Highly efficient subcloning of rodent malaria parasites by injection of single merosomes or detached cells

This protocol describes a method for obtaining rodent Plasmodium parasite clones with high efficiency, which takes advantage of the normal course of Plasmodium in vitro exoerythrocytic development. At the completion of development, detached cells/merosomes form, which contain hundreds to thousands of merozoites. As all parasites within a single detached cell/merosome derive from the same sporozoite, we predicted them to be genetically identical. To prove this, hepatoma cells were infected simultaneously with a mixture of Plasmodium berghei sporozoites expressing either GFP or mCherry. Subsequently, individual detached cells/merosomes from this mixed population were selected and injected into mice, resulting in clonal blood stage parasite infections. Importantly, as a large majority of mice become successfully infected using this protocol, significantly less mice are necessary than for the widely used technique of limiting dilution cloning. To produce a clonal P. berghei blood stage infection from a non-clonal infection using this procedure requires between 4 and 5 weeks.

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the Development of Liver-Stage Malarial Parasites

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Inhibition of apoptosis by intracellular protozoan parasites

Protozoan parasites which reside inside a host cell avoid direct destruction by the immune system of the host. The infected cell, however, still has the capacity to counteract the invasive pathogen by initiating its own death, a process which is called programmed cell death or apoptosis. Apoptotic cells are recognised and phagocytosed by macrophages and the parasite is potentially eliminated together with the infected cell. This potent defence mechanism of the host cell puts strong selective pressure on the parasites which have, in turn, evolved strategies to modulate the apoptotic program of the host cell to their favour. Within the last decade, the existence of cellular signalling pathways which inhibit the apoptotic machinery has been demonstrated. It is not surprising that intracellular pathogens subvert these pathways to ensure their own survival in the infected cell. Molecular mechanisms which interfere with apoptotic pathways have been studied extensively for viruses and parasitic bacteria, but protozoan parasites have come into focus only recently. Intracellular protozoan parasites which have been reported to inhibit the apoptotic program of the host cell, are Toxoplasma gondii, Trypanosoma cruzi, Leishmania sp., Theileria sp., Cryptosporidium parvum, and the microsporidian Nosema algerae. Although these parasites differ in their mechanism of host cell entry and in their final intracellular localisation, they might activate similar pathways in their host cells to inhibit apoptosis. In this respect, two families of molecules, which are known for their capacity to interrupt the apoptotic program, are currently discussed in the literature. First, the expression of heat shock proteins is often induced upon parasite infection and can directly interfere with molecules of the cellular death machinery. Secondly, a more indirect effect is attributed to the parasite-dependent activation of NF-kappaB, a transcription factor that regulates the transcription of anti-apoptotic molecules.

Dynamics of Apis mellifera filamentous virus (AmFV) infections in honey bees and relationships with other parasites

Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.

Protective efficacy and safety of liver stage attenuated malaria parasites

During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(-) or uis3(-) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(-) parasites protected better than uis3(-) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy.

Regulation of host cell survival by intracellular Plasmodium and Theileria parasites

Plasmodium and Theileria parasites are obligate intracellular protozoa of the phylum Apicomplexa. Theileria infection of bovine leukocytes induces transformation of host cells and infected leukocytes can be kept indefinitely in culture. Theileria-dependent host cell transformation has been the subject of interest for many years and the molecular basis of this unique phenomenon is quite well understood. The equivalent life cycle stage of Plasmodium is the infection of mammalian hepatocytes, where parasites reside for 2-7 days depending on the species. Some of the molecular details of parasite-host interactions in P. berghei-infected hepatocytes have emerged only very recently. Similar to what has been shown for Theileria-infected leukocytes these data suggest that malaria parasites within hepatocytes also protect their host cell from programmed cell death. However, the strategies employed to inhibit host cell apoptotic pathways appear to be different to those used by Theileria. This review discusses similarities and differences at the molecular level of Plasmodium- and Theileria-induced regulation of the host cell survival machinery.

Cellular and molecular interactions between the apicomplexan parasites Plasmodium and Theileria and their host cells

Apicomplexan parasites of the genera Theileria and Plasmodium have complicated life cycles including infection of a vertebrate intermediate host and an arthropod definitive host. As the Plasmodium parasite progresses through its life cycle, it enters a number of different cell types, both in its mammalian and mosquito hosts. The fate of these cells varies greatly, as do the parasite and host molecules involved in parasite-host interactions. In mammals, Plasmodium parasites infect hepatocytes and erythrocytes whereas Theileria infects ruminant leukocytes and erythrocytes. Survival of Plasmodium-infected hepatocytes and Theileria-infected leukocytes depends on parasite-mediated inhibition of host cell apoptosis but only Theileria-infected cells exhibit a fully transformed phenotype. As the development of both parasites progresses towards the merozoite stage, the parasites no longer promote the survival of the host cell and the infected cell is finally destroyed to release merozoites. In this review we describe similarities and differences of parasite-host cell interactions in Plasmodium-infected hepatocytes and Theileria-infected leukocytes and compare the observed phenotypes to other parasite stages interacting with host cells.

Intracellular survival of apicomplexan parasites and host cell modification

The intracellular stages of apicomplexan parasites are known to extensively modify their host cells to ensure their own survival. Recently, considerable progress has been made in understanding the molecular details of these parasite-dependent effects for Plasmodium-, Toxoplasma- and Theileria-infected cells. We have begun to understand how Plasmodium liver stage parasites protect their host hepatocytes from apoptosis during parasite development and how they induce an ordered cell death at the end of the liver stage. Toxoplasma parasites are also known to regulate host cell survival pathways and it has been convincingly demonstrated that they block host cell major histocompatibility complex (MHC)-dependent antigen presentation of parasite epitopes to avoid cell-mediated immune responses. Theileria parasites are the masters of host cell modulation because their presence immortalises the infected cell. It is now accepted that multiple pathways are activated to induce Theileria-dependent host cell transformation. Although it is now known that similar host cell pathways are affected by the different parasites, the outcome for the infected cell varies considerably. Improved imaging techniques and new methods to control expression of parasite and host cell proteins will help us to analyse the molecular details of parasite-dependent host cell modifications.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

Live and let die: manipulation of host hepatocytes by exoerythrocytic Plasmodium parasites

The generation of rodent Plasmodium strains expressing fluorescent proteins in all life cycle stages has had a big impact on malaria research. With this tool in hand, for the first time it was possible to follow in real time by in vivo microscopy the infection route of Plasmodium sporozoites transmitted to the mammalian host by Anopheles mosquitoes. Recently, this work has been extended to the analysis of both hepatocyte infection by Plasmodium sporozoites, as well as liver merozoite transport into blood vessels. The stunning results of these studies have considerably changed our understanding of hepatocyte invasion and parasite liberation. Here, we describe the most important findings of the last years and in addition, we elaborate on the molecular events during the intracellular development of Plasmodium exoerythrocytic forms that give rise to erythrocyte infecting merozoites.

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.

GFP-targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites.

BACKGROUND INFORMATION The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo-erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. RESULTS In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo-erythrocytic schizogony in vitro, leading to impaired parasite maturation. CONCLUSIONS Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red-blood-cell-infective merozoites.