Going live: a comparative analysis of the suitability of the RFP derivatives RedStar, mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed

Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8(+) memory T cells.

Protection against malaria can be achieved by induction of a strong CD8(+) T-cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long-lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8(+) T-cell response and sterile protection, which is short-lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite-specific antibodies that showed protective capacity. The numbers of CSP-specific CD8(+) T cells were not substantially enhanced by sporozoite infections; however, CSP-specific memory CD8(+) T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized-only mice. CD4(+) T cells contributed to protection as well; but CD8(+) memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8(+) effector-memory T cells with increased cytotoxic activity as well as CD4(+) memory T cells and neutralizing antibodies

The Leishmania donovani chaperone cyclophilin 40 is essential for intracellular infection independent of its stage-specific phosphorylation status

During its life cycle, the protozoan pathogen Leishmania donovani is exposed to contrasting environments inside insect vector and vertebrate host, to which the parasite must adapt for extra- and intracellular survival. Combining null mutant analysis with phosphorylation site-specific mutagenesis and functional complementation we genetically tested the requirement of the L. donovani chaperone cyclophilin 40 (LdCyP40) for infection. Targeted replacement of LdCyP40 had no effect on parasite viability, axenic amastigote differentiation, and resistance to various forms of environmental stress in culture, suggesting important functional redundancy to other parasite chaperones. However, ultrastructural analyses and video microscopy of cyp40-/- promastigotes uncovered important defects in cell shape, organization of the subpellicular tubulin network and motility at stationary growth phase. More importantly, cyp40-/- parasites were unable to establish intracellular infection in murine macrophages and were eliminated during the first 24 h post infection. Surprisingly, cyp40-/- infectivity was restored in complemented parasites expressing a CyP40 mutant of the unique S274 phosphorylation site. Together our data reveal non-redundant CyP40 functions in parasite cytoskeletal remodelling relevant for the development of infectious parasites in vitro independent of its phosphorylation status, and provide a framework for the genetic analysis of Leishmania-specific phosphorylation sites and their role in regulating parasite protein function

Alteration of the parasite plasma membrane and the parasitophorous vacuole membrane during exo-erythrocytic development of malaria parasites

The rodent malaria parasite Plasmodium berghei develops in hepatocytes within 48-52h from a single sporozoite into up to 20,000 daughter parasites, so-called merozoites. The cellular and molecular details of this extensive proliferation are still largely unknown. Here we have used a transgenic, RFP-expressing P. berghei parasite line and molecular imaging techniques including intravital microscopy to decipher various aspects of parasite development within the hepatocyte. In late schizont stages, MSP1 is expressed and incorporated into the parasite plasma membrane that finally forms the membrane of developing merozoites by continuous invagination steps. We provide first evidence for activation of a verapamil-sensitive Ca(2+) channel in the plasma membrane of liver stage parasites before invagination occurs. During merozoite formation, the permeability of the parasitophorous vacuole membrane changes considerably before it finally becomes completely disrupted, releasing merozoites into the host cell cytoplasm

Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids

The merozoite stage of the malaria parasite that infects erythrocytes and causes the symptoms of the disease is initially formed inside host hepatocytes. However, the mechanism by which hepatic merozoites reach blood vessels (sinusoids) in the liver and escape the host immune system before invading erythrocytes remains unknown. Here, we show that parasites induce the death and the detachment of their host hepatocytes, followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on the outer leaflet of host plasma membranes, which act as eat me'' signals to phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the migration of parasites into the bloodstream and their protection from host immunity.Bernhard Nocht Inst Trop Med, D-20359 Hamburg, GermanyInst Pasteur, Dept Parasitol, F-75724 Paris 15, FranceUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilUniv Hamburg, Hosp Eppendorf, Dept Hepatobiliary Surg, D-20246 Hamburg, GermanyUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilWeb of Scienc

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

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

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

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