Independent Pathways Can Transduce the Life-Cycle Differentiation Signal in Trypanosoma brucei

African trypanosomes cause disease in humans and livestock, generating significant health and welfare problems throughout sub-Saharan Africa. When ingested in a tsetse fly bloodmeal, trypanosomes must detect their new environment and initiate the developmental responses that ensure transmission. The best-established environmental signal is citrate/cis aconitate (CCA), this being transmitted through a protein phosphorylation cascade involving two phosphatases: one that inhibits differentiation (TbPTP1) and one that activates differentiation (TbPIP39). Other cues have been also proposed (mild acid, trypsin exposure, glucose depletion) but their physiological relevance and relationship to TbPTP1/TbPIP39 signalling is unknown. Here we demonstrate that mild acid and CCA operate through TbPIP39 phosphorylation, whereas trypsin attack of the parasite surface uses an alternative pathway that is dispensable in tsetse flies. Surprisingly, glucose depletion is not an important signal. Mechanistic analysis through biophysical methods suggests that citrate promotes differentiation by causing TbPTP1 and TbPIP39 to interact.


Figure S1
Glucose depletion does not stimulate the differentiation of stumpy form trypanosomes. Stumpy forms were incubated with 10% serum (approximately 0.5mM glucose final concentration) plus 0mM, 2.5mM, 5.0mM, 7.5mM or 10mM glucose in the presence (+CA) or absence (-CA) of 6mM cis-aconitate. In low glucose, no differentiation was observed, though the cells underwent efficient differentiation in the presence of CA. Without serum, the cells rapidly underwent cell death (not shown). Procyclin expression was monitored after 4h and 24h. Control assays were carried out also with stumpy form cells (red, right hand panel; negative control) or procyclic form cells (blue, right hand panel, positive control).

Figure S2
Western blots detecting TbPIP39 from samples derived from TbPIP39-RNAi stumpy cells, induced or uninduced with doxycycline during in vivo growth, and then exposed to cis-aconitate, mild acid, and pronase. Induction was retained after differentiation was stimulated by inclusion of tetracycline in the cell culture medium, with samples being prepared at the indicated times (in hours) thereafter. Western blots were reacted with the antibody against αtubulin (Tub) to show the relative loading in each case, and with antibody detecting TbPIP39.

Figure S3
Trypanosomes in tsetse midgut extracts treated (+STI) or untreated (-STI) with Soybean trypsin inhibitor and probed for EP procyclin expression after 4 h. Controls of cultured procyclic forms and bloodstream forms are also shown. Exposures in the EP procyclin panels in each case were identical to allow comparison of relative staining. Bar= 10 µm.

PIP39
ATGGTGAGGACGACACGCTTTTCACGCAACAGTTCCAAACCACTGTTTCAGCAATTACAG 60 PIP39s atggttcgcacaacacgctttagccgcaacagcagcaagccactttttcagcagctgcag M V R T T R F S R N S S K P L F Q Q L Q 20 PIP39 CTTATTACATCGTATGGACTCTTTCAGGAGTATCCACCCATTTGGTCACTTCCCCTTCCA 120 PIP39s ctgattacgagctacggtctttttcaagagtacccgccaatttggagccttccacttcca L I T S Y G L F Q E Y P P I W S L P L P 40 Gene sequence of TbPIP39 and recoded TbPIP39 mutants where predicted citrate binding residues (in bold and underlined) are mutated

Figure S5
Citrate and isocitrate allow interaction between TbPIP39 and TbPTP1.
A. Purified TbPTP1 and TbPIP39wt used in these interaction assays B. 2-fold dilution of PIP39wt from 59 µM -3.7 µM in the absence of either citrate or iso-citrate subjected to surface plasmon resonance on a TbPTP1 surface and analysed using a T200 BIAcore. C. 2-fold dilution of citrate alone from 2 mM -0.125 mM subjected to surface plasmon resonance on a TbPTP1 surface and analysed using a T200 BIAcore. D. 2-fold dilution of TbPIP39wt from 59 µM -3.7 µM in the presence of 2 mM citrate subjected to surface plasmon resonance on a TbPTP1 surface and analysed using a T200 BIAcore. E. 2-fold dilution of iso-citrate alone from 2 mM -0.125 mM subjected to surface plasmon resonance on a TbPTP1 surface and analysed using a T200 BIAcore. F. 2-fold dilution of PIP39wt in from 59 µM -3.7 µM in the presence of 2 mM iso-citrate subjected to surface plasmon resonance on a TbPTP1 surface and analysed using a T200 BIAcore.

Figure S6
Calorimetric titration of citrate into recombinant TbPIP39 wt, D, DD and 6364 mutants.
TbPIP39 wt, D, DD or 6364 mutants were desalted into ITC buffer (10 mM Hepes, pH7.3; 0.05% P20; 20 mM MgCl 2 and 2mM DTT) on a pre-equilibrated 5ml HiTrap desalting column (GE Healthcare). After a preliminary injection of 2 µl, nineteen 15 µl aliquots of 2 mM citrate in ITC buffer were injected into 1.41 ml of 5 µM TbPIP39 wt, D, DD or 6364 mutants at 25 o C using an AutoITC instrument (MicroCal). The experimental parameters were as follows: injection time 30s; 200s inter-injection delay, stirring 250 rpm and power setting 5. Control experiments were carried out in which 2 mM citrate in ITC buffer was injected into ITC buffer to determine heat of dilution for 2 mM citrate in ITC buffer. Each peak represents the injection of a 15 µl aliquot of 2 mM citrate into 1.41 ml of 5 µM TbPIP39 at 25 o C in 10 mM Hepes, pH7.3; 0.05% P20; 20 mM MgCl 2 and 2mM DTT. No difference was observed in the presence of TbPIP39 or buffer only, demonstrating an absence of detectable citrate binding.