Fig 1.
Selection of negative regulators of stumpy enriched transcript expression.
A. Schematic representation of the selection schema to isolate negative regulators of ESAG9 expression in slender forms; NR denotes a negative regulator. The selection construct used to transfect the RNAi library is shown beneath. The NeoR gene is flanked by the 5’UTR of the trypanosome aldolase gene, whilst the 3’UTR is derived from 1057bp downstream of the ESAG9-EQ stop codon (long form reporter, blue). B. Titration of the sensitivity of trypanosome lines with NeoR flanked by either the ESAG9-EQ 3’UTR or aldolase 3’UTR. Error bars symbolize standard deviations of biological triplicates. C. RNAi library selection of parasites containing NeoR flanked by the ESAG9 3’UTR (diamonds). The right hand panel shows the library insert amplicon profile from cells with NeoR flanked by the ESAG9 3’UTR at different stages of selection in G418 when RNAi was either induced, or not. The gel shows gDNA isolated from untreated (no tetracycline, no G418) parasites at days 2 and 3 (as a control), and treated parasites at days 11 and 14 after exposure to 1μg/ml of tetracycline and 10μg/ml G418, when parasites were outgrowing as a resistant population. D. Genome-wide distribution of selected RNAi inserts determined by ion torrent sequencing, vertical dashes representing the boundaries of each chromosome (with Chromosome 1 at the left). Targets highlighted comprise those with a high enrichment in the selected population and a predicted RNA binding domain. The full dataset is available in S1 Table.
Fig 2.
Validation of RNAi lines silencing REG9.1 selected from the RNAi library.
A. Northern blot demonstrating the inducible gene silencing of REG9.1. Two independent RNAi clones were analysed (REG9.1, clone 3 and 4).); rRNA was used as a loading control. Smearing is caused by the transcript being larger than 6 kb. B. G418 resistance (at 10μg/ml) of the respective RNAi clones when RNAi was induced or not. RNAi resulted in decreased sensitivity to G418. Error bars (obscured by the symbols) symbolize standard deviations of the triplicate assays of each clone ±tetracycline. C. Northern blot of NeoR transcript levels when each REG9.1 RNAi line was induced or not; rRNA was used as a loading control. D. Western blot of NeoR protein when each REG9.1 RNAi line was induced or not; EF1α was used as a loading control. E. Northern blot of ESAG9-EQ transcript levels when each REG9.1 RNAi line was induced or not; rRNA was used as a loading control.
Fig 3.
REG9.1 developmental expression profile and REG9.1 RNAi in pleomorphic parasites.
A. Schematic representation of REG9.1 showing the position of its single predicted RNA binding domain (RBD). B. Protein expression of REG9.1. The antibody detects three bands, the lower band being detected inconsistently between blots. The abundance of the two upper bands differ in different life cycle stages, with the uppermost band being less abundant in stumpy forms. Procyclic forms express more REG9.1 than bloodstream forms. C. Northern blot for REG9.1 and ESAG9-EQ in the parental line (T. brucei AnTat90:13) or two replicates of the pleomorphic RNAi lines induced or not to silence REG9.1. Smearing is caused by the REG9.1 transcript being larger than 6 kb. rRNA provides the loading control. ESAG9-EQ mRNA is elevated upon REG9.1 depletion in each replicate. D. Western blot of REG9.1 expression in pleomorphic cells induced to deplete expression via RNAi. The loading control was an antibody detecting EF1α.
Fig 4.
Phenotype of REG9.1 gene silencing in vivo.
A. Growth of REG9.1 pleomorphic RNAi lines in vivo, with RNAi induced or not by doxycycline. The inset shows the respective abundance of REG9.1 transcript in each case after cells were harvested on day 4 post infection and RNA prepared. Two mice replicates (R1 and R2) were used for each condition (–DOX and +DOX). B. Morphology of cells on day 4 of infection. Two cells are shown for the uninduced population, both being ‘intermediate’ in morphology, although the upper cell is somewhat more stumpy with the nucleus more proximal to the kinetoplast; in the induced population a stumpy cell is shown, with the nucleus moved toward the cell posterior. Hence although at a much lower parasitaemia (see panel A), the stumpy morphology of induced cells was equivalent to, or greater than, uninduced cells. The DAPI panel at the left hand side shows the positions of the nucleus (N) and kinetoplast (K) in each case Bar = 10μm. C. Cell cycle profile of pleomorphic RNAi lines (R1, R2) induced or not to silence REG9.1 expression. The kinetoplast/nuclear configuration was assessed on day 3 and day 4 of infection, with the associated parasitaemia shown above each bar. At least 250 cells were counted for each condition/replicate/day. D. PAD1 expression profile determined by flow cytometry in the REG9.1 RNAi lines (R1, R2) induced or not, with slender or stumpy T. brucei AnTat1.1 cells as controls. The parasitaemia of each population at harvest is shown. PAD positivity was determined by gating with respect to the Slender (negative) and Stumpy (positive) control samples. Below the chart is the PAD1 expression profile determined by western blotting in the REG9.1 RNAi lines (R1, R2) induced or not, when harvested, with stumpy AnTat1.1 cells as control (‘ST’). EF1α abundance was used as a loading control. E. Flow cytometry of the expression of EP procyclin during differentiation to procyclic forms of each REG9.1 RNAi line (R1, R2) 0h, 4h and 24h after exposure to 6mM cis aconitate. EP procyclin expression on slender, stumpy and procyclic forms provide controls. A low level of EP procyclin expression is detected on stumpy cells, this being induced during isolation and cell preparation procedures. Although at lower parasitaemia (panel A) the induced cells differentiate as effectively as uninduced cells demonstrating the physiological relevance of the stumpy forms generated.
Fig 5.
Global transcriptome changes associated with REG9.1 RNAi.
A. Transcripts up and down regulated upon REG9.1 RNAi in pleomorphic trypanosomes. The data represents an analysis of biological replicates whether cells were induced or not to deplete REG9.1 (two replicates each). The down regulation of histones and the upregulation of key regulated molecules, including ESAG9 (lilac), Family 5 (red) and Family 7 members (blue) is highlighted. B. Relative LogFC (Log2 fold change) expression of different members of T. brucei surface protein Family 5 (red) Family 7 (blue) and ESAG9 (green) after REG9.1 depletion by RNAi. C. Northern blots of ESAG9, Family 5 and family 7 transcripts in slender (SL), stumpy (ST) and procyclic forms (PF). The similarity within family 5 and 7 results in the detection of multiple family members by the riboprobes, generating a smear of bands. Northerns for GP63 and RHS7 are also shown, both being upregulated in the RNA-seq dataset. rRNA provides the loading control.
Fig 6.
REG9.1 RNAi in procyclic forms.
A. Growth of procyclic form AnTat1.1 90:13 RNAi lines induced and uninduced to deplete REG9.1. B. Morphology of T. brucei AnTat 90:13 procyclic form REG9.1 RNAi lines, induced and uninduced. DAPI stained and phase-contrast/DAPI stained cells (‘Merge’) are shown. Bar = 10μm. C. On the left panel is shown a Western blot of REG9.1 in AnTat1.1 90:13 procyclic forms whether induced or not to deplete REG9.1 transcript by RNAi for 96 h. Established T. brucei AnTat 29:13 procyclic forms were used as a control; the loading control was an antibody detecting EF1α. On the right are northern blots of REG9.1 and ESAG9-EQ mRNAs in stumpy forms, procyclic AnTat1.1 29:13 control cells and the AnTat1.1 90:13 procyclic form REG9.1 RNAi line. Upon REG9.1 depletion (for 48h), ESAG9-EQ levels are elevated. Smearing for REG9.1 is caused by the transcript being larger than 6 kb.
Fig 7.
Pleomorphic trypanosomes expressing REG9.1 ectopically using pLEW express procyclic form mRNAs.
A. Ectopic overexpression of REG9.1 in pleomorphic bloodstream forms. The panel shows the expressed REG9.1 detected with an antibody to the protein in bloodstream forms (induced or uninduced for overexpression using pLEW), with procyclic form (PF) samples included to demonstrate that similar levels of expression to procyclic forms were generated upon induction. B. In vitro growth of parasites induced to express REG9.1 from the pLEW expression vector. C. Transcripts up and down regulated upon REG9.1 ectopic expression using pLEW in pleomorphic trypanosomes. The data represents an analysis of two biological replicates whether cells were induced or not to ectopically express REG9.1.
Fig 8.
Ectopic expression of REG9.1 in pleomorphic trypanosomes potentiates differentiation.
A. Immunofluorescence showing EP procyclin expression on pleomorphic parasites induced to express REG9.1 ectopically using pLEW. αVSG AnTat1.1 is used as a marker of the bloodstream forms surface. B. Flow cytometry showing EP procyclin expression on pleomorphic slender parasites induced to express REG9.1 ectopically using pLEW. 24h after induction parasites were resuspended in procyclic form culture medium (SDM79) and induced to form procyclic forms in the presence of cis aconitate (+CCA). Induced cells differentiated faster than the uninduced (**, P<0.01; ***, P<0.001).
Fig 9.
Differential cellular location of REG9.1 in the trypanosome life cycle.
A. Cellular location of REG9.1 in slender, stumpy and procyclic forms. Kinetoplast and nuclear DNA was visualised with DAPI staining (pseudocoloured blue), whereas the cell outline was visualised via Phase contrast microscopy. The REG9.1 signal is cytoplasmic in slender and procylic forms but concentrated at the cell posterior in stumpy forms (arrowed). Bar = 10μm. B. Co staining of two stumpy cells with REG9.1 (Green), AMCA (Red, staining the flagellar pocket) and propidium iodide (pseudocoloured Blue, staining the nucleus and kinetoplast). The REG9.1 staining is close to but not coincident with the AMCA staining at the flagellar pocket.