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PTEX is an essential nexus for protein export in malaria parasites

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Abstract

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol1,2,3,4,5,6. These proteins have a variety of roles that are essential to virulence or parasite growth7. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins8,9. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX)10. Although PTEX has been linked to the export of one class of exported proteins10,11, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D)1,2 and PEXEL-negative exported proteins (PNEPs)6. Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite’s capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

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Figure 1: Inducible knockdown of P. berghei HSP101 (i101 KD).
Figure 2: Knockdown of P. berghei HSP101 blocks export of PEXEL and PNEP proteins.
Figure 3: Generation of a PTEX150 knockdown line in P. falciparum.
Figure 4: PTEX150 knockdown blocks protein export in P. falciparum.

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Acknowledgements

We thank T. Templeton, B. Franke-Fayard, C. Janse, A. Cowman, J. Boddey, B. Cooke, M. Duffy, L. Tilley, R. Anders, F. Fowkes, A. McLean and D. Bursac for reagents and/or other assistance with aspects of this study; D. Stanisic, F. Baiwog and I. Mueller for contributions to clinical studies of pregnant women; and P. Siba. We also thank the Australian Red Cross Blood Bank for the provision of human blood and serum. This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia (1021560, 1025665 and 637406) and the Victorian State Government Operational Infrastructure Support Scheme. T.F.d.K.-W. is an NHMRC Career Development Fellow, and J.G.B. is a NHMRC Senior Research Fellow. B.E. and K.M. are the recipients of Australian Postgraduate Awards.

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Authors and Affiliations

Authors

Contributions

B.E., K.M,. P.R.G. and T.F.d.K.-W. designed, performed and interpreted much of the experimental work. B.S.C. designed and interpreted the work and, along with P.R.G. and T.F.d.K.-W., wrote the manuscript. C.Q.N., M.K., S.C.C., P.R.S., S.A.C. and N.A.C. performed experiments and provided intellectual insight into aspects of this study. P.J.S., P.P., J.C., M.F.A., J.G.B. and S.J.R. provided reagents and intellectual input into study design. All authors commented on the manuscript.

Corresponding authors

Correspondence to Brendan S. Crabb, Paul R. Gilson or Tania F. de Koning-Ward.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Disruption of P. berghei TRX2 leads to reduced protein export.

a, IFA of fixed infected erythrocytes using P. berghei semi-immune sera reveals TRX2 knockout parasites (TRX2 KO) show reduced surface labelling compared with wild-type P. berghei ANKA parasites (WT), indicative of a reduction in expression of parasite antigens on the surface of erythrocytes infected with the TRX2 KO. Pre-bleed sera were used as a negative control. b, Quantitative FACS analysis of erythrocytes harvested from asynchronously infected mice (n = 6) show that two independent clonal populations of TRX2 KO parasites exhibit significantly reduced levels of surface labelling with P. berghei semi-immune sera compared with wild-type parasites (*P < 0.05; **P < 0.01; ***P < 0.001, unpaired t-test). c, As b, except that synchronous mouse infections were initiated by injecting purified merozoites into the tail veins of mice, and surface labelling of infected erythrocytes with semi-immune sera was performed at time points relative to when the wild-type line reinvaded erythrocytes for the second cycle (left); p.i., post invasion. Even taking into consideration that disruption of TRX2 leads to slower growth by about 6 h, the surface labelling of TRX2 KO parasites at a stage of growth comparable to that of wild-type parasites is also significantly reduced (right) (n = 3 independent experiments). d, Giemsa smears showing the stages of parasite development at time points relative to when wild-type parasites had invaded erythrocytes.

Extended Data Figure 2 Generation of a HSP101 knockdown line in P. berghei.

a, Schematic representation used to construct Pbi101 KD parasites. PCR primers used to detect 5′ integration (a/b), 3′ integration (c/d) and wild-type locus (a/d) are indicated. E, EcoRI. b, Representative experiments (n = 3) showing parasitaemias in mice that were (upper panel) or were not (lower panel) pre-exposed to ATc in their drinking water before infection. At day 4 after infection, the treatment regimens in both experiments were switched. Error bars show s.e.m. for three mice per condition performed in parallel.

Extended Data Figure 3 Knockdown of HSP101 blocks protein export.

a, Representative IFA of intraerythrocytic stages showing that export of three different P. berghei proteins across the parasitophorous vacuole membrane is blocked when Pbi101 KD, but not wild-type, is exposed to ATc. Samples were harvested at the times indicated by the asterisks in Fig. 1d and e. b, IFAs show that correct localization of EXP2 and ACP is unaffected in Pbi101 KD parasites treated with ATc (right panels). In these samples, cells were permeabilized after fixation with 0.5% Triton X-100. Because the PbANKA_114540, EXP2 and ACP antibodies were all raised in rabbits, sequential labelling with anti-PbANKA_114540, anti-rabbit AlexaFluor488, anti-EXP2 or anti-ACP, and anti-rabbit AlexaFluor568 had to be performed. Control IFAs were therefore performed in which anti-EXP2 or anti-ACP were omitted (left panels).

Extended Data Figure 4 Diagnostic PCR analysis shows the ptex150 gene has been appended with a HA tag in the PTEX150-HA parasites and a HAglmS tag in the PTEX150-HAglmS parasites.

a, Diagram of the targeted genetic crossovers and binding sites of the PCR primers. b, Using the indicated primer combinations, correct 3′- recombination has occurred in the PTEX150-HA and PTEX-HAglmS parasites using primers A/C, with a band specific to the integrated locus (1.7 kb) only observed in HA-tagged parasite lines. c, Diagram showing how the glmS ribozyme after glucosamine binding is stimulated to cleave its mRNA, resulting in message destabilization and a decrease in protein levels.

Extended Data Figure 5 Growth assays of PTEX150-HA and PTEX150-HAglmS parasites show that growth of the latter declines substantially after treatment with glucosamine (GlcN).

CS2 PTEX150-HA and CS2 PTEX150-HA-glmS parasites were treated with different concentrations of glucosamine from 24–30 h after invasion (hpi) and then allowed to invade fresh erythrocytes for 4 h. At the times after invasion indicated, the cells were stained with ethidium bromide to measure DNA content as a marker for parasite growth. Representative histograms show the levels of ethidium bromide intensity (x axis) and cell number (y axis). Infected and uninfected erythrocytes (uE) are shown as black and grey, respectively. Those parasites to the right of the red line are the strongly staining trophozoites; those to the left are the younger, weakly staining ring stages. Assays were performed at least three times independently.

Extended Data Figure 6 PTEX150-HAglmS protein levels are markedly reduced on induction of the glmS ribozyme with GlcN.

a, Western blots of PTEX150-HAglmS and control PTEX150-HA mid-ring-stage parasites (12 hpi) probed with the antibodies indicated on the right. GlcN was added at the concentration indicated above the blots, halfway through the previous cell cycle. b, Western blots were performed in duplicate and densitometry of the bands has been graphed showing the mean ± s.d. relative to no GlcN. Top: PTEX150 levels in the PTEX150-HAglmS (150-glmS) decrease with increasing concentrations of GlcN to a minimum 17% of the level without GlcN. The levels of PTEX150 in the control PTEX150-HA (150-HA) parasites does not decrease in GlcN. Middle and bottom: the levels of co-regulated HSP101 and RESA proteins and cytoplasmic constitutive HSP70-1, GAPDH and ERC proteins also decline in the PTEX150-HAglmS parasites after treatment with GlcN to about 50–60%, indicative of slowed growth due to loss of PTEX150 function. c, Western blot of infected erythrocytes treated with trypsin to cleave off surface-exposed PfEMP1. The blot has been probed with a monoclonal antibody against the intracellular C-terminal tail of PfEMP1, and the densitometry of the 350 kDa VAR2CSA band (arrow) has been compared between trypsin-treated and untreated infected erythrocytes to calculate the percentage cleaved in the presence or absence of 0.3 mM GlcN. d, IFAs of PTEX150-HAglmS probed for PfEMP1 and EXP2 after treatment with GlcN indicate a decrease in the export of PfEMP1-containing structures to the periphery of the infected erythrocyte. Scale bar, 5 μm.

Extended Data Figure 7 Export of KAHRP in PTEX150-HAglmS (glms) is decreased after treatment with GlcN.

The mean fluorescence intensity (MFI) of the erythrocyte compartment in infected erythrocytes stained with rabbit anti-KAHRP always declines after the addition of GlcN halfway through the previous cell cycle. In comparison, treatment with GlcN does not consistently decrease KAHRP export in the control PTEX150-HA (HA) parasites; the variation is possibly due to inconsistencies in sample preparation. In the graphs, the boxes and whiskers delineate the 25–75th and 10–90th centiles, respectively. Outlying data points are shown as dots. Significances: *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired t-test. The number of cells (n) counted is indicated below the graph. Example immunofluorescence images of only PTEX150-HAglmS are shown. The regions occupied by the parasite are indicated by staining with DAPI and staining for EXP2. Scale bar, 5 μm.

Extended Data Figure 8 Export of SBP1 in PTEX150-HAglmS (glms) is decreased after treatment with GlcN.

The number of punctate Maurer’s clefts (MCs) present in the erythrocyte compartment in infected erythrocytes stained with rabbit anti-SBP1 nearly always declines after the addition of GlcN halfway through the previous cell cycle. In comparison, treatment with GlcN does not consistently decrease SBP1 export in the control PTEX150-HA (HA) parasites; the variation is possibly due to inconsistencies in sample preparation. In the graphs, the boxes and whiskers delineate the 25–75th and 10–90th centiles, respectively. Outlying data points are shown as dots. Significances: *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired t-test. The number of cells (n) counted is indicated below the graph. Example immunofluorescence images of only PTEX150-HAglmS are shown. The regions occupied by the parasite are indicated by staining with DAPI and staining for EXP2. Scale bar, 5 μm.

Extended Data Figure 9 Export of Hyp8 in PTEX150-HAglmS (glms) is reduced following glucosamine treatment.

The number of punctate Maurer’s Clefts (MCs) present in the erythrocyte compartment in infected erythrocytes stained with rabbit anti-Hyp8 nearly always declines following addition of GlcN half way through the previous cell cycle. In comparison, GlcN treatment does not consistently reduce Hyp8 export in the control PTEX150-HA (HA) parasites and the variation is possibly due to inconsistencies in sample preparation. In the graphs, the boxes and whiskers border the 25–75th and 10–90th percentiles, respectively. Outlying data points are shown as dots. Significances: *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired t-test. The number of cells (n) counted is indicated below the graph. Example immunofluorescence images of only PTEX150-HAglmS are shown. The regions occupied by the parasite are indicated by staining with DAPI and staining for EXP2. Scale bar, 5 μm.

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Elsworth, B., Matthews, K., Nie, C. et al. PTEX is an essential nexus for protein export in malaria parasites. Nature 511, 587–591 (2014). https://doi.org/10.1038/nature13555

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