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

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

Generation of transgenic rodent malaria parasites by transfection of cell culture-derived merozoites

Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector(®) electroporation technology. Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8-18 days

The SIB Swiss Institute of Bioinformatics' resources: focus on curated databases

The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) provides world-class bioinformatics databases, software tools, services and training to the international life science community in academia and industry. These solutions allow life scientists to turn the exponentially growing amount of data into knowledge. Here, we provide an overview of SIB's resources and competence areas, with a strong focus on curated databases and SIB's most popular and widely used resources. In particular, SIB's Bioinformatics resource portal ExPASy features over 150 resources, including UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt, STRING, UniCarbKB, SugarBindDB, SwissRegulon, EPD, arrayMap, Bgee, SWISS-MODEL Repository, OMA, OrthoDB and other databases, which are briefly described in this article

Host cell cytosolic immune response during Plasmodium liver stage development

Recent years have witnessed a great gain in knowledge regarding parasite-host cell interactions during Plasmodium liver stage development. It is now an accepted fact that a large percentage of sporozoite invading a hepatocyte fail to form infectious merozoites. There appears to be a delicate balance between parasite survival and elimination and we now start to understand why this is so. Plasmodium liver stages replicate within the parasitophorous vacuole (PV), formed during invasion by invagination of the host cell plasma membrane. The main interface between the parasite and hepatocyte is the parasitophorous vacuole membrane (PVM) that surrounds the PV. Recently, it was shown that autophagy marker proteins decorate the PVM of Plasmodium liver stages and eliminate a proportion of parasites by an autophagy-like mechanism. Successfully developing Plasmodium berghei parasites are initially also labeled but in the course of development, they are able to control this host defense mechanism by shedding PVM material into the tubovesicular network (TVN), an extension of the PVM that releases vesicles into to the host cell cytoplasm. Better understanding of the molecular events at the PVM/TVN during parasite elimination could be the basis of new anti-malarial measures

Deciphering host lysosome-mediated elimination of Plasmodium berghei liver stage parasites.

Liver stage Plasmodium parasites reside in a parasitophorous vacuole (PV) that associates with lysosomes. It has previously been shown that these organelles can have beneficial as well as harmful effects on the parasite. Yet it is not clear how the association of lysosomes with the parasite is controlled and how interactions with these organelles lead to the antagonistic outcomes. In this study we used advanced imaging techniques to characterize lysosomal interactions with the PV. In host cells harboring successfully developing parasites we observed that these interaction events reach an equilibrium at the PV membrane (PVM). In a population of arrested parasites, this equilibrium appeared to shift towards a strongly increased lysosomal fusion with the PVM witnessed by strong PVM labeling with the lysosomal marker protein LAMP1. This was followed by acidification of the PV and elimination of the parasite. To systematically investigate elimination of arrested parasites, we generated transgenic parasites that express the photosensitizer KillerRed, which leads to parasite killing after activation. Our work provides insights in cellular details of intracellular killing and lysosomal elimination of Plasmodium parasites independent of cells of the immune system

Generation of transgenic rodent malaria parasites by transfection of cell culture-derived merozoites

Abstract Background Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. Methods HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector® electroporation technology. Results Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. Conclusion An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8–18 days

LC3-association with the parasitophorous vacuole membrane of Plasmodium berghei liver stages follows a noncanonical autophagy pathway.

Eukaryotic cells can employ autophagy to defend themselves against invading pathogens. Upon infection by Plasmodium berghei sporozoites, the host hepatocyte targets the invader by labelling the parasitophorous vacuole membrane (PVM) with the autophagy marker protein LC3. Until now, it has not been clear whether LC3 recruitment to the PVM is mediated by fusion of autophagosomes or by direct incorporation. To distinguish between these possibilities, we knocked out genes that are essential for autophagosome formation and for direct LC3 incorporation into membranes. The CRISPR/Cas9 system was employed to generate host cell lines deficient for either FIP200, a member of the initiation complex for autophagosome formation, or ATG5, responsible for LC3 lipidation and incorporation of LC3 into membranes. Infection of these knockout cell lines with P. berghei sporozoites revealed that LC3 recruitment to the PVM indeed depends on functional ATG5 and the elongation machinery, but not on FIP200 and the initiation complex, suggesting a direct incorporation of LC3 into the PVM. Importantly, in P. berghei-infected ATG5(-/-) host cells, lysosomes still accumulated at the PVM, indicating that the recruitment of lysosomes follows an LC3-independent pathway

A Cysteine Protease Inhibitor of <i>Plasmodium berghei</i> Is Essential for Exo-erythrocytic Development

<div><p><i>Plasmodium</i> parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the <i>Plasmodium berghei</i> ICP (PbICP). Excision of the <i>pbicb</i> gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a <i>pbicp-gfp</i> construct fully reversed these defects. Taken together, in <i>P. berghei</i> this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.</p></div

PbICP is not essential for parasite blood stage development.

<p>(A) Assessment of SSR at the <i>pbicp</i> locus in <i>pbicp</i>-transgenic parasites (PbICP_cond, PbICP_KO, and PbICP_comp) by PCR of genomic DNA using primers P1 and P3 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004336#ppat.1004336.s001" target="_blank">Figure S1</a>). PbICP_KO erythrocytic stages were generated by subcloning of PbICP_cond parasites via single merosome injection into mice. PbICP_comp erythrocytic stages were generated by transfection of PbICP_KO parasites with the pL0017-<i>pbicp</i>-<i>gfp</i> plasmid <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004336#ppat.1004336-Rennenberg1" target="_blank">[29]</a> as a complementation (add-back) for <i>pbicp</i>. The sizes of the DNA fragments amplified from <i>pbicp</i> excised (SSR+) or non-excised (SSR−) loci are shown. As a control, primers specific for <i>pbicp</i> were used (bottom panel). (B) Western blot analysis of extracts from blood stage parasites. <i>In vitro</i> cultured parasites were collected at the schizont stage. Analysis included the following strains: PbICP_control (UIS4/Flp(−)), PbICP_cond, PbICP_KO, and PbICP_comp parasites. PbICP-C was detected using a specific mouse anti-PbICP-C antiserum <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004336#ppat.1004336-Rennenberg1" target="_blank">[29]</a>. Rat anti-MSP1 was used as a control. Molecular masses are indicated in kDa. The expected mass of full-length PbICP is 55 kDa in PbICP_control and 57 kDa in PbICP_cond parasites and 23 kDa after processing (PbICP-C). PbICP-GFP has an expected molecular mass of 81 kDa and 49 kDa after processing (PbICP-C-GFP). (*: non-specific protein bands, probably Ig subunits remaining in the blood culture detected by the secondary HRP-labeled anti-mouse antibody). (C) Blood stage development of PbICP_control (UIS4/Flp(−)), PbICP_KO and PbICP_comp parasites. Mice were infected by i.p. injection of 100 µl blood from infected mice, adjusted to a parasitemia of 5% with PBS. The onset and development of a blood stage infection was determined by observation of blood smears. The two graphs represent two separate sets of experiments. In the left graph, parasitemia of PbICP_control and PbICP_KO parasites were compared and, in the right graph, parasitemia of PbICP_control and PbICP_comp parasites were compared. For statistical evaluation of the difference in parasitemia at day 3 see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004336#ppat.1004336.s002" target="_blank">Figure S2D</a>.</p

PbICP is Important for EEF Maturation.

<p>(A) IFA of infected, detached cells 65 hpi (mature EEFs) of either PbICP_control (first panel), PbICP_cond (second panel), or PbICP_comp (third panel) parasites. Cells were stained with mouse anti-MSP1 or mouse anti-GFP (green), rat anti-PbICP-C (red), and chicken anti-ExpI (cyan). Secondary antibodies were: anti-mouse Alexa488, anti-rat Alexa594, or anti-chicken Cy5. DNA was stained with DAPI (blue), Scale bars: 10 µm. (B) Quantification of infected, detached cells 65 hpi (mature EEFs) of the different parasite strains in relation to infected cells 48 hpi. HepG2 cells were infected with either PbICP_control (control), PbICP_cond (cond), or PbICP_comp (comp) sporozoites and infected cells were quantified 48 hpi (values normalized to 100%). At 65 hpi, supernatant was collected and stained with Hoechst 33342. The number of infected, detached cells was quantified and the ratio between infected cells at 48 hpi and detached cells at 65 hpi was calculated. Results are the means ± S.D. from three independent measurements. Differences between PbICP_control and <i>pbicp</i>-transgenic parasites (PbICP_cond, PbICP_comp) were compared using Student's t test (**** = P<0.0001; ns, not significant). PbICP_KO parasites were excluded from this experiment as no invasion could be achieved. n.a.: not applicable.</p