Replicative slender bloodstream forms complete transmission of Trypanosoma brucei without prior differentiation into stumpy forms
- Department of Cell and Developmental Biology, Biocenter, Julius-Maximilians-Universitaet, Germany
- Helmholtz Institute for RNA-based Infection Research, Helmholtz-Center for Infection Research, Germany
Figures
Figure 1
Infection rates (%) of slender T. brucei in teneral tsetse flies, with and without N-acetylglucosamine (NAG) supplementation.
Flies of both genders were fed 200 slender cells per ml, translating to 4 slender cells per bloodmeal, estimating a 20 µl drinking volume for each tsetse fly (Gibson and Bailey, 2003), with or without 60 mM NAG. Slender cells were harvested and checked for PAD1 signal to confirm slender identity before infection (<1% PAD1 positive). Infections were performed in triplicates with approximately 20 flies/replicate. The midgut (MG), proventriculus (PV), and salivary glands (SG) of all flies were dissected after 35 days to check for infection. Bar graphs show mean infection rates across replicates, with individual dots representing infection percentages per replicate. Fisher’s exact test was used on the mean infection rates to determine significance. ns = not significant (p>0.05). Infection rates are shown for both sexes (A), male (B), and female flies (C).
Figure 2 with 2 supplements
Infection rates (%) of slender and stumpy T. brucei in non-teneral tsetse flies.
Non-teneral flies were infected between 144 and 168 hr post eclosion (hpe) with either slender (orange) or stumpy (blue) parasites, after receiving two non-infectious bloodmeals beforehand. The tdTomato NLS-GFP:PAD1 3′UTR cell line enabled fluorescence-activated cell sorting (FACS) to separate stumpy (PAD1 positive, GFP in nucleus) and slender (PAD1 negative, no nuclear fluorescence) prior to infection. With the confidence of pure slender or pure stumpy cultures after sorting, infection numbers were increased to 1×106 cells/ml or 2×104 cells per bloodmeal per fly, considering a 20 µl drinking volume per fly (Gibson and Bailey, 2003). Infections were performed in quadruplets with roughly 20 flies/replicate. Midgut (MG), proventriculus (PV), and salivary glands (SG) of all flies were dissected after 30–35 days to check for parasite presence. Bar graphs show mean infection rates across replicates, with individual dots representing infection percentages per replicate. Fisher’s exact test was used on the mean infection rates to determine significance; ns = not significant (p>0.05). Infection rates are presented for both sexes (A), males (B), and females (C).
Figure 2—figure supplement 1
Slender T. brucei cells of the tdTomato NLS-GFP:PAD13′UTR line do not express PAD1 or exhibit stress responses following fluorescence-activated cell sorting (FACS).
FACS was used to ensure pure slender populations (pad1 negative) prior to infection. (A) Immunofluorescence (IF) images of slender cells immediately after FACS, confirming sorting success. Parasites were fixed in 4% paraformaldehyde (PFA), stained with DAPI (blue), and labelled with an anti-PAD1 antibody (orange); scalebar: 20 μm. (B) High-resolution IF image of a single slender trypanosome post sorting, showing clear absence of nuclear pad1 signal; scalebar: 10 μm. (C) Growth curves comparing slender cells post sorting (green) and untreated slender cells (purple), indicating no growth impairment due to sorting.
Figure 2—figure supplement 2
Fluorescence-activated cell sorting (FACS) was used to isolate stumpy cells of the tdTomato NLS-GFP:PAD1 3′UTR line, ensuring a pure stumpy population prior to infection.
(A) Stumpy cells from an SIF-induced stumpy culture (grown to 5×105 cells/ml and kept for 48 hr), after FACS to confirm sorting success. Cells were fixed in 4% paraformaldehyde (PFA) immediately after sorting, stained with DAPI (blue), and labelled with an anti-PAD1 antibody (orange); scalebar: 20 μm. (B) High-resolution immunofluorescence (IF) image of a single stumpy trypanosome displaying characteristic stumpy morphology and strong pad1 signal (orange); scalebar: 10 μm.
Figure 3 with 5 supplements
RNA sequencing of slender and stumpy trypanosomes differentiating into procyclic forms, performed in biological triplicates.
(A) Principal component analysis (PCA) showing the transcriptional progression to procyclic forms for slender (blue/green) and stumpy (orange/red) cells. The trajectories remain distinct until converging at 72 hr. (B) Number of differentially expressed genes that are upregulated in slender compared to stumpy forms at corresponding time points during differentiation. Genes with an absolute log2FC>2 and an adjusted p-value<0.01 were classified as differentially expressed. Corresponding volcano plots and detailed gene counts can be found in Figure 3—figure supplement 1. (C) Differentially expressed genes that are upregulated between offset time points during slender and stumpy differentiation. The offset comparison aligns slender 15 hr with stumpy 0 hr, where only 22 genes are differentially expressed. Genes with an absolute log2FC>2 and an adjusted p-value<0.01 were classified as differentially expressed. Corresponding volcano plots and detailed gene counts can be found in Figure 3—figure supplement 2.
Figure 3—figure supplement 1
Volcano plots showing differential gene expression between stumpy (red) and slender (blue) T. brucei forms during in vitro differentiation to the procyclic form at 0, 8, 15, 24, and 72 hrs after induction.
Differentiation was initiated by adding cis-aconitate, lowering the temperature to 27°C, and depleting glucose in the medium. Each black dot represents one gene. Genes within the coloured boxes show 2× upregulation in slender (blue dotted line) or 2× upregulation in stumpy (red dotted line) cells, with a p-value≤0.01 (grey dotted line). Red and blue boxes highlight significantly upregulated genes in stumpy or slender, respectively, with the exact gene counts listed in the accompanying table. logFC = log2 fold change; hrs = hours after the addition of cis-aconitate.
Figure 3—figure supplement 2
Volcano plots showing differential gene expression of stumpy (red) and slender (blue) T. brucei forms following in vitro differentiation to the procyclic form.
Differentiation was initiated by adding cis-aconitate, lowering the temperature to 27°C, and depleting glucose. An offset comparison – based on proximity in the principal component analysis (PCA) plot (Figure 3A) – aligns slender cells at 15 hours (hrs) with stumpy cells at 0 hrs. Each black dot represents one gene. Genes within the coloured boxes show 2× upregulation for slender (blue dotted line) or 2× upregulation for stumpy (red dotted line) cells, with a p-value≤0.01 (grey dotted line). Red and blue boxes highlight significantly upregulated genes in stumpy or slender, respectively, with the exact numbers of differentially expressed genes listed in the accompanying table. Notably, slender cells at 15 hrs exhibit a similar gene expression profile to that of stumpy trypanosomes after 0 hrs, before diverging again at later time points. logFC = log2 fold change; hrs = hours after the addition of cis-aconitate.
Figure 3—figure supplement 3
During differentiation into the procyclic form, slender and stumpy parasites exhibit gene expression profiles associated with distinct biological processes and molecular functions.
Gene Ontology (GO) enrichment analysis between corresponding time points identified genes with a log2 fold change of at least 1 (indicating 2× expression) and a p-value of ≤0.01 for either slender (orange) or stumpy (purple) forms at 0, 8, 15, 24, and 72 hr (Figure 3—figure supplement 1). GO annotations were sourced from the TriTryp.org database (TriTryp.org) and refined using Revigo. The most significantly enriched GO terms for each time point are shown.
Figure 3—figure supplement 4
Dot plots showing expression levels of genes associated with the stumpy form – PAD1 (A) and PAD2 (B) – in slender (orange) and stumpy (blue) forms during in vitro differentiation.
Differentiation was induced by the addition of cis-aconitate, reduction of temperature to 27°C, and glucose depletion. RNA sequencing was performed in triplicates; each coloured dot represents one replicate (1000 cells), and the black line indicates the mean log2 counts per million (CPM) value. A statistical significance assessment using Welch’s t-test showed no significant differences in PAD1 expression between stumpy and slender forms at 0 hr (p-value=0.059), nor in PAD1 expression trajectory between 0 to 72 hr (p-value=0.102). In contrast, PAD2 expression differed significantly between slender and stumpy forms at the baseline (0 hr; p-value<0.001) and showed significant changes in expression from 0 to 72 hr (p-value<0.003), suggesting differential regulation prior to the transition to the procyclic stage.
Figure 3—figure supplement 5
Dot plots showing expression levels of genes associated with the procyclic form – EP1 (A), EP2 (B), and pyruvate phosphate dikinase (PPDK) (C) – in slender (orange) and stumpy (blue) forms during in vitro differentiation.
Differentiation was induced by the addition of cis-aconitate, reduction of temperature to 27°C, and glucose depletion. Stumpy forms reach procyclic-like expression levels by approximately 8 hr, whereas slender forms display a more gradual increase. RNA sequencing was performed in triplicates; each coloured dot represents one replicate (1000 cells), and the black line indicates the mean log2 counts per million (CPM) value.
Figure 4
Slender and stumpy bloodstream forms must activate distinct pathways to transform into the procyclic form in the tsetse fly.
Differentiation to the PAD1-positive (green), cell-cycle-arrested, short stumpy form can be triggered by either SIF or ES attenuation (Zimmermann et al., 2017). Stumpy forms have a 2- to 3-day window to be ingested by a tsetse fly before they perish. When a tsetse fly takes a bloodmeal, it can ingest both slender and stumpy forms. Once in the fly’s midgut, both forms begin their transformation into the procyclic fly form. During the initial 15 hr, slender forms shift towards stumpy gene expression before diverging again. Stumpy forms need to reactivate the cell cycle, fully switch to proline metabolism, and elongate both their cytoskeleton and flagella (Figure 3—figure supplement 3A). Slender forms must activate the essential PAD1 pathway, complete the switch to proline metabolism, and change to a procyclin coat (Figure 3—figure supplement 3B). By 72 hr into differentiation, both slender and stumpy forms have transitioned into the procyclic form. This figure was adapted from Schuster et al., 2021.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/108688/elife-108688-mdarchecklist1-v1.pdf
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Supplementary file 1
Absolute numbers of tsetse fly infections using slender bloodstream forms of T. brucei.
(A) Absolute numbers of tsetse fly infections using slender bloodstream forms of T. brucei, with or without the addition of N-acetylglucosamine (NAG). Both male and female flies were infected with blood containing 200 slender cells per ml of blood, either untreated or supplemented with the immune-suppressing chemical, NAG (60 mM). Tsetse flies have an estimated drinking volume of 20 µl (Gibson and Bailey, 2003), which results in an uptake of 4 parasites per bloodmeal. All flies were dissected 35 days post infection, and their midgut (MG), proventriculus (PV), and salivary glands (SG) examined for parasite presence. Prior to infection, slender cells of the tdTomato NLS-GFP:PAD1 3′UTR line were verified to lack pad1 expression, confirming pure slender identity (<0.05% PAD1 positive). (B) Absolute numbers of infections in non-teneral tsetse flies with either slender or stumpy T. brucei cells. Male and female flies were infected with untreated blood containing 1×106 cells/ml. The tdTomato NLS-GFP:PAD1 3′UTR cell line enabled FACS-based separation of stumpy (PAD1 positive, GFP in nucleus) and slender (PAD1 negative, no nuclear fluorescence) forms prior to infection. All non-teneral flies were 144–168 hr post eclosion (hpe) and had already received two non-infectious bloodmeals prior to the infectious feed. Flies were dissected 35 days post infection, and their midgut (MG), proventriculus (PV), and salivary glands (SG) examined for parasite presence.
- https://cdn.elifesciences.org/articles/108688/elife-108688-supp1-v1.docx
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Replicative slender bloodstream forms complete transmission of Trypanosoma brucei without prior differentiation into stumpy forms
eLife 14:RP108688.
https://doi.org/10.7554/eLife.108688.4
