The Plasmodium berghei translocon of exported proteins reveals spatiotemporal dynamics of tubular extensions

Abstract

The erythrocyte is an extraordinary host cell for intracellular pathogens and requires extensive remodelling to become permissive for infection. Malaria parasites modify their host red blood cells through protein export to acquire nutrients and evade immune responses. Endogenous fluorescent tagging of three signature proteins of the Plasmodium berghei translocon of exported proteins (PTEX), heat shock protein 101, exported protein 2 (EXP2), and PTEX88, revealed motile, tubular extensions of the parasitophorous vacuole that protrude from the parasite far into the red blood cell. EXP2 displays a more prominent presence at the periphery of the parasite, consistent with its proposed role in pore formation. The tubular compartment is most prominent during trophozoite growth. Distinct spatiotemporal expression of individual PTEX components during sporogony and liver-stage development indicates additional functions and tight regulation of the PTEX translocon during parasite life cycle progression. Together, live cell imaging and correlative light and electron microscopy permitted previously unrecognized spatiotemporal and subcellular resolution of PTEX-containing tubules in murine malaria parasites. These findings further refine current models for Plasmodium-induced erythrocyte makeover.

Figure 1. Live fluorescent imaging of Plasmodium berghei HSP101 during asexual blood-stage development.

(a) Recombination strategy for the endogenous tagging of HSP101. Double crossover integration into the wild-type locus yields recombinant parasites with their endogenous locus tagged by mCherry-3xMyc. For details see Supplementary Fig. S1 and Supplementary Table S1. (b) Intravital competition assay of WT and hsp101-mCherry parasites. Parasite multiplication rates for WT and hsp101-mCherry parasites were 10.7 and 9.6, respectively (non-significant). (c) Western blot analysis of hsp101-mCherry parasites. The predicted size for tagged HSP101 is 133 kDa and was identified correctly using an anti-mCherry antibody. (d) Fluorescent micrographs of live hsp101-mCherry-infected erythrocytes. Shown are representative images of the fluorescent signal of HSP101-mCherry (top), a merge of HSP101-mCherry and cytoplasmic GFP (middle), and differential interference contrast images (DIC, bottom) for three asexual developmental stages. Inset, free merozoite; scale bar, 5 μm.

Figure 2. Spatiotemporal analysis of extraparasitic HSP101.

(a) Quantification of HSP101-mCherry localization throughout a synchronized infection at 4 h intervals according to four categories indicated by representative images (top). The localization categories are: punctate cytoplasmic (white), additional periphery (light brown), one tubular extension (light blue), and two or more tubular extensions (dark blue). White outlines, erythrocyte; green, parasite cytoplasm; red, HSP101-mCherry. The proportions of extraparasitic HSP101-mCherry are indicated for six time points (n = 100 per time point) of the 24 h asexual blood-stage cycle. (b) Quantification of tubular length in relation to the red blood cell (RBC) diameter indicated by representative images (top). The length categories are: ≤0.5 RBC diameter (light brown), 0.5–1 RBC diameter (light blue), and ≥1 RBC diameter (dark blue). White outlines, erythrocyte; green, parasite cytoplasm; red, HSP101-mCherry. Length distributions of extraparasitic HSP101-mCherry are indicated for the same six time points (n = 100 per time point) as in (a).

Figure 3. HSP101 delineates a tubular subcompartment of the parasitophorous vacuole.

(a) Live co-localization of HSP101-mCherry (centre left) with a marker protein of the parasitophorous vacuole (GFP^PV, centre). The line in the merge (centre right) indicates profiling of the fluorescent signal (right). Shown are three representative trophozoites demonstrating vacuolar tubules, loops, and vesicles. The blue arrowhead denotes a detached, vacuole-derived, and HSP101-mCherry negative lumen. The red arrowheads denote budding structures at the site of a vacuolar tubule. Note that HSP101-mCherry is excluded from these compartments. (b) FRAP analysis reveals free diffusion from the parasitophorous vacuole to the tubular extensions. Erythrocytes infected with mCherry^PV parasites were analysed by confocal microscopy before (pre bleach) and after (post bleach) photo bleaching (red area, bleach location). Shown is a representative trophozoite and the respective temporal fluorescence analysis in the erythrocyte cytoplasm (blue dotted line); black arrowhead indicates time of the bleaching pulse. Scale bar, 5 μm.

Figure 4. Ultrastructure of the vacuolar tubules.

(a) Representative transmission electron micrograph (TEM) of an hsp101-mCherry-infected erythrocyte, obtained by correlative light and electron microscopy (left). The red arrowhead denotes a tubular extension. Two representative high magnification images of the compartment are shown (right). Scale bar, 1 μm. (b) Tubules were visualized by fluorescence microscopy (inset) and correlated with multiple transmission electron microscopic (TEM) sections of the same cell. The 3D-reconstruction was generated by parasite membrane alignment of 29 consecutive TEM sections. Green, parasite surface; red, tubule. (c) TEM section of a WT-infected erythrocyte. Shown is a representative high magnification image of a vacuolar tubule. (d) 3D-reconstruction generated by parasite membrane alignment of 19 TEM sections of a WT-infected erythrocyte. Green, parasite surface; red, tubule.

Figure 5. Live fluorescent imaging of the PTEX components EXP2 and PTEX88.

(a) Recombination strategy for the endogenous tagging of EXP2. Double crossover integration into the wild-type locus yields recombinant parasites with their endogenous locus tagged by mCherry-3xMyc. (b) Intravital competition assay of WT and exp2-mCherry parasites. Parasite multiplication rates for WT and exp2-mCherry parasites were 10.0 and 11.2, respectively (non-significant). (c) Western blot analysis of exp2-mCherry parasites. The predicted size for tagged EXP2 is 62 kDa and was identified correctly using an anti-mCherry antibody. (d) Purified exp2-mCherry × GFP^PV-infected erythrocytes were lysed with hypotonic buffer (input) and spun at 100 000 × g. The supernatant (hypotonic lysate sup) along with proteins released from the pellet after Triton X-100 treatment (TX-100 sup) and the remaining insoluble pellet were analyzed by SDS-PAGE and Western blotting using anti-mCherry (EXP2-mCh) and anti-GFP (GFP^PV) antibodies. (e) Micrographs of live exp2-mCherry-infected erythrocytes. Shown are representative images for three asexual developmental stages including the fluorescent EXP2-mCherry signal (top), a merge of EXP2-mCherry and cytoplasmic GFP (middle), and differential interference contrast images (DIC, bottom). Inset, free merozoite. (f) Co-localization of EXP2-mCherry (centre left) with the parasitophorous vacuole (GFP^PV, centre). The line in the merge (centre right) indicates profiling of the fluorescent signal (right). (g) Co-localization of HSP101-mCherry (centre left) with PTEX88-GFP (centre). The line in the merge (centre right) indicates profiling of the fluorescent signal (right). Scale bars, 5 μm.

Figure 6. Live imaging of four PTEX components during Plasmodium berghei life cycle progression.

Micrographs of live midgut-associated oocysts (a), salivary gland sporozoites (b), and liver stages 24 h (c) and 48 h after infection (d). Shown are representative images including the fluorescent signal of the tagged protein (top), a merge of tagged protein, cytoplasmic GFP, and Hoechst 33342 DNA dye (middle) and differential interference contrast images (DIC, bottom). Scale bars, 10 μm.