Abstract
Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.
Highlights
Trypanosomatids, the causative agents of human sleeping sickness and Nagana cattle disease in Africa (Trypanosoma brucei species), South-American Chagas’ disease (T. cruzi) and the different forms of leishmaniasis (Leishmania species), possess an unusual thiol redox metabolism
Our work is the first report on T. brucei thioredoxin-2 (Trx2)
We show that Trx2 is located in the mitochondrion and its absence affects parasite proliferation and infectivity
Summary
Trypanosomatids, the causative agents of human sleeping sickness and Nagana cattle disease in Africa (Trypanosoma brucei species), South-American Chagas’ disease (T. cruzi) and the different forms of leishmaniasis (Leishmania species), possess an unusual thiol redox metabolism. The dithiol is kept in the reduced form by the NADPH-dependent trypanothione reductase which catalyzes the only reaction directly connecting the thiol and dinucleotide redox metabolism in these parasitic protozoa. An important mediator for T(SH)2-dependent reactions is tryparedoxin (Tpx), an essential and parasite-specific distant member of the thioredoxin (Trx)-type protein family [10]. Members of the Trx protein family have since been shown to exhibit multiple physiological functions. In addition to their role as general thiol-disulfide oxidoreductases, they can function in transcription factor regulation, protein binding, protein folding facilitation and chaperone-type activities [16]. This Trx (TriTrypDB gene ID Tb927.9.3370) has the canonical WCGPC motif of classical Trxs and catalyzes the electron transfer from T(SH) onto
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