Shedding of host autophagic proteins from the parasitophorous vacuolar membrane of Plasmodium berghei

Abstract

The hepatic stage of the malaria parasite Plasmodium is accompanied by an autophagy-mediated host response directly targeting the parasitophorous vacuolar membrane (PVM) harbouring the parasite. Removal of the PVM-associated autophagic proteins such as ubiquitin, p62, and LC3 correlates with parasite survival. Yet, it is unclear how Plasmodium avoids the deleterious effects of selective autophagy. Here we show that parasites trap host autophagic factors in the tubovesicular network (TVN), an expansion of the PVM into the host cytoplasm. In proliferating parasites, PVM-associated LC3 becomes immediately redirected into the TVN, where it accumulates distally from the parasite's replicative centre. Finally, the host factors are shed as vesicles into the host cytoplasm. This strategy may enable the parasite to balance the benefits of the enhanced host catabolic activity with the risk of being eliminated by the cell's cytosolic immune defence.

Figure 1

Comparison of the PVM–associated LC3 clearance during liver stage development in vivo and in vitro. (a–c) Liver intravital time-lapse of gfp-lc3 transgenic mice infected with schizonts of different developmental stages. (a) Young PbmCherry schizont 25 hpi (red) shows LC3 (green) incorporated into the PVM and the dynamic TVN (white arrowhead). Movie interval 2 min. (b) Proliferating liver schizonts 30 hpi accumulating LC3 in the evolving TVN. Lower panel shows magnification of the depicted inset. Arrowhead highlights increasing GFP-LC3 signal in the TVN. Acquisition interval 5 s. (c) Mature PbmCherry schizont 44 hpi (red) entirely cleared from PVM-associated LC3 (green). Movie interval 2 s. (d) Wide field in vitro time-lapse microscopy of GFP-LC3 expressing HeLa cell (green) infected with mCherry expressing parasites (red). Long-term movie of the P. berghei liver stage showing the accumulation of GFP-LC3 in the evolving TVN (white arrowhead) and a progressive loss from the PVM. With completion of parasite division, LC3 is completely lost from the PVM. Imaging interval 30 min. (e–h) In vivo quantification of the LC3 accumulation at the PVM at indicated times of the liver stage development. (e) Stacked column graph of the overall percentage of LC3-positive and -negative parasites. (f) LC3-coverage of the PVM normalized to parasite surface area. (g) Still image of a liver intravital movie showing the elimination of mature schizonts. Green area outlines the PVM. The necrotic schizont shows release of parasite mCherry into the host cytoplasm (white arrowhead) (h) Quantification of the parasite survival rate in correlation to the LC3-load measured at the PVM. Time stamps, min:s (a–c) and h:min (d). Scale bars, 10 µm.

Figure 2

TVN expansion and dynamics promote sequestration of LC3. (a) Long-term imaging of GFP-LC3 HeLa cells infected with schizonts expressing EXP1-mCherry under the late liver stage promoter LISP2 (red, 33 hpi). Translocation of PVM-bound LC3 (red arrowhead) coincides with the TVN expansion (white arrowhead). Acquisition interval 5 min. (b, c) Fast-iterative movie of an infected GFP-LC3 cell displaying the TVN-dynamics. (b) A young EXP1-mCherry schizont (red, 30 hpi) shows rather static cisternae-like clusters, but motile tubules and vesicles. Arrowhead emphasizes LC3 accumulation in the TVN-cluster. (c) Focus on basal TVN region of the EXP1-mCherry schizonts in (b) shows a fast moving tubular structure (white arrowhead) and a PVM-derived LC3-vesicle (red arrowhead). Acquisition interval 10 s. (d) Liver intravital microscopy of gfp-lc3 transgenic mice infected with a mature EXP1-mCherry schizont (red, 41 hpi). Residual LC3 (green) locates to the EXP1-positive TVN, highlighted in the inset. Acquisition interval 5 s. Time stamps, h:min (a), min:s (b–d). Scale bars, 10 µm (a, d), 5 µm (b, c).

Figure 3

Compartment-specific LC3 mobility reveals trapping of LC3 in the TVN. (a–c) Fluorescence Recovery After Photobleaching (FRAP) of GFP-LC3 HeLa cells infected with PbmCherry 33-36 hpi. Fluorescence recovery reveals a distinctive LC3 motility at the PVM (orange ROI) versus TVN (green ROI). (b) Pseudo-coloured heat maps visualizing the dynamics of LC3-recovery at the bleached area (arrowhead). (c) FRAP-quantification at the PVM (orange) and the TVN (green) (n = 40; mean ± s.d.). (d–f) Fluorescence Loss In Photobleaching (FLIP) shows loss of LC3 at the PVM and retention at the TVN. (d) Repetitive bleaching (black ROI) performed on an adjacent, uninfected GFP-LC3 cell for imaging-induced fluorescence decay (control), or on PVM-localized LC3 (FLIP). Mean fluorescence intensity of GFP-LC3 measured around PVM (orange ROI) and TVN (green ROI). (e) Pseudo-coloured heat maps of GFP-LC3 intensity at the PVM (white arrowhead) or TVN (yellow arrowhead). (f) FLIP-quantification at the PVM (orange) and TVN (green) (n = 40; mean ± s.d.). (g) Photoconversion of Dendra2-LC3 at the PVM (orange ROI) or TVN (green ROI) showing pre-activation, post-activation and final image of young schizonts (24 hpi, Pbwt). Insets display the green channel (non-converted Dendra2-LC3). Arrowheads point out the re-distribution of converted Dentra2-LC3 at the PVM. (h) Representative, pseudo-coloured kymograph of activated LC3 in (g) measured at ROI. (i) Quantification of LC3 at the PVM (orange) and TVN (green) based on kymographs (n = 10; mean ± s.d.). Time stamps in s. Scale bars, 10 µm (a, d, g) and 5 µm (b, e).

Figure 4

Control of the autophagic components coincides with TVN development. (a) Fast-iterative wide field microscopy of PbmCherry sporozoites (1 hpi) outlined by parasite-associated LC3. Arrowheads indicate LC3-accumulation in the TVN. Acquisition interval 30 s. (b) Photoconversion of Dendra2-LC3 at the tip of a Pbwt sporozoite (3 hpi) comprising a nascent TVN. Activated LC3 (black ROI) is tracked at the PVM and accumulates in membrane patches (black arrowhead). White arrowhead points out the accumulation of non-activated LC3 in the TVN. Red asterisk indicates the tip of the sporozoite. Inset displays non-activated LC3 in the green channel. (c) Photoactivation of PAGFP-Ub at the PVM of sporozoites (2 hpi, black ROI) in HeLa cells co-transfected with pmRFP-LC3. Activated ubiquitin (Ub) and LC3 share patches and relocate together from the anterior tip into the posterior TVN-accumulation (black arrowhead). Insets display mRFP-LC3. Time stamps, min:s. Scale bars, 5 µm.

Figure 5

TVN-architecture and vesicle shedding in liver schizonts. (a) 3D reconstructions of the PVM/TVN architecture. Young liver schizonts (36 hpi) were labelled for the indicated PVM proteins by immunofluorescence and images acquired by confocal laser scanning microscopy (3D-CLSM) with 0.22 µm z-increments. The TVN consists of branched tubular structures, several node-like clusters and vesicles. iii, iv, Magnifications of a single z-plane. Potential budding sites or “growth cones” (arrowheads) are visible along protrusions and TVN-clusters. (b, c) The origin of the parasite-derived vesicles was determined based on the scattering pattern in the infected host cell. (b) Schematic of the analysis performed on PbmCherry schizonts (36 hpi) either stained for UIS4 or EXP1. LC3 vesicles were determined on GFP-LC3 HeLa cells infected with PbmCherry. (c) Quantification of the minimal distance of PVM-/LC3-vesicles from the TVN (green) and PVM (orange). n > 200 vesicles. ***P < 0.0001, two-way ANOVA. (d, e) In vivo quantification of the mean LC3-vesicle distribution. (d) Representative images from intravitals performed on the liver of gfp-lc3 mice infected with PbmCherry at different stages of the infection. (e) The percentage of vesicles scored within a defined area relative to the parasite surface, represented in a stacked column graph. Host cytoplasm of infected hepatocytes was divided into 3 consecutive sections, referred to as proximal, intermediate and distal. (f, g) Fast iterative 3D-movies displaying TVN dynamics. (f) GFP-LC3 positive schizont (24 hpi) recorded with 0.25 µm z-increments and 30 s intervals. White arrowhead highlights formation and budding of a LC3-vesicle. Red arrowhead tracks a LC3-positive protrusion. (g) P. berghei-EXP1-mCherry schizonts 33 hpi recorded with 0.4 µm z-increments and 15 s intervals. Inset shows maximum projection of tracked vesicles. Arrowheads highlight the dynamics of an EXP1-protrusion (red) and EXP1-vesicle (white). (h) Photoconversion of Dendra2-LC3 at the PVM of PbGFPcon schizonts (33 hpi). Converted LC3 accumulates in a vesicular structure (black arrowhead), which is shed from the PVM. At the same time converted LC3 is lost from the original activation site (white arrowhead). Insets display the green channel (parasite cytoplasm and non-converted LC3). Time stamps, min:s (f, g) and h:min (h). Scale bars, 10 µm (d, f, g), 5 µm (h, i, ii) and 2 µm (iii, iv).

Figure 6

Model of the putative shedding mechanism during the hepatic stage of P. berghei. Progressive removal of PVM-associated LC3 during the P. berghei liver stage development. (a) LC3 incorporation into the PVM, (b) accumulation into PVM-patches, (c) transfer and entrapment in the TVN, and (d) vesicle shedding into the host cytoplasm.