The RNAi-Competent Malaria Parasite: A Novel Strategy to Knock Down Plasmodium Genes via Non-Canonical RNAi

Malaria, caused by apicomplexan parasites of the Plasmodium species, is one of the deadliest infectious diseases worldwide. Despite the urgent need to identify new drug targets and vaccine candidates, a large proportion of the Plasmodium genes are uncharacterized, as tools to study gene function are limited. In many eukaryotes, genes can be silenced via RNA interference (RNAi) using artificial short hairpin RNAs (shRNAs). However, Plasmodium parasites lack the machinery required for RNAi. In this study, I therefore engineered a non-canonical RNAi machinery into the rodent parasite Plasmodium berghei (P. berghei). To this end, I exploited a non-canonical RNAi pathway which requires only a single protein, Argonaute 2 (Ago2), and a specifically designed shRNA, a so-called AgoshRNA, for gene silencing. I generated a P. berghei line constitutively expressing Ago2, named PbAgo2, and demonstrated that this parasite can complete its life cycle through the mammalian and insect host, despite exhibiting a reduced growth in blood and mosquito stages. Expression of AgoshRNAs targeting the mRNA of the green fluorescent protein GFP (constitutively expressed by PbAgo2) induced a potent knockdown of GFP both in blood and in non-erythrocytic stages. As different AgoshRNAs mediated gene silencing to various levels, target gene expression could be fine-tuned. AgoshRNA-mediated gene knockdown was also possible for endogenous genes, and the knockdown of a non-essential gene phenocopied the full knockout. Additionally, the expression of a blood-stage-essential gene was reduced using RNAi. The analysis of the transcriptome of PbAgo2 by RNA sequencing suggested a possible interaction between Ago2 and a Plasmodium mRNA storage protein as a putative reason for the growth impairment. To further increase the potential applications of the RNAi-competent parasite, Ago2 expression was restricted to the liver stage using a stage-specific promoter. This transgenic line behavee indistinguishable from wild type and the expression of an AgoshRNA targeting GFP silenced fluorescence exclusively in late liver stages. In summary, PbAgo2 is a potent tool to modulate gene expression without the need to alter the genetic locus. In contrast to existing tools, PbAgo2 provides the option to target genes exclusively in a single life cycle stage, to multiplex different AgoshRNAs enabling the simultaneous knockdown of multiple genes, or to screen for phenotypes using a library of AgoshRNAs. This novel, RNAi-competent parasite line opens a wealth of new options to annotate genes in Plasmodium

Plasmodium asexual growth and sexual development in the haematopoietic niche of the host

Plasmodium spp. parasites are the causative agents of malaria in humans and animals, and they are exceptionally diverse in their morphology and life cycles. They grow and develop in a wide range of host environments, both within blood-feeding mosquitoes, their definitive hosts, and in vertebrates, which are intermediate hosts. This diversity is testament to their exceptional adaptability and poses a major challenge for developing effective strategies to reduce the disease burden and transmission. Following one asexual amplification cycle in the liver, parasites reach high burdens by rounds of asexual replication within red blood cells. A few of these blood-stage parasites make a developmental switch into the sexual stage (or gametocyte), which is essential for transmission. The bone marrow, in particular the haematopoietic niche (in rodents, also the spleen), is a major site of parasite growth and sexual development. This Review focuses on our current understanding of blood-stage parasite development and vascular and tissue sequestration, which is responsible for disease symptoms and complications, and when involving the bone marrow, provides a niche for asexual replication and gametocyte development. Understanding these processes provides an opportunity for novel therapies and interventions

No evidence for Ago2 translocation from the host erythrocyte into the Plasmodium parasite

Background: Plasmodium parasites rely on various host factors to grow and replicate within red blood cells (RBC). While many host proteins are known that mediate parasite adhesion and invasion, few examples of host enzymes co-opted by the parasite during intracellular development have been described. Recent studies suggested that the host protein Argonaute 2 (Ago2), which is involved in RNA interference, can translocate into the parasite and affect its development. Here, we investigated this hypothesis. Methods: We used several different monoclonal antibodies to test for Ago2 localisation in the human malaria parasite, P. falciparum and rodent P. berghei parasites. In addition, we biochemically fractionated infected red blood cells to localize Ago2. We also quantified parasite growth and sexual commitment in the presence of the Ago2 inhibitor BCI-137. Results: Ago2 localization by fluorescence microscopy produced inconclusive results across the three different antibodies, suggesting cross-reactivity with parasite targets. Biochemical separation of parasite and RBC cytoplasm detected Ago2 only in the RBC cytoplasm and not in the parasite. Inhibition of Ago2 using BCl-137 did not result in altered parasite development. Conclusion: Ago2 localization in infected RBCs by microscopy is confounded by non-specific binding of antibodies. Complementary results using biochemical fractionation and Ago2 detection by western blot did not detect the protein in the parasite cytosol, and growth assays using a specific inhibitor demonstrated that its catalytical activity is not required for parasite development. We therefore conclude that previous data localising Ago2 to parasite ring stages are due to antibody cross reactivity, and that Ago2 is not required for intracellular Plasmodium development

Revisiting gametocyte biology in malaria parasites

Gametocytes are the only form of the malaria parasite that is transmissible to the mosquito vector. They are present at low levels in blood circulation and significant knowledge gaps exist in their biology. Recent reductions in the global malaria burden have brought the possibility of elimination and eradication, with renewed focus on malaria transmission biology as a basis for interventions. This review discusses recent insights into gametocyte biology in the major human malaria parasite, Plasmodium falciparum and related species

Host cell maturation modulates parasite invasion and sexual differentiation in Plasmodium berghei

Malaria remains a global health problem causing more than 400,000 deaths annually. Plasmodium parasites, the causative agents of malaria, replicate asexually in red blood cells (RBCs) of their vertebrate host, while a subset differentiates into sexual stages (gametocytes) for mosquito transmission. Parasite replication and gametocyte maturation in the erythropoietic niches of the bone marrow and spleen contribute to pathogenesis and drive transmission, but the mechanisms underlying this organ enrichment remain unknown. Here, we performed a comprehensive analysis of rodent P. berghei infection by flow cytometry and single-cell RNA sequencing. We identified CD71 as a host receptor for reticulocyte invasion and found that parasites metabolically adapt to the host cell environment. Transcriptional analysis and functional assays further revealed a nutrient-dependent tropism for gametocyte formation in reticulocytes. Together, we provide a thorough characterization of host-parasite interactions in erythropoietic niches and define host cell maturation state as the key driver of parasite adaptation

Gene knockdown in malaria parasites via non-canonical RNAi

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Raman spectroscopic analysis of skin as a diagnostic tool for Human African Trypanosomiasis

Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT)

Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection

International audiencePlasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections

Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.

The most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in " - ", another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 " - " genes whose expression was spleen dependent. To determine their role in cytoadherence, two " - " transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five " - " spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in " - " biology toward deeper studies of the spleen during infection