RESUMO
Malaria vaccination approaches using live Plasmodium parasites are currently explored, with either attenuated mosquito-derived sporozoites or attenuated blood-stage parasites. Both approaches would profit from the availability of attenuated and avirulent parasites with a reduced blood-stage multiplication rate. Here we screened gene-deletion mutants of the rodent parasite P. berghei and the human parasite P. falciparum for slow growth. Furthermore, we tested the P. berghei mutants for avirulence and resolving blood-stage infections, while preserving sporozoite formation and liver infection. Targeting 51 genes yielded 18 P. berghei gene-deletion mutants with several mutants causing mild infections. Infections with the two most attenuated mutants either by blood stages or by sporozoites were cleared by the immune response. Immunization of mice led to protection from disease after challenge with wild-type sporozoites. Two of six generated P. falciparum gene-deletion mutants showed a slow growth rate. Slow-growing, avirulent P. falciparum mutants will constitute valuable tools to inform on the induction of immune responses and will aid in developing new as well as safeguarding existing attenuated parasite vaccines.
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Microtubules are a ubiquitous eukaryotic cytoskeletal element typically consisting of 13 protofilaments arranged in a hollow cylinder. This arrangement is considered the canonical form and is adopted by most organisms, with rare exceptions. Here, we use in situ electron cryo-tomography and subvolume averaging to analyse the changing microtubule cytoskeleton of Plasmodium falciparum, the causative agent of malaria, throughout its life cycle. Unexpectedly, different parasite forms have distinct microtubule structures coordinated by unique organising centres. In merozoites, the most widely studied form, we observe canonical microtubules. In migrating mosquito forms, the 13 protofilament structure is further reinforced by interrupted luminal helices. Surprisingly, gametocytes contain a wide distribution of microtubule structures ranging from 13 to 18 protofilaments, doublets and triplets. Such a diversity of microtubule structures has not been observed in any other organism to date and is likely evidence of a distinct role in each life cycle form. This data provides a unique view into an unusual microtubule cytoskeleton of a relevant human pathogen.
Assuntos
Culicidae , Pavilhão Auricular , Parasitos , Humanos , Animais , Microtúbulos , CitoesqueletoRESUMO
One hundred and twenty five years ago, in August 1897, Ronald Ross discovered forms of the malaria parasite in the gut of two mosquitoes. Shortly afterwards, Giovanni Battista Grassi established how the transmissive forms of the parasite, sporozoites, develop in these Plasmodium oocysts. Today, we still understand surprisingly little about the molecular processes governing oocyst biology.
Assuntos
Anopheles , Plasmodium , Animais , Anopheles/parasitologia , Oocistos , EsporozoítosRESUMO
Malaria-causing parasites rely on an actin-myosin-based motor for the invasion of different host cells and tissue traversal in mosquitoes and vertebrates. The unusual myosin A of Plasmodium spp. has a unique N-terminal extension, which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19. Here, using the rodent-infecting P. berghei we show that phosphorylation of serine 19 increases ookinete but not sporozoite motility and is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry. S19A along with E6R mutations slow ookinetes and salivary gland sporozoites in both 2D and 3D environments. In contrast to data from purified proteins, both E6R and S19D mutations lower force generation by sporozoites. Our data show that the phosphorylation cycle of S19 influences parasite migration and force generation and is critical for optimal migration of parasites during transmission from and to the mosquito.
Assuntos
Culicidae , Malária Falciparum , Miosina não Muscular Tipo IIA , Animais , Miosina não Muscular Tipo IIA/metabolismo , Fosforilação , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Proteínas de Protozoários/metabolismo , Serina/metabolismo , Esporozoítos/metabolismoRESUMO
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.
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Culicidae , Malária , Parasitos , Animais , Culicidae/parasitologia , Malária/parasitologia , Plasmodium berghei/genética , Diferenciação SexualRESUMO
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).
Assuntos
Pele/patologia , Análise Espectral Raman/métodos , Trypanosoma brucei brucei/fisiologia , Trypanosoma brucei gambiense/fisiologia , Tripanossomíase Africana/diagnóstico , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pele/parasitologia , Tripanossomíase Africana/parasitologiaRESUMO
Plasmodium 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.
Assuntos
Antígenos de Protozoários/imunologia , Genes de Protozoários , Malária Vivax/imunologia , Plasmodium vivax/imunologia , Baço/metabolismo , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos de Protozoários/genética , Aotidae , Células CHO , Adesão Celular/genética , Adesão Celular/imunologia , Criança , Cricetulus , Modelos Animais de Doenças , Fibroblastos , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Malária Vivax/sangue , Malária Vivax/parasitologia , Família Multigênica , Papua Nova Guiné , Plasmodium vivax/genética , Baço/citologia , Baço/parasitologia , Esplenectomia , Análise Serial de TecidosRESUMO
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.
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Diferenciação Celular , Divisão Celular , Eritrócitos/parasitologia , Plasmodium/crescimento & desenvolvimento , Animais , Medula Óssea/parasitologia , Humanos , Fígado/parasitologia , Malária/parasitologiaRESUMO
The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species.
Assuntos
Proteínas Argonautas/genética , Engenharia Genética/métodos , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Anopheles/parasitologia , Proteínas Argonautas/metabolismo , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/antagonistas & inibidores , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Estágios do Ciclo de Vida/genética , Camundongos , Camundongos Endogâmicos C57BL , Mosquitos Vetores/parasitologia , Organismos Geneticamente Modificados , Perforina/genética , Perforina/metabolismo , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , RNA Interferente Pequeno/metabolismo , TransgenesRESUMO
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.
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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.
Assuntos
Estágios do Ciclo de Vida/fisiologia , Malária/parasitologia , Malária/transmissão , Plasmodium/fisiologia , Animais , Culicidae/parasitologia , Humanos , Plasmodium/crescimento & desenvolvimentoRESUMO
The increasing use of screening technologies in malaria research has substantially expanded our knowledge on cellular factors hijacked by the Plasmodium parasite in the infected host, including those that participate in the clinically silent liver stage. This rapid gain in our understanding of the hepatic interaction partners now requires a means to validate and further disentangle parasite-host networks in physiologically relevant liver model systems. Here, we outline seminal work that contributed to our present knowledge on the intrahepatic Plasmodium host factors, followed by a discussion of surrogate models of mammalian livers or hepatocytes. We finally describe how Adeno-associated viruses could be engineered and used as hepatotropic tools to dissect Plasmodium-host interactions, and to deliberately control these networks for antimalaria vaccination or therapy.
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Dependovirus/fisiologia , Plasmodium/fisiologia , Animais , Engenharia Genética , Interações Hospedeiro-Patógeno , Humanos , Fígado/parasitologia , Fígado/patologia , Fígado/virologia , Modelos BiológicosRESUMO
Malaria, caused by protozoan Plasmodium parasites, remains a prevalent infectious human disease due to the lack of an efficient and safe vaccine. This is directly related to the persisting gaps in our understanding of the parasite's interactions with the infected host, especially during the clinically silent yet essential liver stage of Plasmodium development. Previously, we and others showed that genetically attenuated parasites (GAP) that arrest in the liver induce sterile immunity, but only upon multiple administrations. Here, we comprehensively studied hepatic gene and miRNA expression in GAP-injected mice, and found both a broad activation of IFNγ-associated pathways and a significant increase of murine microRNA-155 (miR-155), that was especially pronounced in non-parenchymal cells including liver-resident macrophages (Kupffer cells). Remarkably, ectopic upregulation of this miRNA in the liver of mice using robust hepatotropic adeno-associated virus 8 (AAV8) vectors enhanced GAP's protective capacity substantially. In turn, this AAV8-mediated miR-155 expression permitted a reduction of GAP injections needed to achieve complete protection against infectious parasite challenge from previously three to only one. Our study highlights a crucial role of mammalian miRNAs in Plasmodium liver infection in vivo and concurrently implies their great potential as future immune-augmenting agents in improved vaccination regimes against malaria and other diseases.