Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum

Abstract

Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx's ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx's chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection.

Keywords: Leishmania; chaperone; peroxiredoxin.

Fig. 1.

Reduced mTXNPx prevents thermal aggregation of luciferase. Influence of mTXNPx_red (A) or mTXNPx_ox (B) on the thermal aggregation of luciferase. Native luciferase (0.1 µM) was incubated in the absence (0:1) or presence of different ratios of mTXNPx at 41.5 °C, and light scattering (expressed in arbitrary units, A.U.) was monitored at 360 nm. Upon dilution of mTXNPx_red into the assay buffer, the concentration of residual DTT was 0.2 mM. The same amount of DTT was added to the respective controls. (C) Influence of a 10-fold molar excess of reduced or oxidized mTXNPxC81S mutant variant on the in vitro aggregation of thermally unfolding luciferase. The addition of 0.2 mM DTT did not significantly affect luciferase aggregation in the absence of chaperones. The ratio of chaperone to client protein was calculated based on the monomeric form of mTXNPx. Arrows indicate the time point at which luciferase was added to the reactions.

Fig. 2.

mTXNPx_red maintains luciferase in a refolding competent state. (A) Luciferase (0.1 µM) was incubated in the absence (Inset) or presence of a 40:1 molar ratio of mTXNPx_red for 20 min at 42 °C. Samples were cooled to 25 °C. After 10 min (indicated by the arrow), the samples were supplemented with 2 mM MgATP, 0.1 mg/mL BSA, and, where indicated, with a 20:4:20:1 ratio of DnaK:DnaJ:GrpE (KJE) system to luciferase. At the indicated time points, aliquots were removed, and luciferase activity was determined. (B) Luciferase (0.1 µM) was inactivated in the absence of any chaperone system (−), in the presence of a 10:1, 20:1, or 40:1 molar ratio of mTXNPx_red/mTXNPx_ox to luciferase, or in the presence of a 20:4:20:1 ratio of DnaK:DnaJ:GrpE (KJE) to luciferase at 42 °C. All samples shown in the left panel contained 0.2 mM DTT, whereas samples shown in the right panel did not contain any reducing agents. After 20 min, the temperature was shifted to 25 °C for 10 min, and 2 mM MgATP and 0.1 mg/mL BSA were added to all samples. In addition, all samples except those that already contained the KJE system were supplemented with a 20:4:20:1 ratio of DnaK:DnaJ: GrpE:luciferase. After 120 min of incubation at 25 °C, luciferase activity was measured. Luciferase activity before incubation at 42 °C was set to 100%.

Fig. 3.

mTXNPx displays general chaperone activity. (A) Aliquots of a soluble extract (T) from the chaperone-deficient E. coli rpoH mutant strain were prepared, supplemented with increasing amounts of mTXNPx_red (in the presence of 1.5 mM DTT) or mTXNPx_ox (in the absence of DTT), and heat treated for 60 min at 45 °C. Samples without mTXNPx served as controls. The insoluble proteins then were separated from the soluble supernatant by centrifugation and were analyzed on reducing SDS/PAGE. The order of the lanes in the gel was rearranged electronically to facilitate data interpretation. (B) Wild-type, mtxnpx⁻, or mtxnpx⁻/+mTXNPx L. infantum promastigotes were incubated at 25 °C or 37 °C. Parasites were collected at the indicated time points, and their aggregated proteins were isolated and analyzed by SDS/PAGE. The major protein band that is present in all lanes is likely GP63, a GPI-anchored membrane protein and one of the most abundant proteins in Leishmania.

Fig. 4.

mTXNPx undergoes temperature-mediated structural changes. (A) Bis-ANS binding to mTXNPx_red (red lines) or mTXNPx_ox (black lines) at either 25 °C (broken lines) or 42 °C (solid lines). (Inset) Bis-ANS binding of mTXNPx_red or mTXNPx_ox as a function of temperature monitored at 500 nm. (B) Far-UV circular dichroism spectra of mTXNPx_red (red lines) or mTXNPx_ox (black lines) at either 25 °C (dashed lines) or 42 °C (solid lines). (Inset) Temperature-dependent changes in the secondary structure of mTXNPx_red or mTXNPx_ox recorded at 197 nm. Temperature was increased at a rate of 1 °C/min. All spectra were buffer corrected.

Fig. 5.

The mTXNPx_red decamer binds thermally unfolded luciferase in the center of its ring. (A and B) Representative EM micrographs and selected single-particle images of negatively stained mTXNPx_red decamers alone (A) or after incubation with thermally unfolded luciferase (B). (C and D) Reference-free 2D projection averages showing top-down and side views of the mTXNPx_red decamer alone or with luciferase. The percentage of mTXNPx_red decamers in complex with luciferase varied between experiments. (Scale bars: 200 Å for the micrograph images and 50 Å for the boxed particles and averages.)

Fig. 6.

Chaperone activity of mTXNPxs is critical for Leishmania virulence. (A) Influence of a 20-fold molar excess of His.THR-mTXNPx_red on the in vitro thermal aggregation of luciferase. See the legend of Fig. 1 for details. (B) Analysis of mTXNPx expression in mtxnpx⁻ (lane 1), mtxnpx⁻/+mTXNPx (lane 2), and two mtxnpx⁻/+MTS.His.THR-mTXNPx (lanes 3 and 4, respectively) promastigote clones using a polyclonal antibody raised against the recombinant His.THR-MTS.mTXNPx. Purified mTXNPx and His.THR-mTXNPx were used as controls for the expected size of the proteins, and their position on the blot is indicated. Ponceau S staining of the blot is shown as loading control. (C) Localization of the His.THR-mTXNPx chimera in mtxnpx⁻ promastigotes using indirect immunofluorescence with the anti-mTXNPx antibody (green) merged with DAPI (blue). Controls as described above are included. (D) Growth rate of mtxnpx⁻ (black circles), mtxnpx⁻/+mTXNPx (black squares), and two mtxnpx⁻/+MTS.His.THR-mTXNPx clones (dark gray triangles). (E) Parasite burden of BALB/c mice after 2 and 8 wk of infection. BALB/c mice were inoculated i.p. with stationary-phase promastigotes of the mtxnpx⁻/+mTXNPx clone (squares) or two mtxnpx⁻/+MTS.His.THR-mTXNPx clones (triangles). After 2 and 8 wk of infection, the number of parasites per gram of spleen (parasite burden, PB) was determined. Each point represents one animal. The red line indicates the detection limit of the technique (log₁₀ = 2.7). Animals with parasite burdens below this limit are “not detected” (ND) and are represented by red symbols.

Fig. 7.

Physiological role of mTXPNx’s chaperone function in Leishmania. Exposure of Leishmania promastigotes (the insect stage) to the mammalian body temperature of 37 °C leads to the protein unfolding. This change in temperature is sensed by reduced mTXNPx decamers and translated into structural rearrangements, which likely contribute to the activation of its chaperone function. Once they are chaperone active, mTXNPx decamers bind the client proteins in the center of their ringlike structure, preventing the client proteins from forming cytotoxic aggregates. Client proteins are maintained in a refolding-competent state and can be reactivated by members of the Hsp70 system in the presence of ATP. The working cycle of mTXNPx chaperone appears to be crucial for Leishmania to adapt to and survive the temperature encountered in the mammalian host and hence to generate viable amastigotes.