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PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes

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Abstract

To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte1. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive2,3,4. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell.

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Figure 1: HSP101 is essential for development of asexual and sexual blood stages.
Figure 2: HSP101 is required for export of PEXEL and PNEP proteins.
Figure 3: HSP101 is required for activation of PSAC but not trafficking of CLAG3 to the RBC periphery.
Figure 4: Inactivated HSP101DDD dissociates from the PTEX complex.

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  • 31 July 2014

    A panel label in Fig. 2e has been corrected

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Acknowledgements

This work was supported by National Institutes of Health grants AI047798 to D.E.G., T32-AI007172 to J.R.B. and AI099156 to V.M. We thank J. McBride, D. Cavanagh and EMRR for anti-EXP2 antibody, J. Adams and ATCC (MR4) for anti-BiP antibody, D. Taylor for anti-HRP2 antibody, R. Anders for anti-RESA antibody, C. Braun-Breton for anti-SBP1 antibody, K. Williamson for anti-PfGECO and anti-Pfs16 antibodies, T. Spielmann for anti-REX2, anti-REX3 and anti-MSRP6 antibodies, L. Tilley for anti-REX1 and anti-PfEMP1 antibodies, S. Desai for anti-CLAG3 antibody, A. Cowman for anti-KAHRP antibody, J. Przyborski and K. Lingelbach for anti-SERP antibody, W. Beatty for assistance with electron microscopy, B. Vaupel and T. Butler for technical assistance and P. Sigala and N. Spillman for suggestions.

Author information

Authors and Affiliations

Authors

Contributions

J.R.B., V.M. and D.E.G. conceived and designed experiments. J.R.B. performed the majority of the experiments and V.M. performed some experiments. V.M. and A.O. generated the HSP101DDD strains. J.R.B and A.O. performed the gametocyte analysis. J.R.B. and D.E.G. analysed the data and wrote the manuscript. All authors discussed and edited the manuscript.

Corresponding author

Correspondence to Daniel E. Goldberg.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Generation of HSP101DDD strains.

a, Schematic of strategy to generate HSP101DDD parasites. 3′F, 3′ flank for homologous recombination; BSD, blasticidin S deaminase; TMP, trimethoprim; HA, haemagglutinin tag; DDD, DHFR destabilization domain. b, Diagnostic PCR showing integration of the DDD fusion in the two independent clones 13F10 and 14G11. Primers shown as black arrows in a. Image is representative of two independent experiments. c, Southern blot showing integration of plasmid pHSP101-HDB occurred at the intended genomic locus. Expected NcoI digestion products and sizes are indicated in blue, red and orange in a. The 7.3-kb band in 13F10 and 14G11 indicates the presence of concatemers commonly observed in P. falciparum. Image is representative of one experiment. d, Western blot with anti-HA antibodies detects a 124 kDa band in clones 13F10 and 14G11. Image is representative of two independent experiments. e, IFA of acetone-fixed 13F10 parasites showing colocalization of HSP101DDD and EXP2 at the PVM. Scale bar, 5 µm. Images are representative of two independent experiments.

Extended Data Figure 2 Characterization of HSP101DDD strains.

a, TMP dose-response for 13F10 and 14G11 parasites grown for 96 h was measured. Error bars represent s.d. of three technical replicates. b, Western blot on lysates from asynchronous 13F10 parasites grown with or without TMP for 24 h. BiP serves as a loading control. Blotting with anti-HA antibody shows no decrease in HSP101DDD protein levels relative to the BiP loading control. Images are representative of two independent experiments. c, Quantification of new ring parasitaemia with or without TMP. In a parallel experiment to that shown in Fig. 1d (but beginning with lower parasitaemia cultures), new rings were counted in Giemsa-stained smears of synchronous 13F10 parasites where TMP was removed or not during the proceeding trophozoite stage. No significant difference in the resulting new ring parasitaemia was observed with or without TMP, indicating no difference in re-invasion efficiency–TMP. Error bars represent s.d. of three technical replicates. Data are representative of two independent experiments. d, Giemsa-stained smears of synchronous 13F10 parasites grown with or without TMP from Fig. 1d. Reintroduction of TMP after 24 or 48 h restored progression through the intraerythrocytic cycle. After 72 h without TMP, most parasites appeared as dead, pyknotic forms. Images are representative of three independent experiments. Original magnification × 1,000.

Extended Data Figure 3 HSP101 function is critical for early stage gametocyte development.

Giemsa-stained smears from day nine post gametocyte induction as quantified in Fig. 1e. TMP was removed from parallel samples at 24-h intervals beginning just before reinvasion (0 h) following gametocytogenesis induction. Data are representative of 4 independent experiments. While control (+TMP) gametocytaemia varied between experiments, a similar effect on gametocyte formation was observed in each experiment following TMP removal. Original magnification × 1,000.

Extended Data Figure 4 Localization and quantification of exported proteins following HSP101DDD inactivation.

af, IFA and immunoelectron microscopy of ring-stage 13F10 parasites with or without TMP. TMP was removed in late schizont stage and parasites were allowed to reinvade and grow 18–24 h before fixation with paraformaldehyde (a) or acetone (bd, f). Immunoelectron microscopy fixation (e) is detailed in methods. A similar export block with accumulation of the exported protein at the parasite periphery was observed in each case when TMP was removed. The soluble PEXEL-containing protein REX3 (a) is normally exported into the host RBC cytosol. The PEXEL-containing KAHRP protein (b) is normally exported through Maurer’s clefts to knob structures at the cytoplasmic face of the infected RBC membrane. The PEXEL-containing protein RESA (c) is normally exported to the RBC periphery. SERP is a marker for the PV. SBP1 (d) is an integral membrane PNEP normally exported to the Maurer’s clefts. In the absence of TMP, blocked SBP1 colocalizes with EXP2. e, Immunoelectron microscopy showing localization of SBP1 in ring-stage parasites with or without TMP. MC, Maurer’s cleft. Scale bars, 500 nm. Images are representative of one experiment. REX2 (f) is an integral membrane PNEP normally exported to the Maurer’s clefts. g, Quantification of export block by IFA for exported proteins shown here and in Fig. 2c–f. Export was scored as complete (all signal in the host cell), partial (signal within the host cell but also within the PV) or no export (signal only seen within the PV and not in the host cell). Example images of each scoring scenario are given for REX1 in Extended Data Fig. 5a. In the case of PfEMP1, cells were scored as having PfEMP1 signal at the RBC periphery (complete export) or not (no export) due to the fact that some PfEMP1 signal is always seen within the PV under normal export conditions (see Fig. 2e, +TMP). Error bars represent s.d. of three technical replicates. Data are representative of at least two independent experiments. h, IFA of PfGECO in paraformaldehyde-fixed, stage I gametocytes 36 h post invasion with or without TMP. Pfs16 is a gametocyte-specific PVM marker. All IFA scale bars, 5 µm. All IFA images (ad, f, h) are representative of two independent experiments.

Extended Data Figure 5 Reactivation of export does not require new protein synthesis.

a, b, TMP was removed in late schizonts and parasites were allowed to reinvade and develop for 18 h before TMP add back with or without 10 µg ml−1 cycloheximide (CHX). Parasites were acetone-fixed 24 h later and processed for IFA. Export was scored as complete (no REX1 retained within the EXP2-labelled PVM), partial (REX1 in the host cell and retained within the PVM) or no export (no REX1 signal beyond the PVM). Error bars represent s.d. of three technical replicates. Data are representative of two independent experiments. Scale bar, 5 µm. c, Metabolic labelling with [35S]methionine/cysteine, performed as previously described44, confirms that CHX treatment conditions inhibit new protein synthesis. Parasite proteins were TCA-precipitated and incorporated radioactivity was determined through scintillation counting. Error bars represent s.d. of three technical replicates.

Extended Data Figure 6 Maturation of plasmepsin 2 is not affected by inactivation of HSP101.

Western blot showing normal maturation of plasmepsin 2 (PM2) in asynchronous parasites after 24 h –TMP. BiP serves as a loading control. Maturation requires proPM2 trafficking through the PV before internalization to the digestive vacuole where maturation occurs25. Images are representative of two independent experiments.

Extended Data Figure 7 DDD targeted to the PV independent of HSP101 does not interfere with parasite growth or export.

a, Schematic of the PV-targeted GFP–DDD fusion protein consisting of a signal peptide appended to a GFP–DDD fusion with a C-terminal HA epitope tag and expressed under the control of the HSP86 promoter. The predicted size of the fusion protein after signal peptide cleavage is 46 kDa. Live imaging of GFP demonstrates PV targeting of the fusion protein. Images are representative of two independent experiments. b, Western blot showing GFPDDD is not degraded in the absence of TMP. Synchronized GFPDDD parasites were grown 72 h with or without TMP before purification over Percoll and treatment with tetanolysin to release the RBC cytosol but not the PV contents. The GFPDDD fusion was detected with anti-HA antibodies. BiP serves as a loading control. Images represent one experiment. c, Growth analysis of asynchronous GFPDDD parasites shows no growth defect −TMP. Error bars represent s.d. of three technical replicates. Data are representative of two independent experiments. d, IFA showing no defect in export in GFPDDD parasites in the absence of TMP. No difference was observed in export of ring-specific (REX1) and the trophozoite-specific (MSRP6) exported proteins in the presence or absence of TMP. All scale bars, 5 µm. Images represent one experiment.

Extended Data Figure 8 Localization of PTEX components is unchanged under export blocking conditions.

a, Immunoelectron microscopy showing localization of HSP101DDD in ring-stage parasites with or without TMP. TMP was removed or not from synchronous late schizonts and parasites were allowed to re-invade and develop for 18 h before fixation. Scale bars, 500 nm. Images represent one experiment. b, c, IFA of 13F10 parasites showing co-localization between HSP101DDD or EXP2 and the PV marker SERP with or without TMP, indicating that localization of these PTEX components is not altered under export blocking conditions. TMP treatment was performed as in a before fixation with acetone and processing for IFA. d, e, IFA of ring-stage 13F10 parasites expressing a PTEX150–Flag fusion. The upper panel (d) shows co-localization with HSP101DDD. The lower two panels (e) show that PTEX150 remains at the PVM during export block and partially colocalizes with blocked SBP1. TMP treatment and fixation as in b, c. All IFA scale bars, 5 µm. All IFA images (be) are representative of two independent experiments.

Extended Data Figure 9 Immunoprecipitation bead controls indicate target-specific interactions.

Replicate IP experiments to those shown in Fig. 4c, e with bead controls included. a, b, Purification of RESA by HSP101DDD or EXP2 (a) and purification of EXP2 by PTEX150Flag (b) is specific to the target antibodies. IP experiments were performed in parallel by incubating equivalent portions of lysate supernatant input with beads alone or beads and the indicated IP antibodies. S, supernatant input. E, elution. Data are representative of two independent experiments.

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Beck, J., Muralidharan, V., Oksman, A. et al. PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes. Nature 511, 592–595 (2014). https://doi.org/10.1038/nature13574

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