Hostile Takeover by Plasmodium: Reorganization of Parasite and Host Cell Membranes during Liver Stage Egress

The protozoan parasite Plasmodium is transmitted by female Anopheles mosquitoes and undergoes obligatory development within a parasitophorous vacuole in hepatocytes before it is released into the bloodstream. The transition to the blood stage was previously shown to involve the packaging of exoerythrocytic merozoites into membrane-surrounded vesicles, called merosomes, which are delivered directly into liver sinusoids. However, it was unclear whether the membrane of these merosomes was derived from the parasite membrane, the parasitophorous vacuole membrane or the host cell membrane. This knowledge is required to determine how phagocytes will be directed against merosomes. Here, we fluorescently label the candidate membranes and use live cell imaging to show that the merosome membrane derives from the host cell membrane. We also demonstrate that proteins in the host cell membrane are lost during merozoite liberation from the parasitophorous vacuole. Immediately after the breakdown of the parasitophorous vacuole membrane, the host cell mitochondria begin to degenerate and protein biosynthesis arrests. The intact host cell plasma membrane surrounding merosomes allows Plasmodium to mask itself from the host immune system and bypass the numerous Kupffer cells on its way into the bloodstream. This represents an effective strategy for evading host defenses before establishing a blood stage infection

Physiological variables in association with spreading depolarizations in the late phase of ischemic stroke

Physiological effects of spreading depolarizations (SD) are only well studied in the first hours after experimental stroke. In patients with malignant hemispheric stroke (MHS), monitoring of SDs is restricted to the postoperative ICU stay, typically day 2-7 post-ictus. Therefore, we investigated the role of physiological variables (temperature, intracranial pressure, mean arterial pressure and cerebral perfusion pressure) in relationship to SD during the late phase after MHS in humans. Additionally, an experimental stroke model was used to investigate hemodynamic consequences of SD during this time window. In 60 patients with MHS, the occurrence of 1692 SDs was preceded by a decrease in mean arterial pressure (-1.04 mmHg; p = .02) and cerebral perfusion pressure (-1.04 mmHg; p = .03). Twenty-four hours after middle cerebral artery occlusion in 50 C57Bl6/J mice, hypothermia led to prolonged SD-induced hyperperfusion (+2.8 min; p < .05) whereas hypertension mitigated initial hypoperfusion (-1.4 min and +18.5%Delta rCBF; p < .01). MRI revealed that SDs elicited 24 hours after experimental stroke were associated with lesion progression (15.9 vs. 14.8 mm(3); p < .01). These findings of small but significant effects of physiological variables on SDs in the late phase after ischemia support the hypothesis that the impact of SDs may be modified by adjusting physiological variables

A key role for lipoic acid synthesis during Plasmodium liver stage development.

The successful navigation of malaria parasites through their life cycle, which alternates between vertebrate hosts and mosquito vectors, requires a complex interplay of metabolite synthesis and salvage pathways. Using the rodent parasite Plasmodium berghei, we have explored the synthesis and scavenging pathways for lipoic acid, a short-chain fatty acid derivative that regulates the activity of α-ketoacid dehydrogenases including pyruvate dehydrogenase. In Plasmodium, lipoic acid is either synthesized de novo in the apicoplast or is scavenged from the host into the mitochondrion. Our data show that sporozoites lacking the apicoplast lipoic acid protein ligase LipB are markedly attenuated in their infectivity for mice, and in vitro studies document a very late liver stage arrest shortly before the final phase of intra-hepaticparasite maturation. LipB-deficient asexual blood stage parasites show unimpaired rates of growth in normal in vitro or in vivo conditions. However, these parasites showed reduced growth in lipid-restricted conditions induced by treatment with the lipoic acid analogue 8-bromo-octanoate or with the lipid-reducing agent clofibrate. This finding has implications for understanding Plasmodium pathogenesis in malnourished children that bear the brunt of malarial disease. This study also highlights the potential of exploiting lipid metabolism pathways for the design of genetically attenuated sporozoite vaccines

Identification of New PNEPs Indicates a Substantial Non-PEXEL Exportome and Underpins Common Features in <i>Plasmodium falciparum</i> Protein Export

<div><p>Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite <i>Plasmodium falciparum</i>. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.</p></div

Transcription profile based screen for new PNEPs.

<p>(A) Venn diagram of selection of PNEPs based on a transcription profile similar to genes encoding known PNEPs and ETRAMPs. (B, C) Fluorescence pattern of non-exported (B) or exported (C) GFP fusion proteins. Protein structure of each candidate is indicated before each panel (red bars with hydrophobic regions indicated in black, not to scale). For PF08_0003-GFP two panels showing a trophozoite stage (troph) and a ring stage (ring) parasite are shown to demonstrate the different localisations in these stages. For PF14_0045-GFP two panels are shown to demonstrate cells with (yellow arrows) and without additional foci of fluorescence in the host cell (ratio indicated in %, at least 50 cells were analysed on 3 occasions, standard deviation in brackets). Nuclei were stained with DAPI. Size bars: 5 µm. (D) The fluorescence in the host cell of PF13_0194-GFP represents full length soluble protein as determined by Western blot using anti-GFP antibodies with extracts from saponin lysed infected RBCs separated into pellet (P) and supernatant (SN). Parasite cytosolic aldolase was used to control for parasite integrity; REX3 (found soluble in infected RBCs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat.1003546-Spielmann2" target="_blank">[17]</a>) was used as a control for release of infected host cell cytosol.</p

Genomic position based screen for new PNEPs.

<p>(A) Venn diagram of selection of PNEPs based on a subtelomeric gene location (<200 kb from chromosome end). (B, C) Fluorescence pattern of non-exported (B) or exported (C) GFP fusion proteins. Protein structure of each candidate is indicated to the left of each panel as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat-1003546-g001" target="_blank">Figure 1</a>. Nuclei were stained with DAPI. Size bars: 5 µm. (D) The fluorescence in the host cell of PF08_0005-GFP represents full length soluble protein as determined by Western blot as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat-1003546-g001" target="_blank">Figure 1D</a>, except that GAPDH was used as the parasite internal control.</p

N-termini of new PNEPs without a signal peptide promote export of a reporter with a PNEP TM.

<p>(A) Parasites expressing the 20 first amino acids of PF07_0007 (top two rows, wild type and scrambled version of N-termini) or PFF0090w (bottom rows, wild type and scrambled version of N-termini) fused N-terminally to R^REX2-TM (truncated mTRAP-GFP containing the REX2TM). For the scrambled PF07_0007 N-terminus the most typical phenotype (mixed) is shown. For the PFF0090w scrambled N-terminus a non-exported and a mixed phenotype is shown. Red bar, PNEP domain; striated bar, randomly scrambled version; grey bar, mTRAP part; green bar, GFP. Export levels are shown to the right for each cell line and were assessed by counting (blinded) the number of cells showing export only (export), export together with parasite periphery and/or internal fluorescence (mixed), or parasite periphery and/or internal fluorescence only (no export). Graphs represent counting of at least 50 cells on three different occasions; error bars represent SD. (B) As in A but parasites expressing R^REX2-TM with the first 20 amino acids of PF11_0505 (yellow bar). Nuclei were stained with DAPI. Size bars: 5 µm.</p

Soluble PNEPs need to be unfolded to reach the host cell.

<p>(A) Double transgenic parasites expressing MSRP6-GFP as well as MSRP6 fused to mDHFR and mCherry (constructs indicated on top) grown either with (+wr) or without (control) WR99210. The DIC image with DAPI stained nuclei (DIC/DAPI) and the merged GFP and mCherry signal (merge) are also presented. (B) As in (A) but with parasites expressing MSRP7-GFP and MSRP7-mDHFR-mCherry. The fluorescence internal to the parasite represents re-internalised protein in the food vacuole. Size bars: 5 µm. (C) Western blots of Percoll enriched double transgenic parasites shown in (A) and grown in the absence (control, exported) or presence (+wr, fluorescence remaining in parasite periphery) of WR99210 that were treated with saponin and separated into supernatant (SN) and pellet (P). SERP is found soluble in the PV and was used to demonstrate the action of saponin. GAPDH is the parasite internal control. The intensity of the mCherry, GFP and SERP signals in the extracts of parasites treated with wr is less than that of parasites not treated with wr, which may reflect either a somewhat slower growth cycle or slight differences in the stage composition of the parasite population after sample preparation. MSRP7-mDHFR-mCherry in the PV may also be more efficiently re-internalised and degraded in the food vacuole, reducing its amount in the blocked compared to the unblocked state.</p

Further PNEPs encoded by genes at the <i>msp7</i> gene locus.

<p>(A) Fluorescence pattern of PF13_0192-GFP. (B) Co-localisation IFA of PF13_0192-GFP with SBP1. (C) Fluorescence pattern of PF13_0191-GFP. Two panels are shown to demonstrate cells with (yellow arrows) and without additional foci of fluorescence in the host cell (ratio indicated in %, at least 50 cells were analysed on 3 occasions, standard deviation in brackets). (D) Bodipy-TR-C₅-ceramide (Bodipy) stained parasites expressing PF13_0191-GFP. Protein structure in A and C indicated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat-1003546-g001" target="_blank">Figure 1</a>. Nuclei were stained with DAPI. Size bars: 5 µm.</p