Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei

Abstract

African trypanosomes are protozoan parasites transmitted by a tsetse fly vector to a mammalian host. The life cycle includes highly proliferative forms and quiescent forms, the latter being adapted to host transmission. The signaling pathways controlling the developmental switch between the two forms remain unknown. Trypanosoma brucei contains two target of rapamycin (TOR) kinases, TbTOR1 and TbTOR2, and two TOR complexes, TbTORC1 and TbTORC2. Surprisingly, two additional TOR kinases are encoded in the T. brucei genome. We report that TbTOR4 associates with an Armadillo domain-containing protein (TbArmtor), a major vault protein, and LST8 to form a unique TOR complex, TbTORC4. Depletion of TbTOR4 caused irreversible differentiation of the parasite into the quiescent form. AMP and hydrolysable analogs of cAMP inhibited TbTOR4 expression and induced the stumpy quiescent form. Our results reveal unexpected complexity in TOR signaling and show that TbTORC4 negatively regulates differentiation of the proliferative form into the quiescent form.

Fig. 1.

TbTOR4 associates with both TbLST8 and Armtor. (A) Gel filtration elution profile of TbTOR4. Whole-cell lysates prepared in lysis buffer containing 0.3% CHAPS from wild-type bloodstream cells were loaded onto a Superose 6 sizing column. One-milliliter fractions were collected and processed for TbTOR4 immunoblotting. (B). Detection of TbLST8-interacting proteins in affinity purifications of TAP-LST8. Copurified material was resolved by SDS PAGE and visualized using Coomassie Brilliant Blue staining. An untagged cell line was used as negative control. (C) Endogenous TbTOR4 interacts with TbArmtor. Co-IP experiments were carried out using anti-TbTOR4 and anti-TbArmtor antibodies and conditions previously described (5). Western blotting of coimmunoprecipitated material revealed the binding of TbArmtor to TbTOR4. (D) TbTOR4 depletion halts cell proliferation. Growth curves of TbTOR4-depleted bloodstream trypanosomes after RNAi induction with doxycycline (Ind.) is compared with either uninduced (Unind.) or parental cell lines (Parent.) as controls. Cultures were diluted daily to 2.5 × 10⁴ cells/mL to maintain cell density within a range that supports exponential growth. (E) Western blot analysis of TbTOR4 expression in total cell extracts (5 ×10⁶ cells per lane) upon RNAi induction. Tubulin was used as a loading control.

Fig. 2.

TbTOR4 knockdown induces irreversible cell-cycle arrest. (A) FACS analysis of DNA content in TbTOR4-depleted cells (TbTOR4 ind.) and in the uninduced control (TbTOR4 unind.) 48 h after RNAi induction with 1μg/mL doxycycline. (B) FACS analysis showing forward scatter of cells (FSC) with reduced expression of TbTOR4 (TbTOR4 ind.), TbTOR1 (TbTOR1 ind.), and the uninduced control (TbTOR4 unind). (C) Relative plating efficiency measured after induction of TbTOR1, TbRaptor, and TbTOR4 RNAi, compared with uninduced and parental cell lines. RNAi was induced by adding doxycycline to the medium for 48 h. Later, cells were washed to recover protein expression, and wells positive for cell growth were counted (SI Materials and Methods). (D) Western blotting analysis using anti-TbTOR4 antisera shows recovery of TbTOR4 expression after doxycycline removal. Cytosolic marker was used as loading control (31). (E) Cell morphology remodeling in bloodstream trypanosomes upon TbTOR4 loss of function. A stumpy morphology is visualized by DIC optics on live cells 96 h after TbTOR4 RNAi induction.

Fig. 3.

TbTOR4 regulates processes involved in slender-to-stumpy differentiation including transcription remodeling. (A) The percentage of NADH dehydrogenase (diaphorase)-positive cells after the induction of TbTOR1 and TbTOR4 RNAi was determined at 8-h intervals for 96 h. Cells were visualized using DIC optics (Fig. S4). (B) The effect of mild acidic extracellular conditions on cell survival was assayed in TbTOR4- and TbTOR1-depleted cells. Cells were harvested at 8-h intervals for 96 h and incubated for 2 h at pH 5.5 and 37 °C. The number of remaining motile cells after the incubation was assessed with a hemocytometer. *P < 0.05, **P < 0.001, unpaired Student’s t test. (C) TbTOR4 and TbTOR1 are localized in distinct subcellular compartments. 3D microscopy localization was performed using anti-TbTOR1 rabbit antiserum and anti-TOR4 monoclonal antibody (Materials and Methods). Cells were counterstained with DAPI to locate the nuclear (N) and kinetoplast (K) mitochondrial DNA. (D) TbTOR4 loss of function induces changes in the transcriptome similar to stumpy differentiation. qRT-PCR analysis of TbTOR4-depleted cells versus parental cells was compared with slender-versus-stumpy RNA samples as a control. The genes analyzed by qRT-PCR were ESAG 11 (Tb427.BES40.19), histone H4 (H4) (Tb927.5.4240), TAO (Tb10.6k15.3640), PAD8 (Tb927.7.6000), control (C1) (Tb10.389.0540), PAD1 (Tb927.7.5930), VSG 221 (MITat 1.2/VSG2), procyclin (Tb927.6.480), myosin (MYO) (Tb11.01.7990), tubulin (TUB) (Tb927.1.2370), and PAD 2 (Tb927.7.5940). The histogram shows gene expression levels expressed as fold changes based on comparative cycle threshold (Ct) calculations using PIK-related (Tb927.2.2260) as house-keeping gene.

Fig. 4.

TbTOR4 loss of function triggers the quiescent stumpy form and preadapts bloodstream trypanosomes for differentiation to the insect form. (A) In the proliferative slender form, TbRPA1 (the largest subunit or RNA polymerase I) (21) is restricted to the nucleolus (No) and the ESB, whereas in the quiescent stumpy form TbRPA1 nuclear localization is scattered in multiple foci within the nucleoplasm. TbTOR4 depletion induced RNA polymerase I delocalization from the nucleolus and the ESB similar that detected in the stumpy form. Maximum intensity projections of three-channel 3D representative stacks show the anti-TbRPA1 signal in green and DAPI staining in blue. (Scale bars, 1 μm.) (B) PAD1 protein expression specific for the stumpy form (St) also is up-regulated upon TbTOR4 depletion as assessed by Western blotting using specific antibodies (22). Cytosolic marker was used as loading control (31). (C) Responsiveness to cis-aconitate during bloodstream-to-procyclic differentiation was analyzed in TbTOR4-depleted cells vs. uninduced control. Differentiation to the procyclic form was triggered in DTM medium at 28 °C using increasing concentrations of cis-aconitate (SI Materials and Methods). (D and E) TbTOR4 loss of function promotes a rapid differentiation to the procyclic form. Shown are the results of a time-course experiment following changes in the expression of stage-specific markers during in vitro bloodstream-to-procyclic differentiation. The expression on the surface of the procyclin and VSG was followed by indirect immunofluorescence (SI Materials and Methods).

Fig. 5.

AMP and hydrolysable analogs of cAMP inhibited TbTOR4 expression and induced the stumpy-like quiescent form. (A) The membrane-permeable and hydrolysable cAMP analog (8-pCPT-2′-O-Me-cAMP) (10 μM) down-regulates TbTOR4 expression and induces stumpy-like (pH5.5-resistant) cells in monomorphic trypanosomes; TbTOR1 expression is unaffected. Anti-tubulin was used as a loading control. (B) The hydrolysis-resistant cAMP analog (Sp-8-pCPT-2′-O-Me-cAMPS) (10 μM) does not affect TbTOR4 expression levels or induction of stumpy-like cells. Anti-tubulin was used as a loading control. (C) Membrane permeable 5′-AMP (8-pCPT-2′-O-Me-5′-AMP) (5 μM) promotes rapid TbTOR4 down-regulation and induces stumpy-like monomorphic trypanosomes. Anti-TbRRP4 was used as a loading control (31).