the procyclic insect stage is routinely induced by the addition of cis-aconitate, removal of glucose, and a temperature drop from 37°C to 27°C (Brun et al., 1981; Czichos et al., 1986; Engstler and Boshart, 2004; Qiu et al., 2018; Ziegelbauer et al., 1990).

We used this protocol to further investigate the developmental potential of cultivated pleomorphic slender bloodstream stage in vitro using the same cell lines and analysis as above (Figure 7A). Slender trypanosomes activated the PAD1 pathway rapidly after receiving the trigger, with 9.8% of all parasites being PAD1-positive within 2 hr, and 83.2% after 10 hr. PAD1 expression peaked after one day (98.3%), and declined thereafter (Figure 7A; Figure 7—figure supplement 1). Shortly after PAD1 reporter expression, EP1 appeared on the cell surface of 19.6% of all parasites within 8 hr, increasing to 98.3% after 3 days (Figure 8). PAD1 and EP protein appearance on the cell surface was monitored throughout the timecourse using immunofluorescence (Figure 7—figure supplement 2). Throughout the timecourse, PAD1-positive 2K1N and 2K2N cells were continually observed, demonstrating that the PAD1-positive slender parasites did not arrest in the cell cycle, and continued dividing throughout in vitro differentiation to the procyclic stage (Figure 7B, blue). The acquisition of procyclic identity was further confirmed by measuring the distance between the nucleus and cell posterior, with lengths matching those of procyclic cells being reached within 72 hr (Figure 7—figure supplement 3). After 3 days of differentiation treatment in vitro, slender trypanosomes had established a proliferating procyclic parasite population, which was further confirmed using morphological analysis (Figure 7—figure supplement 3).

Figure 7 with 3 supplements see all
Slender trypanosomes activate the PAD1 pathway in vitro with a continuously dividing population.

Cultured slender or stumpy trypanosomes were differentiated in vitro by the addition of cis-aconitate and a temperature reduction to 27°C. At the times indicated, trypanosomes were analysed for the expression of the fluorescent stumpy reporter GFP:PAD1UTR, as in Figure 5. Slender cells (n=1653) are shown in blue and stumpy cells (n=1798) in red. Data were compiled from five independent experiments, with each time point being analysed in at least two separate experiments. (A) Percentages of PAD1-positive slender and stumpy cells over time. Points indicate the individual experiments for either slender (blue) or stumpy (red) trypanosomes. Point sizes correspond to the total number of cells counted per experiment. These data were fed into a point estimate model, and are shown as solid lines, indicating the predicted percentage of PAD1-positive cells, based on time vs. cell type. Transparent colours indicate the associated 95% confidence bands. The difference between slender and stumpy cells over time was strongly significant (p<0.001). (B, C) Slender and stumpy trypanosomes scored as PAD1-positive or -negative were also stained with DAPI, and the cell cycle position determined based on the configuration of kinetoplast (K) to nucleus (N) at the timepoints indicated. The dividing slender population (B) and dividing stumpy population (C) are shown. As seen, the percentage of PAD1-positive slender cells steadily increased (B, blue) and the percentage of PAD1-negative cells steadily decreased (B, grey). This shows that slender cells can turn on the PAD1 pathway without apparent cell cycle arrest. Although a small portion of the stumpy population was observed to to divide throughout the time points (C, red), cells did not return to a normal cell cycle profile until 48 hr after the addition of cis-aconitate. As the cells became more procyclic, they began to lose their PAD1 signal and an increase in PAD1-negative dividing cells was seen (B, C, grey). Data are shown as mean +/- SD. Points without SD were the result of two measurements at those timepoints. Figure 7—figure supplement 1. Percentage of slender and stumpy PAD1-positive and negative cell populations in each stage of the cell cycle. Figure 7—figure supplement 2. Exemplary images of an antibody staining against proteins PAD1 and EP in slender cells at various timepoints, after the addition of cis-aconite. The timing of PAD1 expression matched that of the GFP:PAD1UTR reporter. Figure 7—figure supplement 3. Morphological measurements of the distance from the nucleus to the posterior end of the cell for stumpy and slender cells at various timepoints after the addition of cis-aconitate. Slender and stumpy trypanosomes showed similar timing in the morphological transition to the procyclic insect stage.

Slender trypanosomes show delayed expression of EP while they differentiate to the procyclic life cycle stage in vitro.

Cultured slender or stumpy trypanosomes were differentiated in vitro by the addition of cis-aconitate and a temperature reduction to 27°C. At the times indicated, trypanosomes were analysed for the expression of the procyclic fluorescent reporter EP1:YFP, as in Figure 6. Stumpy cells (n=1798) are shown in red and slender cells (n=1653) in blue. Points indicate the individual experiments for either slender or stumpy. Point sizes correspond to the total number of cells counted per experiment. These data were fed into a point estimate model and are shown as solid lines, indicating the predicted percentage of EP-positive cells, based on time vs. cell type. Shading above and below the lines indicates the associated 95% confidence bands. The difference between slender and stumpy cells over time was strongly significant (p<0.001).

By comparison, stumpy parasites (Figure 7A, red) responded to in vitro cis-aconitate treatment with rapid expression of the EP1:YFP marker, with 28.6% of all cells being positive within 2 hr (Figure 8). After 1 day, EP1 was present on almost all (96.7%) stumpy trypanosomes. The cell cycle analysis revealed that these parasites were not dividing, however (Figure 7C). The first cells re-entered the cell cycle only after 15 hr, and a normal procyclic cell cycle profile was not reached until day 3. Morphological analysis using the distance between the nucleus and the posterior of the cell confirmed acquisition of procyclic identity within 72 hr (Figure 7—figure supplement 3). Thus, the in vitro differentiation supported the in vivo observations, demonstrating that pleomorphic slender trypanosomes are able to directly differentiate to the procyclic stage without becoming cell cycle-arrested stumpy cells. The surface expression of EP1 is also of note: it has been shown that in slender bloodstream parasites, ectopically expressed EP1 does not enter the cell surface, but is retained in endosomes and the flagellar pocket (Engstler and Boshart, 2004). Hence, as in stumpy trypanosomes, lifting of the cell surface access block for EP1 in the slender trypanosome correlates with activation of the PAD1 pathway. To summarise, the overall developmental capacity of the two life cycle stages - slender and stumpy cells - is comparable both in vitro and in vivo.

Discussion

Our observations suggest a revised view of the life cycle of African trypanosomes (Figure 9). We have shown that one trypanosome suffices to produce robust infections of the tsetse vector, and that the stumpy stage is not essential for tsetse transmission. Slender parasites can complete the complex life cycle in the fly with comparable overall success rates and kinetics as the stumpy stage. Interestingly, the stumpy stage appears more able to establish initial infections in the fly midgut (Figure 2A, column 5, MG), while slender-derived parasites appear to produce salivary gland infections slightly more efficiently (higher amount of salivary gland infections compared to midgut infections, TI) than stumpy-derived counterparts (Figure 2A, column 8, TI). At first sight, this discrepancy may be related to a greater resistance of the stumpy stage to the digestive environment in the fly’s gut, as has been suggested (Matetovici et al., 2019; Nolan et al., 2000). This, however, is not supported by our data. We have not observed cell death of monomorphic or pleomorphic slender cells in infected tsetse midguts (Figure 5—figure supplement 1). And even if so, why then should slender-derived cells perform better in the second part of the life cycle? As there will not be a difference between slender- and stumpy-derived procyclic cells, the difference observed must be based on the behaviour of bloodstream parasites in the midgut. It is tempting to speculate that one decisive factor could be trypanosome motility. Slender trypanosomes exhibit significantly higher motility compared to stumpy trypanosomes (Bargul et al., 2016). Thus, the mean square displacement in the midgut will be much larger for slender parasites. While stumpy trypanosomes probably never reach the ‘midgut exit’ before differentiation to the insect stage, slender trypanosomes could already be located close to the proventriculus before starting to differentiate to the procyclic stage. Thus, passage through the proventriculus could occur immediately, and the slender-derived trypanosomes could rapidly progress to the mesocyclic stage. This faster mesocyclic progression would result in a less-pronounced infection of the midgut, and a higher TI-value for the slender-derived trypanosomes. While the above hypothesis is consistent with our data, experimental proof would be extremely challenging to obtain. The recent demonstration that glucose levels are a developmental trigger in addition to the well-characterised ones of cold shock and cis-aconitate adds another layer of complexity to the early events during tsetse infection (Qiu et al., 2018). It is also of note that a recent review has suggested that stumpy trypanosomes may have evolved as a stress response, with procyclic pathways already activated in order to increase chances of survival in the fly (Quintana et al., 2021).

A revised life cycle for the parasite Trypanosoma brucei.

Cell-cycle-arrested (G0) metacyclic trypanosomes are injected by the tsetse fly into the mammalian host’s skin. There, the parasites re-enter the cell cycle, and proliferate as () slender forms in the blood, while disseminating into the interstitium and various tissues, including fat and brain. At least two triggers (SIF or ES) launch the PAD1-dependent differentiation pathway (light green boxes) to the cell cycle-arrested () stumpy bloodstream stage. Stumpy trypanosomes can establish a fly infection when taken up with the bloodmeal of a tsetse. The work described here reveals that proliferating slender stage trypanosomes are equally effective for tsetse transmission, that a single parasite suffices, and that the population can continuously divide while differentiating to the procyclic insect stage. The triggers that initiate further developmental transitions are temperature (°C), cis-aconitate (CA) and glucose deprivation (Glc).

The dogma that cell cycle-arrested stumpy cells are the only trypanosomes that infect the tsetse fly has never been experimentally challenged, although there are quite a number of reports that point against an exclusive role for stumpy parasites in the life cycle. Koch’s detailed report on the activities of the German sleeping sickness commission sent to East Africa in 1906/7 states that the trypanosome numbers in the blood of human sleeping sickness infections was always very low (Koch, 1909). From his data, we have calculated an average blood parasitaemia between 10 and 100 trypanosomes cells/ml (see Appendix 1). This means that two or fewer trypanosomes would be present in an average tsetse bloodmeal, again highlighting the rarity of a tsetse taking up a stumpy cell. In 1930, Duke discussed the evidence for the essential status of the stumpy stage for tsetse transmission, and his data did not support it (Duke, 1930). Further, Baker and Robertson in 1957 compared the infection capability of T. rhodesiense and T. brucei using guinea pig feeding (Baker and Robertson, 1957). They concluded: 'Neither the morphology nor the intensity of the parasitaemia in the infecting mammal was obviously related to the subsequent infection rates in the tsetse-flies'. In 1990, Bass and Wang suggested that in fact the stumpy stage may be dispensable for development to the insect stage (Bass and Wang, 1991). The experiments, however, were in part inconclusive, mainly because a molecular marker for the stumpy stage was missing. The discovery of SIF in the 1990s and the realisation that quorum sensing underpinned the differentiation to the stumpy stage led to an assumption that the slender stage had no role to play in the transmission event. Subsequent research has been focused on the details of stumpy formation, while the developmental role of the stumpy cell has not undergone further examination. The above publications all relate to what is nowadays referred to as the transmission paradox, the persistence and circulation of trypanosomiasis in a population even when parasitaemia levels in individuals are low or close to elimination (Capewell et al., 2019). When parasitaemia is low, stumpy trypanosomes are characteristically absent, making the probability of being ingested by a tsetse fly (which on average ingests 20 µl of blood) extremely low. Yet trypanosomiasis persists, even when statistically it should be eliminated. Solutions to the paradox have long been hypothesised and variously include flawed diagnostic testing, asymptomatic cases, and animal reservoirs (Alvar et al., 2020). Recent work using theoretical modelling suggests that for T. gambiense, trypanosomes residing in the skin of humans could solve the problem (Capewell et al., 2019). However, there are currently no data available on the number of trypanosomes located in asymptomatic human skin, nor have the kinetics of fly uptake of skin-localised trypanosomes been explored. Also, different tsetse-transmitted trypanosome species reveal rather distinct distributions in the host, such as Trypanosoma congolense preferentially residing in small blood vessels (Banks, 1978). Thus, while trypanosomes in the skin may be important for the persistence of the parasites, their existence alone does not automatically solve the transmission paradox. As tsetse are blood pool feeders, it actually does not matter if the trypanosomes reside in the skin, fat tissue, or blood.

Furthermore, stumpy cells will inevitably run into an age-related problem. They are not replicative, and their lifetime is limited to roughly 3 days (Turner et al., 1995). In the fly, re-entry into the cell cycle is by no means immediate, but takes at least one day. Following induction of cell cycle arrest, the stumpy cells would need to be taken up by the fly within 1 day. Thus, only a subset of rather young stumpy cells would prove successful in the midgut. It is important to note that this is not the case in our experiments, as only freshly differentiated stumpy cells were used for tsetse infection. Thus, our experiments in fact overestimate the success rate of stumpy stage trypanosomes.

It should be stressed that our data provide a possible solution to the transmission paradox without challenging any of the extensive published work on stumpy trypanosomes. We have shown that slender and stumpy trypanosomes are equally competent for fly passage. The PAD1 pathway has an essential role in preparing both bloodstream stages for differentiation to the procyclic cell stage. For successful passage through the tsetse fly, however, the stumpy stage is not uniquely required. Along similar lines, it is worth noting that Trypanosoma congolense, the principal causative agent of the cattle plague nagana, infects tsetse flies without manifesting a cell cycle-arrested stumpy stage (Rotureau and Van Den Abbeele, 2013). Thus, the essential biological function of the stumpy life cycle stage in T. brucei may not be transmission, but rather quorum sensing (SIF)-dependent control of population size in the host. This pathway can be triggered in other ways, and even at low levels of parasitaemia, for example by VSG expression site attenuation (ES)(Zimmermann et al., 2017). The capacity for inducing cell cycle arrest at the single cell level might actually have been important for the evolution of antigenic variation. As not all trypanosome species develop a stumpy life cycle stage (Rotureau and Van Den Abbeele, 2013), density-dependent differentiation at the population level may well be a later innovation in evolution, and specific to the T. brucei group. In conclusion, our work evidences a high degree of plasticity in the life cycle of an important parasite. It shows that the trypanosome life cycle is not rigid, and we have proposed a revised and more flexible view of the trypanosome life cycle that may help solve a longstanding puzzle in parasitology.

Materials and methods

Key resources table
Reagent type
(species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line (Trypanosoma brucei brucei)‘SIF’ cell line, EATRO 1125 Antat1.1(GFP:PAD1UTR)The GFP:PAD1UTR reporter was verified in:
Zimmermann et al., 2017
DOI:10.1371/journal.ppat.1006324Transfected with: p4231 (containing an NLS-GFP reporter fused to the 3’ UTR of the PAD1 gene, which inserts into the β-tubulin locus; provided by Mark Carrington)
Cell line (Trypanosoma brucei brucei)‘ES’ cell line, Antat1.1 13–90 (GFP:PAD1UTR)This cell line was verified in: Zimmermann et al., 2017DOI:10.1371/journal.ppat.1006324Transfected with: pLew13 (Addgene plasmid 24007, G. Cross); pLew90 (Addgene plasmid 24008, G. Cross); p4231 (containing an NLS-GFP reporter fused to the 3’ UTR of the PAD1 gene, which inserts into the β-tubulin locus; provided by Mark Carrington)
Cell line (Trypanosoma brucei brucei)‘SIF’ cell line, EATRO 1125 Antat1.1(GFP:PAD1UTR) with an EP1:YFP fusion proteinThe GFP:PAD1UTR reporter was verified in:
Zimmermann et al., 2017
The EP1:YFP reporter was verified in:
Engstler and Boshart, 2004
DOI:10.1371/journal.ppat.1006324
DOI: 10.1101/gad.323404
Transfected with: p4231 (containing an NLS-GFP reporter fused to the 3’ UTR of the PAD1 gene, which inserts into the β-tubulin locus; provided by Mark Carrington); pGaprone(ble)_EPGFG_rev GPY PARPYFP
Cell line (Trypanosoma brucei brucei)Monomorphic cell line,T. brucei 427 MITat 1.2 13–90This cell line was verified in: Wirtz et al., 1999DOI: 10.1016/s0166-6851(99)00002-x
AntibodyAnti-PAD1
(Trypanosoma brucei brucei) (Rabbit Polyclonal)
This antibody was generated and verified in: Dean et al., 2009DOI: 10.1038/nature07997IF (1:100)
AntibodyAnti-EP1
(Trypanosoma brucei brucei) (Mouse monoclonal)
CEDARLANEProduct code: CLP001AIF (1:500)
Chemical compound, drugMethylcelluloseSigma AldrichProduct code: 94378Methocel A4M
Software, algorithmRR core teamPackage ‘mgcv’
Software, algorithmImageJNIHDOI: 10.1038/nmeth.2089

Trypanosome culture

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Pleomorphic Trypanosoma brucei brucei strain EATRO 1125 (serodome AnTat1.1)(Le Ray et al., 1977) bloodstream stages were grown in HMI-9 medium (Hirumi and Hirumi, 1989), supplemented with 10% (v/v) foetal bovine serum and 1.1% (w/v) methylcellulose (Sigma 94378, Munich, Germany)(Vassella et al., 2001) at 37°C and 5% CO2. Slender stage parasites were maintained at a maximum cell density of 5x105 cells/ml. For cell density-triggered differentiation to the stumpy stage, cultures seeded at 5x105 cells/ml were cultivated for 48 hr without dilution. Within this period, the stumpy induction factor (SIF) accumulated and caused developmental transition of slender to stumpy trypanosomes. For expression site attenuated differentiation (ES stumpy cells) to the stumpy stage (ES stumpy), cells with a construct which overexpresses VSG121 upon tetracycline induction were grown to 5x105 cells/ml and incubated with 1 μg/ml tetracycline for 56 hr (Zimmermann et al., 2017). Pleomorphic parasites were harvested from the viscous medium by 1:4 dilution in trypanosome dilution buffer (TDB; 5 mM KCl, 80 mM NaCl, 1 mM MgSO4, 20 mM Na2HPO4, 2 mM NaH2PO4, 20 mM glucose, pH 7.6), followed by filtration (MN 615 ¼, Macherey-Nagel, Dueren, Germany) and centrifugation (1400xg, 10 min, 37°C)(Zimmermann et al., 2017). For infections with very low trypanosomes per bloodmeal (0.2, 1), cells were pipetted directly from their culture medium and placed in blood. Monomorphic T. brucei 427 MITat 1.2 13–90 bloodstream stage (Wirtz et al., 1999) were grown in HMI-9 medium (Hirumi and Hirumi, 1989), supplemented with 10% (v/v) foetal bovine serum at 37°C and 5% CO2.

For in vitro differentiation to the procyclic insect stage, bloodstream stage trypanosomes were pooled to a cell density of 2x106 cells/ml in DTM medium with 15% fetal bovine serum immediately before use (Overath et al., 1986). Cis-aconitate was added to a final concentration of 6 mM (Brun et al., 1981; Overath et al., 1986) and temperature was adjusted to 27°C. Procyclic parasites were grown in SDM79 medium (Brun and Schönenberger, 19791979), supplemented with 10% (v/v) foetal bovine serum (Hirumi and Hirumi, 1989) and 20 mM glycerol (Schuster et al., 2017; Vassella et al., 2000).

Genetic manipulation of trypanosomes

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Transfection of pleomorphic trypanosomes was done as previously described (Zimmermann et al., 2017), using an AMAXA Nucleofector II (Lonza, Basel, Switzerland). Transgenic trypanosome clones were selected by limiting dilution in the presence of the appropriate antibiotic. The GFP:PAD1UTR reporter construct (Zimmermann et al., 2017) was used to transfect AnTat1.1 trypanosomes to yield the cell line ‘SIF’. The trypanosome ‘ES’ line was described previously (Zimmermann et al., 2017). It contains the reporter GFP:PAD1UTR construct and an ectopic copy of VSG gene MITat 1.6 under the control of a tetracycline-inducible T7-expression system. The EP1:YFP construct was integrated into the EP1-procyclin locus as described previously (Engstler and Boshart, 2004).

Immunofluorescence

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Cells were harvested as stated above, concentration was measured using a Neubauer chamber, and 106 cells per coverslip were taken. The cells were transferred to a 1.5 ml tube, washed twice with 1 ml of phosphate buffered saline (PBS), resuspended in 500 μl of PBS, and fixed by addition of paraformaldehyde to a final concentration of 4% at room temperature (RT) for 20 min. The cells were pelleted by centrifugation (750 xg, RT, 10 min), the supernatant removed, and the cell pellet resuspended in PBS and transferred to poly-L-lysine-coated coverslips in a 24-well plate. Cells were attached to coverslips by centrifugation (750 xg, RT, 4 min). Cells were either permeabilised with 0.25% TritonX-100 in PBS (RT, 5 min) and subsequently washed twice with PBS or not permeabilised, so as to allow only surface labelling. Cells were then blocked with 3% BSA in PBS (RT, 30 min), followed by incubation with the primary antibody (1:100 rabbit anti-PAD1; 1:500 IgG1 mouse anti-Trypanosoma brucei procyclin, Ascites, Clone TBRP1/247, CEDARLANE, Ontario, Canada) followed by secondary antibodies (Alexa488- and Alexa 594-conjugated anti-rabbit and anti-mouse, 1:100, ThermoFisher Scientific, Massachusetts, USA) diluted in PBS (1 hr, RT for each), with three PBS wash steps after each incubation. After the final wash, coverslips were rinsed with ddH2O, excess fluid removed by wicking, and mounted on glass slides using antifade mounting media with DAPI (Vectashield, California, USA).

Tsetse maintenance

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The tsetse fly colony (Glossina morsitans morsitans) was maintained at 27°C and 70% humidity. Flies were kept in Roubaud cages and fed three times a week through a silicone membrane, with pre-warmed, defibrinated, sterile sheep blood (Acila, Moerfelden, Germany).

Fly infection and dissection

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Teneral flies were infected 1–3 days post-eclosion during their first meal. It is known that teneral flies (flies that are newly hatched and unfed) are more susceptible to midgut infections compared to older flies, and it is an accepted practice in the field to use teneral flies for infections. While all of our infections were done during the flies’ first bloodmeal, it is of note that 1–3 days is rather old for teneral flies (Walshe et al., 2011; Wijers, 1958). Depending on the experiment, trypanosomes were diluted in either pre-warmed TDB or sheep blood. For infections with low parasite numbers (0.2 and 1 cell/bloodmeal; Figure 2A), the cell density of either stumpy or slender trypanosomes was calculated, and the dilutions made directly in blood. Thus, the parasites were directly taken from culture and added to blood, thereby completely omitting any filtration step.

The infective meals were supplemented with 60 mM N-acetylglucosamine (Peacock et al., 2006). For infection with 2400 monomorphic parasites per bloodmeal, cells were additionally treated for 48 hr with 12.5 mM glutathione (MacLeod et al., 2007) and 100 µM 8-pCPT-cAMP (cAMP) (Vassella et al., 1997).

Tsetse infection status was analysed between 35 and 40 days post-infection. Flies were euthanised with chloroform and dissected in PBS. Intact tsetse alimentary tracts were explanted and analysed microscopically, as described previously (Schuster et al., 2017). For the analysis of early trypanosome differentiation in vivo, slender or stumpy trypanosomes at a concentration of 6x105 cells/ml were resuspended in TDB to the required final concentration and fed to flies. The numbers of flies used and the number of independent experiments carried out are indicated in the figure legends. Results are presented as sample means.

Fluorescence microscopy and video acquisition

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Live trypanosome imaging was performed with a fully automated DMI6000B widefield fluorescence microscope (Leica microsystems, Mannheim, Germany), equipped with a DFC365FX camera (pixel size 6.45 µm) and a 100x oil objective (NA 1.4). For high–speed imaging, the microscope was additionally equipped with a pco.edge sCMOS camera (PCO, Kelheim, Germany; pixel size 6.5 µm). Fluorescence video acquisition was performed at frame rates of 250 fps. For visualisation of parasite cell cycle and morphology, slender and stumpy trypanosomes were harvested and incubated with 1 mM AMCA-sulfo-NHS (Thermo Fisher Scientific, Erlangen, Germany) for 10 min on ice. Cells were chemically fixed in 4% (w/v) paraformaldehyde and 0.05% (v/v) glutaraldehyde overnight at 4°C. DNA was visualised with 1 µg/ml DAPI immediately before analysis.

3D-Imaging was done with a fully automated iMIC widefield fluorescence microscope (FEI-TILL Photonics, Munich, Germany), equipped with a Sensicam qe CCD camera (PCO, Kelheim, Germany; pixel size 6.45 µm) and a 100x oil objective (NA 1.4). Deconvolution of image stacks was performed with the Huygens Essential software (Scientific Volume Imaging B.V., Hilversum, Netherlands). Fluorescence images are shown as maximum intensity projections of 3D-stacks in false colours with green fluorescence in green and blue fluorescence in grey.

Scanning electron microscopy

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Explanted tsetse alimentary tracts were fixed in Karnovsky solution (2% formaldehyde, 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4) and incubated overnight at 4°C. Samples were washed 3 times for 5 min at 4°C with 0.1 M cacodylate buffer, pH 7.4, followed by incubation for 1 hr at 4°C in post-fixation solution (2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4). After additional washing, the samples were incubated for 1 hr at 4°C in 2% tannic acid in cacodylate buffer, pH 7.4, 4.2% sucrose, and washed again in water (3x for 5 min, 4°C). Finally, serial dehydration in acetone was performed, followed by critical point drying and platinum coating. Scanning electron microscopy was done using the JEOL JSM-7500F field emission scanning electron microscope (JEOL, Freising, Germany).

Nuclear – posterior cell pole measurements

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Cells were harvested and fixed as above, before being mounted directly onto slides containing 3 µl antifade mounting medium with DAPI (Vectashield, California, USA). Images were taken using a DMI6000B widefield fluorescence microscope (Leica microsystems, Mannheim, Germany), equipped with a DFC365FX camera (pixel size 6.45 µm) and a 100x oil objective (NA 1.4). Images (DIC and DAPI) were overlaid using FIJI (NIH, Bethesda, Maryland)(Schneider et al., 2012) and measurements taken from the centre of the cell nucleus, along the midline, to the posterior end of the cell.

Statistics

Statistical analyses were performed using the statistical framework R vers 4.02 (R Development Core Team, 2020). The PAD1 and EP -positive and -negative cells were modelled by the different cell types (Stumpy or Slender), a spline for the time parameter (k=4, dimension of the spline basis) and a binomial link function. Finally, the model was fitted using the function ‘gam’ as implemented in the ‘mgcv’ package (Wood et al., 2016). Unpaired, parametric t-tests were performed using Graphpad prism version 8.4.0 for macOS (Graphpad software, San Diego, California USA, http://www.graphpad.com).

Data availability

All original data are in the submission.

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Decision letter

  1. Christine Clayton
    Reviewing Editor; DKFZ-ZMBH Alliance, Germany
  2. Miles P Davenport
    Senior Editor; University of New South Wales, Australia
  3. Christine Clayton
    Reviewer; DKFZ-ZMBH Alliance, Germany
  4. James Morris
    Reviewer; Clemson U, United States

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

Trypanosoma brucei lives as "bloodstream forms" in the blood and tissue fluids of mammals, where they divide as long slender forms which are dependent on substrate-level phosphorylation during glycolysis. Meanwhile, in the Tsetse fly vector midgut, they divide as "procyclic forms", which rely heavily on mitochondrial metabolism. While in the blood, the parasites differentiate – probably via quorum sensing – to a non-dividing "stumpy form" which has gene expression changes that pre-adapt the parasites to life in the fly. Textbook knowledge is that only the stumpy form can differentiate to the procyclic form. This paper overturns this dogma. The authors show that long slender forms can also differentiate – somewhat more slowly – directly into procyclic forms in the fly, by-passing several known aspects of stumpy formation.

Decision letter after peer review:

Thank you for submitting your article "Unexpected plasticity in the life cycle of Trypanosoma brucei" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, including Christine Clayton as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Miles Davenport as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: James Morris (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

Your paper has been reviewed by the reviewing editor and 3 other reviewers, of whom one was less enthusiastic than the other two. Overall it was agreed that:

1. More statistical analysis is essential, everywhere. At present there is very little indeed. This could partly be achieved by presenting the data as box plots (or "beeswarm" plots) using data from individual flies. From Table 1, it is important to display not just the transmission index (as in Figure 2), but also the individual values for Tsetse fly infection (MG, PV, SG) with the 2-parasite inoculum (lines iii, vi, viii, xi and xii). These are quite complicated to compare from the Table and there are clear differences. Similarly for the bar graphs, it is essential overlay with the values obtained in the individual replicates as dots (not SD!). Readers must be able to assess the variability in ALL of the measurements. One referee also suggested replacing the Table in Figure 4 with more plots – the proportions of different N-K conformations could be shown within the PAD columns, or separately as much narrower columns. The actual numbers could then go into the Supplement.

2. The GFP-positive cells should be described as GFP-positive – not PAD1-positive. The caveats for this assay should be mentioned.

3. Some quantitative analysis of cell morphology (e.g. K-N distances) beyond the GFP should be done – this can almost certainly be done using existing images.

4. One new experiment is needed – assessing GFP-PAD expression in the inoculum after the filtration steps to get rid of the methyl cellulose. (Or is this already what was done?)

5. Can the metacyclics be tested for VSG expression? As an alternative I guess you could use morphology and state that strictly speaking, metacyclic identity has not been confirmed. The rabbit VSG117 antibody that my lab has seems to be fairly non-specific but maybe you have anti Antat1.1? Another option would be to stain for PGK. (Though I actually don't know where PGK is in epimastigotes.) This would be nice but is probably not essential.

Further suggestions for improvement are in the individual reviews.

I would also add that there is a really nice new review about stumpies which is highly relevant and warrants citation: Trends Parasitol. 2020 Dec 11:S1471-4922(20)30316-0. doi: 10.1016/j.pt.2020.11.003.

I also seem to remember that Muriel Roberts originally thought that it was the slender forms that were transmissible, but I may be wrong.

Reviewer #1 (Recommendations for the authors):

I agree with the authors that the data in this paper indicate that slender forms can productively infect Tsetse. Although their initial differentiation is delayed, they ultimately are as successful as stumpy forms. One could add that although stumpy morphology is not an essential intermediate, stumpy-like gene expression may well be: essentially, conditions in the fly are sufficient to induce the developmental programme. I don't think the data support the conclusion that no cell-cycle arrest is required – although such an arrest does have to be quite brief.

I have one major criticism, and that is the authors' equation of GFP-positive with "PAD1 positive". Yes, this means that the GFP-PAD1 mRNA has been present. But the GFP could easily persist after the mRNA has gone, and of course the presence of GFP can't be equated with the presence of PAD1 protein. This limitation has to be stated right at the start and the description re-worded throughout. Has to be something like "GFP-PAD reporter positive", or maybe even just "GFP-positive".

Line 80 – The possibility that densities are higher in the skin should be mentioned briefly (I know it's in the Discussion as well.)

Line 99 – for non-experts, the authors should mention that developmental regulation of PAD1 is controlled by the 3'-UTR.

Line 132 and Table 1: the authors should, in the supplement, provide a simple plot showing the probability distribution for numbers of parasites in 20µl at the different parasite densities. The Legend (as well as the text) should briefly define transmission index and the number of flies per group should be stated. Also, say what the different groups are: what are slender ES and slender SIF? How long was ES induced and what exactly are the "SIF" stumpies? (I know it's in the Methods section but the density of the cultures should be given.) Statistical analysis is needed. Are any of the differences really significant?

Video 2A – expand the legends, these were initially difficult to follow. (Just say that they switch from DIC to fluorescence, and which comes first.)

Line 162 – this is a bit misleading. The authors should define what they mean by "infection efficiency"? Indeed the transmission indices for slender and stumpy are similar, but the number of infections per input slender trypanosome is half of that obtained with stumpy. The should state up front (rather than much later) that the slender parasites have more trouble establishing infection in the midgut.

Line 199-120 – a bit over-stated. If there is no midgut infection then there will definitely be no tryps in the salivary glands.

Figure 5 and Figure 7 – titles are not appropriate since cell cycle arrest was not measured. How about "Slender trypanosomes show delayed EP procyclin expression after Tsetse infection / in vitro differentiation"? Please explain why 2h time-ranges are given rather than single times.

I do not think it is possible to conclude that absolutely no cell-cycle arrest is required. From Figure 6, can a 1-2 h arrest while PAD1 is turned on and the rest of the programme is initiated be excluded? I don't think so – the temporal definition and numbers simply don't allow it. Is the sudden drop in positive 2K1N cells from the slender cells at 15-17h significant?

Line 254 – Did the authors stain for GPEET or phosphorylated GPEET, which are often expressed first after differentiation? IF not they should surely mention that the procyclic-morphology cells are most likely expressing GPEET.

Line 292 – clearly not true since the procyclin expression of slender forms was delayed. Also no other markers of procyclics were measured. (From their existing images can authors also include the N-K and N-posterior tip distances of 1K1N cells, which gives procyclic morphology independently of procyclin expression?) For Figures 6 and 7, please also plot the absolute cell densities in addition to showing the percentages.

Discussion

"We have not observed cell death of monomorphic or pleomorphic slender cells in infected tsetse midguts." But how would you detect it?

The authors emphasise that the slender-derived parasites are "more successful in the second part of the life cycle." Are the differences in Table 1, which are reiterated in lines 300-302, really statistically significant?

Reviewer #3 (Recommendations for the authors):

1. The data as presented in the tables is challenging to follow. In table 1, I recommend moving data on short stumpy and slender form related to expression site attenuation to supplemental data. In Tables 2 and 6, the information in the bars of the bar graphs (the different dotted lines, for example) should be developed into stand-alone parts of the figure, as they are difficult to interpret. In the text of the results, it would be helpful to be consistent with directing the reader to the correct column (for example, Line 192, "column 5).

2. It is unclear what tissues were analyzed in Figure 4 – some clarification about what an "explant" is needed.

Reviewer #4 (Recommendations for the authors):

This is a very important finding that challenges the current dogma. As any paper that challenges the dogma, this paper raises many questions that will be tackled in future studies, namely reproducing these results with parasites recently isolated from the wild, instead of tissue culture adapted lines. Another important avenue will be to test the different mutant parasites that are incapable of differentiating from slender to stumpy forms and dissect the molecular machinery that composes the slender-differentiation.

I describe below two points that, in my opinion, could help strengthen the paper:

1. I am concerned about the parasite purification protocol prior to Tsetse infection (which involves change of buffers, filtration to remove the methylcellulose matrix and centrifugation). Could the protocol stress the slender forms such that these non-cell-cycle-arrested-stressed slenders gain the capacity to infect Tsetses? Previous reports have shown that reduced cellular energy promotes parasite differentiation (Barquilla 2012; Saldivia, 2016). Could the authors avoid or minimize the purification steps? Some T. brucei pleomorphic strains grow in vitro without methylcellulose, which would simplify parasite harvesting. Could the parasites be simply diluted in blood prior to infection of flies (without any purification)? When was the IFA done to confirm expression of GFP::PAD1? Ideally, it should have been at the end of purification protocol, just prior to Tsetse feeding. If it is technically impossible to improve/avoid parasite purification protocol, could the authors provide some evidence that after purification parasites are not stressed? Would purified parasites grow exponentially in culture without a lag phase? What are the levels of phosphorylated AMPKa1? This type of experiments would help ruling out that parasites were stressed by a reduced cellular energy during purification protocol.

2. The authors showed that the kinetics of differentiation of slender and stumpy forms to procyclic forms is different both in vitro and in vivo, although both lead to the formation of bonafide procyclic forms. The authors could consider doing a competition experiment between slenders and stumpy forms to test their individual fitness in group.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Unexpected plasticity in the life cycle of Trypanosoma brucei" for further consideration by eLife. Your revised article has been reviewed by 3 peer reviewers and the evaluation has been overseen by Miles Davenport as the Senior Editor, and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer 4 asks for you to clarify one sentence and I agree. I too think that the phrase "without harvesting the cells" is really difficult to understand.

Reviewer #2 (Recommendations for the authors):

My only real comment is that the videos seem a little out of place. M1 of the fly feeding has little obvious value in the present context, and I found he quality of M2 disappointing. M2 could be improved and if this were done it would then be a genuine addition to the paper.

Reviewer #3 (Recommendations for the authors):

The authors have addressed my concerns through revision. I applaud their efforts to improve the visualization of the data while maintaining transparency, as there is a tremendous amount to interpret.

Reviewer #4 (Recommendations for the authors):

The authors have convincingly responded to my question about a putative "stress" effect on harvested parasites. Controls at multiple levels have been used. Thank you.

While their answer is very clear in the rebuttal, these arguments are not clearly described in the manuscript (or did I miss them?). For example, the description of one of the controls: "cells were directly taken from culture and added to blood, thereby completely omitting any filtration step.", in the rebuttal, is described in the paper (line 483) as "the dilutions made directly in blood without harvesting the cells". I suggest the authors include in the paper the exact words used in the rebuttal to answer my question.

The authors state that my second point (competition) is beyond the scope of this paper. I support the editor's decision.

https://doi.org/10.7554/eLife.66028.sa1

Author response

Essential revisions:

Your paper has been reviewed by the reviewing editor and 3 other reviewers, of whom one was less enthusiastic than the other two. Overall it was agreed that:

1. More statistical analysis is essential, everywhere. At present there is very little indeed. This could partly be achieved by presenting the data as box plots (or "beeswarm" plots) using data from individual flies. From Table 1, it is important to display not just the transmission index (as in Figure 2), but also the individual values for Tsetse fly infection (MG, PV, SG) with the 2-parasite inoculum (lines iii, vi, viii, xi and xii). These are quite complicated to compare from the Table and there are clear differences.

For the revised version, we teamed up with an expert mathematician (Tobias Müller) who performed state-of-the-art modelling and statistical analysis of our data. For the figures with bar graphs (Figure 4-7), statistical analysis was run using a point estimate model based on time vs. cell type (stumpy and slender).

For Figure 2A, we now additionally provide a beeswarm plot with results from all individual fly infections (2B), as well as the total number of infections which go with this figure (Figure 2 – table supplement 1).

A plot showing the transmission index for each N, as well as a table with total infection numbers (rather than percent) and number of infections per N, was added to the supplements (Figure 3—figure supplement 1 and – table supplement 1).

All statistical validation supported the interpretation of the data. We hope that these steps have sufficiently addressed the reviewer's concern.

Similarly for the bar graphs, it is essential overlay with the values obtained in the individual replicates as dots (not SD!). Readers must be able to assess the variability in ALL of the measurements.

The tables of cell cycle profiles for PAD1-positive and -negative cells were moved into the supplements (Figure 5 – Supplemental Table 1 and Figure 7 – Supplemental Table 1) and replaced with graphs depicting the PAD1-positive and -negative dividing populations (Figure 5 B, C; Figure 7 B, C). They are shown as mean with standard deviation. A table showing the conversion from trypanosomes/bloodmeal to trypanosomes/ml was added as requested as Figure 2 – table supplement 1.

One referee also suggested replacing the Table in Figure 4 with more plots – the proportions of different N-K conformations could be shown within the PAD columns, or separately as much narrower columns. The actual numbers could then go into the Supplement.

Nucleus (N) – posterior pole measurements have now been conducted for all timepoints and added to the supplements. While N-kinetoplast (K) was an option, we decided for N-posterior pole so that a difference can also be seen between slender and stumpy at the zero hour timepoint (which cannot really be differentiated with N-K). Data were visualised using violin plots, with the line at the median and dotted lines at the quartiles while tables added below show some descriptive measures from the data (Figure 7—figure supplement 2).

2. The GFP-positive cells should be described as GFP-positive – not PAD1-positive. The caveats for this assay should be mentioned.

The caveats are stated in line 97-105:

“Expression of the protein associated with differentiation 1 (PAD1) is accepted as a marker for development to the stumpy stage (Dean et al., 2009). […] The validity of the GFP:PAD1UTR reporter as an indicator for the activation of the PAD1 pathway has been reported previously (Batram et al., 2014; Zimmermann et al., 2017), and further corroborated by co-staining with an antibody against the PAD1 protein (Figure 1—figure supplement 1).”

We also mention this several times in the manuscript but for the sake of clarity and readability we have decided not to include the description each time the reporter has been used. We hope this is acceptable.

3. Some quantitative analysis of cell morphology (e.g. K-N distances) beyond the GFP should be done – this can almost certainly be done using existing images.

As there is no true discernable difference between stumpy and slender cell K-N distances, we chose instead to measure the distance between the nucleus and posterior pole of both slender and stumpy trypanosomes during differentiation to the procyclic insect stage – this distance is more discernable between slender, stumpy, and procyclic, especially for stumpy and slender. A violin plot showing all measurements, with lines at the median and dotted lines at the quartiles, was added to the supplements (Figure 7—figure supplement 2). Included is a table with some descriptive information about the data.

4. One new experiment is needed – assessing GFP-PAD expression in the inoculum after the filtration steps to get rid of the methyl cellulose. (Or is this already what was done?)

We have considered the possibility that the cells might be “stressed” even by a single, brief filtration step, and made sure that this did not result in increased GFP-PAD1 expression.

Almost all experiments were done using the same harvesting protocol. In Figure 2A, however, for 0.2, 1, and 2 cells/bloodmeal, cells were directly taken from culture and added to blood, thereby completely omitting any filtration step. This was not necessary since such a small number of cells were needed. These very low trypanosome numbers per bloodmeal still resulted in infections. Furthermore, all images taken at the start of the experiment (zero hour timepoint) were taken after the harvest. The cells did not express GFP-PAD1. As a further control, we regularly put harvested cells back in culture where they instantaneously resumed normal growth and did not show GFP-PAD1 expression. In conclusion, the experiments have been carefully controlled on multiple levels.

5. Can the metacyclics be tested for VSG expression? As an alternative I guess you could use morphology and state that strictly speaking, metacyclic identity has not been confirmed. The rabbit VSG117 antibody that my lab has seems to be fairly non-specific but maybe you have anti Antat1.1? Another option would be to stain for PGK. (Though I actually don't know where PGK is in epimastigotes.) This would be nice but is probably not essential.

In the course of another project, we have used the same tsetse colony and identical experimental procedures to allow infection of artificial skin equivalents with tsetse-borne metacyclic parasites. The respective manuscript is currently under review and is available on bioRxiv (https://doi.org/10.1101/2021.05.13.443986). In this work, we provide extensive time-resolved single cell RNA-seq analyses, including that of metacyclic and naïve slender parasites. While the metacyclic cells express metacyclic VSGs, the re-activated slender parasites express Antat1.1 VSG in a subset of cells, as expected. Furthermore, the vast majority of cells found freely swimming in the salivary glands after fly infection are metacyclic forms. There are a few epimastigote trypanosomes detectable, which is not so surprising given that they arrive at the salivary glands as freely swimming trypanosomes.

Further suggestions for improvement are in the individual reviews.

I would also add that there is a really nice new review about stumpies which is highly relevant and warrants citation: Trends Parasitol. 2020 Dec 11:S1471-4922(20)30316-0. doi: 10.1016/j.pt.2020.11.003.

We have added this reference (which was published after our initial submission – the only reason for its initial omission). Lines 338-340 now read:

“It is also of note that a recent review has suggested that stumpy trypanosomes may have evolved as a stress response, with procyclic pathways already activated in order to increase chances of survival in the fly (Quintana et al., 2021).”

Reviewer #1 (Recommendations for the authors):

I agree with the authors that the data in this paper indicate that slender forms can productively infect Tsetse. Although their initial differentiation is delayed, they ultimately are as successful as stumpy forms. One could add that although stumpy morphology is not an essential intermediate, stumpy-like gene expression may well be: essentially, conditions in the fly are sufficient to induce the developmental programme. I don't think the data support the conclusion that no cell-cycle arrest is required – although such an arrest does have to be quite brief.

Stumpy trypanosomes are arrested in G0, and no replicative cell cycle is detectable until either cell death, which occurs after 3 days (our own measurements), or development to procyclic cells. We in fact state that slender parasites also launch the PAD1 pathway in the tsetse fly. We have no indication for a G0 arrest and likewise not for a G1 prolongation (as shown for example in Batram et al., 2014 and Zimmermann et al., 2017). We are actually not aware of a bona fide cell cycle arrest that lasts for a shorter period than the cell cycle.

I have one major criticism, and that is the authors' equation of GFP-positive with "PAD1 positive". Yes, this means that the GFP-PAD1 mRNA has been present. But the GFP could easily persist after the mRNA has gone, and of course the presence of GFP can't be equated with the presence of PAD1 protein. This limitation has to be stated right at the start and the description re-worded throughout. Has to be something like "GFP-PAD reporter positive", or maybe even just "GFP-positive".

We now clearly state in line 97-105:

“Expression of the protein associated with differentiation 1 (PAD1) is accepted as a marker for development to the stumpy stage (Dean et al., 2009). […] The validity of the GFP:PAD1UTR reporter as an indicator for the activation of the PAD1 pathway has been reported previously (Batram et al., 2014; Zimmermann et al., 2017), and further corroborated by co-staining with an antibody against the PAD1 protein (Figure 1—figure supplement 1).”

We also mention this several times later but for the sake of clarity and readability we have not included the description each time the reporter has been used. We hope this is an acceptable compromise.

Line 80 – The possibility that densities are higher in the skin should be mentioned briefly (I know it's in the Discussion as well.)

We now mention this in the introduction. Lines 75-79:

“Although trypanosomes might be found in higher densities in the skin (Capewell et al., 2016), chronic trypanosome infections are characterised by low blood parasitemia, meaning that the chance of a tsetse fly ingesting any trypanosomes, let alone short-lived stumpy ones, is also very low (Frezil, 1971; Wombou Toukam et al., 2011).”

The referee might also consider our recent new work on fly infections of artificial skin, which clearly shows that immediately after fly infection, trypanosomes do enter a dormant stage (https://doi.org/10.1101/2021.05.13.443986).

Line 99 – for non-experts, the authors should mention that developmental regulation of PAD1 is controlled by the 3'-UTR.

This is a good point and was added in line 99-105:

“As the 3’UTR of the PAD1 gene regulates the expression of pad1 (MacGregor and Matthews, 2012), cells expressing an NLS-GFP reporter fused to the 3' UTR of the PAD1 gene (GFP:PAD1UTR) will have GFP-positive nuclei when the PAD1 gene is active. […] pathway has been reported previously (Batram et al., 2014; Zimmermann et al., 2017), and further corroborated by co-staining with an antibody against the PAD1 protein (Figure 1—figure supplement 1).”

Line 132 and Table 1: the authors should, in the supplement, provide a simple plot showing the probability distribution for numbers of parasites in 20µl at the different parasite densities.

Instead of a probability plot, we have given a supplemental table with the calculation of the probable number of trypanosomes per 20µl.

The Legend (as well as the text) should briefly define transmission index and the number of flies per group should be stated.

Done. See Lines 585-586 and Figure 2 – table supplement 2.

Also, say what the different groups are: what are slender ES and slender SIF? How long was ES induced and what exactly are the "SIF" stumpies? (I know it's in the Methods section but the density of the cultures should be given.)

Done. See lines 577-583.

Statistical analysis is needed. Are any of the differences really significant?

For the revision, we teamed up with an expert mathematician (Tobias Müller) who performed state-of-the-art modelling and statistical analyses of our data. For the figures with bar graphs (Figure 4-7), statistical analysis was run using a point estimate model based on time vs. cell type (stumpy and slender), including both confidence intervals (seen as transparent colour) and the individual replicates for each experiment (seen as dots).

For Figure 2A, we now additionally provide a beeswarm plot with results from all individual fly infections (2B), as well as the total number of infections which go with this figure (Figure 2 – table supplement 1).

A plot showing transmission index for each N, as well as a table with total infection numbers (rather than percent) and number of infections per N, was added to the supplements (Figure 3—figure supplement 1 and – table supplement 1).

Video 2A – expand the legends, these were initially difficult to follow. (Just say that they switch from DIC to fluorescence, and which comes first.)

Done. See lines 741-742.

Line 162 – this is a bit misleading. The authors should define what they mean by "infection efficiency"? Indeed the transmission indices for slender and stumpy are similar, but the number of infections per input slender trypanosome is half of that obtained with stumpy. The should state up front (rather than much later) that the slender parasites have more trouble establishing infection in the midgut.

We now state:

“The infection efficiency, using TI as a measure, when the flies were fed with either 20 stumpy trypanosomes or 20 pleomorphic slender trypanosomes was similar (Figure 2A, compare TI in column 8 for rows ii and vii). […] This TI of 0.60 was identical for both populations of slender cells (Figure 3)”.

Furthermore, the abundance of slender trypanosomes, when compared to the short-lived stumpy trypanosomes, should be more than 2-fold higher. This would compensate for any initial delay in transformation to the procyclic stage. As we know that this is rather speculative, we do not discuss this point in the manuscript.

Line 199-120 – a bit over-stated. If there is no midgut infection then there will definitely be no tryps in the salivary glands.

We agree with the referee.

Figure 5 and Figure 7 – titles are not appropriate since cell cycle arrest was not measured.

We agree and have changed the title accordingly. The title for figure 5 (now figure 6) reads “Slender trypanosomes show delayed expression of EP compared to stumpy trypanosomes, while they seamlessly differentiate to the procyclic life cycle stage in the tsetse fly.” The title for figure 7 (now figure 8) reads “Slender trypanosomes show delayed expression of EP while they differentiate to the procyclic life cycle stage in vitro.”

How about "Slender trypanosomes show delayed EP procyclin expression after Tsetse infection / in vitro differentiation"? Please explain why 2h time-ranges are given rather than single times.

Two-hour time-ranges were used to account for the time required for fly dissection and examining the explanted organs. Hence, we did not want to imply more accuracy than the experiment offered. Nevertheless, we now give the start time of the interval and state this in the legend at lines 613-614 and 640-641.

I do not think it is possible to conclude that absolutely no cell-cycle arrest is required.

The conclusion that there is no cell cycle arrest in fact cannot be drawn in our paper, that is why we now instead refer to ‘no apparent cell cycle arrest’. Please also see our comment above.

From Figure 6, can a 1-2 h arrest while PAD1 is turned on and the rest of the programme is initiated be excluded? I don't think so – the temporal definition and numbers simply don't allow it.

See above. A 1-2 hours stalling of the cell cycle cannot be regarded as a cell cycle arrest (G0), but rather prolongation of the cell cycle phase (maybe in G1). We are not aware of any example in which bona fide cell cycle arrest was shorter than the cell cycle.

Is the sudden drop in positive 2K1N cells from the slender cells at 15-17h significant?

We now give graphs showing PAD1-positive and -negative cells throughout the time course and the answer is no. It is within the standard deviation of the neighboring points.

Line 254 – Did the authors stain for GPEET or phosphorylated GPEET, which are often expressed first after differentiation? IF not they should surely mention that the procyclic-morphology cells are most likely expressing GPEET.

Lines 261-265 now read:

“GPEET is another procyclic surface protein that is expressed early in the transition from bloodstream stage to procyclic stage cells in the tsetse midgut, before being replaced by EP (Vassella et al., 2000). Whether these early and morphologically procyclic cells expressed GPEET was not checked, and remains a target of future work.”

Line 292 – clearly not true since the procyclin expression of slender forms was delayed.

We agree and have omitted the mention of differentiation kinetics. The line now reads:

“Furthermore, the overall developmental capacity of both life cycle stages is comparable, in vitro and in vivo.”

Also no other markers of procyclics were measured. (From their existing images can authors also include the N-K and N-posterior tip distances of 1K1N cells, which gives procyclic morphology independently of procyclin expression?) For Figures 6 and 7, please also plot the absolute cell densities in addition to showing the percentages.

As there is no true discernable difference between stumpy and slender cell K-N distances, we chose to measure K-cell posterior of both slender and stumpy transitioning to procyclic – this distance is more discernable between slender, stumpy, and procyclic, especially for stumpy and slender. A violin plot showing all measurements, with lines at the median and dotted lines at the quartiles, was added to the supplements (Figure 7—figure supplement 2). Included is a table with some descriptive information about the data.

Discussion

"We have not observed cell death of monomorphic or pleomorphic slender cells in infected tsetse midguts." But how would you detect it?

The referee is right in stating that one cannot easily detect dead trypanosomes in the midguts, thus we did it the opposite way. We counted the number of live trypanosomes at early timepoints of midgut infections. The data is now given as Figure 5—figure supplement 1.

The authors emphasise that the slender-derived parasites are "more successful in the second part of the life cycle." Are the differences in Table 1, which are reiterated in lines 300-302, really statistically significant?

The transmission index suggests that in fact slender and stumpy trypanosomes complete the passage through the fly with almost equal success rates. Why one or the other life cycle stage should be successful in early or late stages of infection is a matter of speculation, and in the discussion lines 318 ff. we suggest one possibility, namely the documented differences in motile behavior of trypanosomes. We have clearly marked this as a theory.

Reviewer #3 (Recommendations for the authors):

1. The data as presented in the tables is challenging to follow. In table 1, I recommend moving data on short stumpy and slender form related to expression site attenuation to supplemental data. In Tables 2 and 6, the information in the bars of the bar graphs (the different dotted lines, for example) should be developed into stand-alone parts of the figure, as they are difficult to interpret. In the text of the results, it would be helpful to be consistent with directing the reader to the correct column (for example, Line 192, "column 5).

We agree with referee number 3, although we had tried our best to present the data as interpretable as possible. As this was obviously not entirely successful, we now have added a beeswarm plot (Figure 2B) so that Figure 2A is easier to navigate.

We have re-worked most figures entirely in the revised version, thereby also implementing the suggestion to extract the dotted lines and put them in separate graphs (now Figure 5 B, C and Figure 7 B, C). We also included mathematical modelling and statistical analysis to figures 5-8. We hope that this has further improved the readability of our paper.

2. It is unclear what tissues were analyzed in Figure 4 – some clarification about what an "explant" is needed.

Done. We added that it is an “explanted tsetse midgut” to the legend.

Reviewer #4 (Recommendations for the authors):

This is a very important finding that challenges the current dogma. As any paper that challenges the dogma, this paper raises many questions that will be tackled in future studies, namely reproducing these results with parasites recently isolated from the wild, instead of tissue culture adapted lines. Another important avenue will be to test the different mutant parasites that are incapable of differentiating from slender to stumpy forms and dissect the molecular machinery that composes the slender-differentiation.

I describe below two points that, in my opinion, could help strengthen the paper:

1. I am concerned about the parasite purification protocol prior to Tsetse infection (which involves change of buffers, filtration to remove the methylcellulose matrix and centrifugation). Could the protocol stress the slender forms such that these non-cell-cycle-arrested-stressed slenders gain the capacity to infect Tsetses? Previous reports have shown that reduced cellular energy promotes parasite differentiation (Barquilla 2012; Saldivia, 2016). Could the authors avoid or minimize the purification steps? Some T. brucei pleomorphic strains grow in vitro without methylcellulose, which would simplify parasite harvesting. Could the parasites be simply diluted in blood prior to infection of flies (without any purification)? When was the IFA done to confirm expression of GFP::PAD1? Ideally, it should have been at the end of purification protocol, just prior to Tsetse feeding. If it is technically impossible to improve/avoid parasite purification protocol, could the authors provide some evidence that after purification parasites are not stressed? Would purified parasites grow exponentially in culture without a lag phase? What are the levels of phosphorylated AMPKa1? This type of experiments would help ruling out that parasites were stressed by a reduced cellular energy during purification protocol.

We have considered that the cells might be “stressed”, even by a single, brief filtration step, and made sure that this did not result in increased GFP-PAD1 expression.

Almost all experiments were done with the same harvesting protocol. In Figure 2A, however, for 0.2, 1, and 2 cells/bloodmeal, cells were directly taken from culture and added to blood, thereby completely omitting any filtration step. This was not necessary since it was such a small number of cells that was needed. These very low trypanosome numbers per bloodmeal still resulted in infections. Further, all images taken at the start of the experiment (zero hour timepoint) were taken after the harvest. The cells did not express GFP:PAD1. As a further control, we regularly put harvested cells back in culture where they instantaneously resumed normal growth and did not show GFP:PAD1 expression. In conclusion, the experiments have been carefully controlled on multiple levels.

2. The authors showed that the kinetics of differentiation of slender and stumpy forms to procyclic forms is different both in vitro and in vivo, although both lead to the formation of bonafide procyclic forms. The authors could consider doing a competition experiment between slenders and stumpy forms to test their individual fitness in group.

These experiments will be done in a follow-up paper. The experimental design is actually more complex than it might appear. For example, we have to tag the parasites with different fluorescent markers, which might differentially influence the parasites at any stage during the 30-day transition to the salivary glands.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer 4 asks for you to clarify one sentence and I agree. I too think that the phrase "without harvesting the cells" is really difficult to understand.

We agree with the reviewer. We have now instead put: "Almost all experiments were done with the same harvesting protocol. In Figure 2A, however, for 0.2, 1, and 2 cells/bloodmeal, cells were directly taken from culture and added to blood, thereby completely omitting any filtration step. This was not necessary since it was such a small number of cells that was needed. These very low trypanosome numbers per bloodmeal still resulted in infections."

Reviewer #2 (Recommendations for the authors):

My only real comment is that the videos seem a little out of place. Figure 1—video 1 of the fly feeding has little obvious value in the present context, and I found he quality of Figure 4—video 1 disappointing. Figure 4—video 1 could be improved and if this were done it would then be a genuine addition to the paper.

Reviewer #2 asks us to improve the quality of Figure 4—video 1. Since this video shows live fluorescent trypanosomes in explants of tsetse flies, the quality is as good as it gets, and the video clearly supports our conclusions. In addition, the reviewer feels that Figure 1—video 1 is out of place. We would like to keep it, as tsetse membrane feeding may be known in a few specialized trypanosome laboratories, but certainly not by the wide readership that eLife attracts. Therefore, it seems well placed from an educational point of view.

https://doi.org/10.7554/eLife.66028.sa2

Article and author information

Author details

  1. Sarah Schuster

    Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum, Julius-Maximilians-Universität, Würzburg, Germany
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft
    Contributed equally with
    Jaime Lisack and Ines Subota
    Competing interests
    No competing interests declared
  2. Jaime Lisack

    Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum, Julius-Maximilians-Universität, Würzburg, Germany
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Sarah Schuster and Ines Subota
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9621-4000
  3. Ines Subota

    Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum, Julius-Maximilians-Universität, Würzburg, Germany
    Contribution
    Conceptualization, Resources, Formal analysis, Supervision, Investigation, Visualization, Methodology
    Contributed equally with
    Sarah Schuster and Jaime Lisack
    Competing interests
    No competing interests declared
  4. Henriette Zimmermann

    Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum, Julius-Maximilians-Universität, Würzburg, Germany
    Contribution
    Validation, Investigation, Visualization, Methodology
    Competing interests
    No competing interests declared
  5. Christian Reuter

    Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum, Julius-Maximilians-Universität, Würzburg, Germany
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Tobias Mueller

    Lehrstuhl für Bioinformatik, Biozentrum, Julius-Maximilians-Universität, Würzburg, Germany
    Contribution
    Software, Formal analysis, Validation, Methodology
    Competing interests
    No competing interests declared
  7. Brooke Morriswood

    Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum, Julius-Maximilians-Universität, Würzburg, Germany
    Contribution
    Data curation, Validation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7031-3801
  8. Markus Engstler

    Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum, Julius-Maximilians-Universität, Würzburg, Germany
    Contribution
    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    markus.engstler@biozentrum.uni-wuerzburg.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1436-5759

Funding

Deutsche Forschungsgemeinschaft (EN305)

  • Markus Engstler

Deutsche Forschungsgemeinschaft (SPP1726)

  • Markus Engstler

German-Israeli Foundation for Scientific Research and Development (ant I-473-416.13/2018)

  • Markus Engstler

Deutsche Forschungsgemeinschaft (GRK2157)

  • Markus Engstler

Deutsche Forschungsgemeinschaft (396187369)

  • Brooke Morriswood

Bundesministerium für Bildung und Forschung (NUM Organostrat)

  • Markus Engstler

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Nicola Jones, Susanne Kramer, Manfred Alsheimer, Christian Janzen and Ricardo Benavente for discussion and critical reading of the manuscript. We thank Thomas Müller for help with data presentation using python. We thank Alyssa Borges for fruitful discussions about statistics. We thank Keith Matthews (Edinburgh) for the anti-PAD1 antibody. BM is supported by DFG grant number 396187369. ME is supported by DFG grants EN305, SPP1726 (Microswimmers – From Single Particle Motion to Collective Behaviour), GIF grant I-473–416.13/2018 (Effect of extracellular Trypanosoma brucei vesicles on collective and social parasite motility and development in the tsetse fly), GRK2157 (3D Tissue Models to Study Microbial Infections by Obligate Human Pathogens), and NUM Organostrat (Bundesministerium für Bildung und Forschung). ME is a member of the Wilhelm Conrad Roentgen Center for Complex Material Systems (RCCM).

Senior Editor

  1. Miles P Davenport, University of New South Wales, Australia

Reviewing Editor

  1. Christine Clayton, DKFZ-ZMBH Alliance, Germany

Reviewers

  1. Christine Clayton, DKFZ-ZMBH Alliance, Germany
  2. James Morris, Clemson U, United States

Publication history

  1. Preprint posted: July 29, 2019 (view preprint)
  2. Received: December 22, 2020
  3. Accepted: August 5, 2021
  4. Accepted Manuscript published: August 6, 2021 (version 1)
  5. Version of Record published: September 17, 2021 (version 2)

Copyright

© 2021, Schuster et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Further reading

Further reading

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    Myxococcus xanthus, a soil bacterium, predates collectively using motility to invade prey colonies. Prey lysis is mostly thought to rely on secreted factors, cocktails of antibiotics and enzymes, and direct contact with Myxococcus cells. In this study, we show that on surfaces the coupling of A-motility and contact-dependent killing is the central predatory mechanism driving effective prey colony invasion and consumption. At the molecular level, contact-dependent killing involves a newly discovered type IV filament-like machinery (Kil) that both promotes motility arrest and prey cell plasmolysis. In this process, Kil proteins assemble at the predator-prey contact site, suggesting that they allow tight contact with prey cells for their intoxication. Kil-like systems form a new class of Tad-like machineries in predatory bacteria, suggesting a conserved function in predator-prey interactions. This study further reveals a novel cell-cell interaction function for bacterial pili-like assemblages.

    1. Medicine
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    Background:

    It remains unclear whether combination antiretroviral therapy (ART) regimens differ in their ability to fully suppress human immunodeficiency virus (HIV) replication. Here, we report the results of two cross-sectional studies that compared levels of cell-associated (CA) HIV markers between individuals receiving suppressive ART containing either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI).

    Methods:

    CA HIV unspliced RNA and total HIV DNA were quantified in two cohorts (n = 100, n = 124) of individuals treated with triple ART regimens consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) plus either an NNRTI or a PI. To compare CA HIV RNA and DNA levels between the regimens, we built multivariable models adjusting for age, gender, current and nadir CD4+ count, plasma viral load zenith, duration of virological suppression, NRTI backbone composition, low-level plasma HIV RNA detectability, and electronically measured adherence to ART.

    Results:

    In both cohorts, levels of CA HIV RNA and DNA strongly correlated (rho = 0.70 and rho = 0.54) and both markers were lower in NNRTI-treated than in PI-treated individuals. In the multivariable analysis, CA RNA in both cohorts remained significantly reduced in NNRTI-treated individuals (padj = 0.02 in both cohorts), with a similar but weaker association between the ART regimen and total HIV DNA (padj = 0.048 and padj = 0.10). No differences in CA HIV RNA or DNA levels were observed between individual NNRTIs or individual PIs, but CA HIV RNA was lower in individuals treated with either nevirapine or efavirenz, compared to PI-treated individuals.

    Conclusions:

    All current classes of antiretroviral drugs only prevent infection of new cells but do not inhibit HIV RNA transcription in long-lived reservoir cells. Therefore, these differences in CA HIV RNA and DNA levels by treatment regimen suggest that NNRTIs are more potent in suppressing HIV residual replication than PIs, which may result in a smaller viral reservoir size.

    Funding:

    This work was supported by ZonMw (09120011910035) and FP7 Health (305522).