RESUMO
Malaria is caused by parasites of the Plasmodium genus and remains one of the most pressing human health problems. The spread of parasites resistant to or partially resistant to single or multiple drugs, including frontline antimalarial artemisinin and its derivatives, poses a serious threat to current and future malaria control efforts. In vitro drug assays are important for identifying new antimalarial compounds and monitoring drug resistance. Due to its robustness and ease of use, the [3H]-hypoxanthine incorporation assay is still considered a gold standard and is widely applied, despite limited sensitivity and the dependence on radioactive material. Here, we present a first-of-its-kind chemiluminescence-based antimalarial drug screening assay. The effect of compounds on P. falciparum is monitored by using a dioxetane-based substrate (AquaSpark ß-D-galactoside) that emits high-intensity luminescence upon removal of a protective group (ß-D-galactoside) by a transgenic ß-galactosidase reporter enzyme. This biosensor enables highly sensitive, robust, and cost-effective detection of asexual, intraerythrocytic P. falciparum parasites without the need for parasite enrichment, washing, or purification steps. We are convinced that the ultralow detection limit of less than 100 parasites of the presented biosensor system will become instrumental in malaria research, including but not limited to drug screening.
Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Malária Falciparum , Malária , Humanos , Antimaláricos/farmacologia , Plasmodium falciparum , Malária/tratamento farmacológico , Malária Falciparum/parasitologia , Antagonistas do Ácido Fólico/farmacologia , Galactosídeos/farmacologia , Galactosídeos/uso terapêuticoRESUMO
Malaria blood stage parasites commit to either one of two distinct cellular fates while developing within erythrocytes of their mammalian host: they either undergo another round of asexual replication or they differentiate into nonreplicative transmissible gametocytes. Depending on the state of infection, either path may support or impair the ultimate goal of human-to-human transmission via the mosquito vector. Malaria parasites therefore evolved strategies to control investments into asexual proliferation versus gametocyte formation. Recent work provided fascinating molecular insight into shared and unique mechanisms underlying the control and environmental modulation of sexual commitment in the two most widely studied malaria parasite species, Plasmodium falciparum and P. berghei. With this review, we aim at placing these findings into a comparative mechanistic context.
Assuntos
Plasmodium berghei , Plasmodium falciparum , Plasmodium falciparum/fisiologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/genética , Animais , Humanos , Plasmodium berghei/fisiologia , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/genética , Malária/parasitologia , Malária/transmissão , Eritrócitos/parasitologiaRESUMO
Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.
Assuntos
Eritrócitos , Plasmodium falciparum , Polissacarídeos , Proteínas de Protozoários , Humanos , Antígenos de Protozoários/metabolismo , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/genética , Eritrócitos/parasitologia , Eritrócitos/metabolismo , Lectinas/metabolismo , Lectinas/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genéticaRESUMO
Plasmodium falciparum accounts for the majority of over 600,000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with alternative modes of action is thus undoubted. The FK506-binding protein PfFKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus). Whilst there is considerable interest in targeting PfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing and its function in parasite biology remains elusive. Here, we show that limiting PfFKBP35 levels are lethal to P. falciparum and result in a delayed death-like phenotype that is characterized by defective ribosome homeostasis and stalled protein synthesis. Our data furthermore suggest that FK506, unlike the action of this drug in model organisms, exerts its antiproliferative activity in a PfFKBP35-independent manner and, using cellular thermal shift assays, we identify putative FK506-targets beyond PfFKBP35. In addition to revealing first insights into the function of PfFKBP35, our results show that FKBP-binding drugs can adopt non-canonical modes of action - with major implications for the development of FK506-derived molecules active against Plasmodium parasites and other eukaryotic pathogens.
Assuntos
Antimaláricos , Malária Falciparum , Humanos , Tacrolimo , Antibacterianos , Sistemas de Liberação de Medicamentos , Homeostase , Proteínas de Ligação a TacrolimoRESUMO
Recent advances in long read technologies not only enable large consortia to aim to sequence all eukaryotes on Earth, but they also allow individual laboratories to sequence their species of interest with relatively low investment. Long read technologies embody the promise of overcoming scaffolding problems associated with repeats and low complexity sequences, but the number of contigs often far exceeds the number of chromosomes and they may contain many insertion and deletion errors around homopolymer tracts. To overcome these issues, we have implemented the ILRA pipeline to correct long read-based assemblies. Contigs are first reordered, renamed, merged, circularized, or filtered if erroneous or contaminated. Illumina short reads are used subsequently to correct homopolymer errors. We successfully tested our approach by improving the genome sequences of Homo sapiens, Trypanosoma brucei, and Leptosphaeria spp., and by generating four novel Plasmodium falciparum assemblies from field samples. We found that correcting homopolymer tracts reduced the number of genes incorrectly annotated as pseudogenes, but an iterative approach seems to be required to correct more sequencing errors. In summary, we describe and benchmark the performance of our new tool, which improved the quality of novel long read assemblies up to 1 Gbp. The pipeline is available at GitHub: https://github.com/ThomasDOtto/ILRA.
Assuntos
Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA , Pseudogenes , CromossomosRESUMO
Critical events in the life cycle of malaria-causing parasites depend on cyclic guanosine monophosphate homeostasis by guanylyl cyclases (GCs) and phosphodiesterases, including merozoite egress or invasion of erythrocytes and gametocyte activation. These processes rely on a single GCα, but in the absence of known signaling receptors, how this pathway integrates distinct triggers is unknown. We show that temperature-dependent epistatic interactions between phosphodiesterases counterbalance GCα basal activity preventing gametocyte activation before mosquito blood feed. GCα interacts with two multipass membrane cofactors in schizonts and gametocytes: UGO (unique GC organizer) and SLF (signaling linking factor). While SLF regulates GCα basal activity, UGO is essential for GCα up-regulation in response to natural signals inducing merozoite egress and gametocyte activation. This work identifies a GC membrane receptor platform that senses signals triggering processes specific to an intracellular parasitic lifestyle, including host cell egress and invasion to ensure intraerythrocytic amplification and transmission to mosquitoes.
Assuntos
Culicidae , Plasmodium , Animais , Sinais (Psicologia) , Plasmodium/fisiologia , Eritrócitos/parasitologia , Merozoítos/fisiologia , Estágios do Ciclo de Vida , Culicidae/parasitologiaRESUMO
Malaria parasites rely on specialized stages, called gametocytes, to ensure human-to-human transmission. The formation of these sexual precursor cells is initiated by commitment of blood stage parasites to the sexual differentiation pathway. Plasmodium falciparum, the most virulent of six parasite species infecting humans, employs nutrient sensing to control the rate at which sexual commitment is initiated, and the presence of stress-inducing factors, including antimalarial drugs, has been linked to increased gametocyte production in vitro and in vivo. These observations suggest that therapeutic interventions may promote gametocytogenesis and malaria transmission. Here, we engineered a P. falciparum reporter line to quantify sexual commitment rates after exposure to antimalarials and other pharmaceuticals commonly prescribed in malaria-endemic regions. Our data reveal that some of the tested drugs indeed have the capacity to elevate sexual commitment rates in vitro. Importantly, however, these effects are only observed at drug concentrations that inhibit parasite survival and only rarely result in a net increase of gametocyte production. Using a drug-resistant parasite reporter line, we further show that the gametocytogenesis-promoting effect of drugs is linked to general stress responses rather than to compound-specific activities. Altogether, we did not observe evidence for mechanistic links between the regulation of sexual commitment and the activity of commonly used pharmaceuticals in vitro. Our data hence does not support scenarios in which currently applied therapeutic interventions would promote the spread of drug-resistant parasites or malaria transmission in general.
Assuntos
Antimaláricos , Malária Falciparum , Malária , Parasitos , Animais , Antimaláricos/farmacologia , Humanos , Malária/parasitologia , Malária Falciparum/parasitologia , Preparações Farmacêuticas , Plasmodium falciparumRESUMO
Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Animais , Domínio Catalítico , Linhagem Celular , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Eritrócitos/parasitologia , Humanos , Malária , Malária Falciparum/parasitologia , Merozoítos , Parasitos/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.
Assuntos
Sistemas CRISPR-Cas , Malária Falciparum/sangue , Plasmodium falciparum/genética , Esporos de Protozoários/genética , Animais , Anopheles/parasitologia , Células Cultivadas , Eritrócitos/parasitologia , Hepatócitos/citologia , Hepatócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Microscopia de Fluorescência , Mosquitos Vetores/parasitologia , Plasmodium falciparum/fisiologia , Esporos de Protozoários/fisiologia , Esporozoítos/genética , Esporozoítos/fisiologiaRESUMO
Casein kinase 2 (CK2) is a pleiotropic kinase phosphorylating substrates in different cellular compartments in eukaryotes. In the malaria parasite Plasmodium falciparum, PfCK2 is vital for asexual proliferation of blood-stage parasites. Here, we applied CRISPR/Cas9-based gene editing to investigate the function of the PfCK2α catalytic subunit in gametocytes, the sexual forms of the parasite that are essential for malaria transmission. We show that PfCK2α localizes to the nucleus and cytoplasm in asexual and sexual parasites alike. Conditional knockdown of PfCK2α expression prevented the transition of stage IV into transmission-competent stage V gametocytes, whereas the conditional knockout of pfck2a completely blocked gametocyte maturation already at an earlier stage of sexual differentiation. In summary, our results demonstrate that PfCK2α is not only essential for asexual but also sexual development of P. falciparum blood-stage parasites and encourage studies exploring PfCK2α as a potential target for dual-active antimalarial drugs.
Assuntos
Caseína Quinase II/metabolismo , Eritrócitos/parasitologia , Gametogênese , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Antimaláricos/farmacologia , Sistemas CRISPR-Cas , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Domínio Catalítico , Edição de Genes , Humanos , Estágios do Ciclo de Vida , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Protozoários/genética , Reprodução AssexuadaRESUMO
The human malaria parasite Plasmodium falciparum encodes a single ortholog of heterochromatin protein 1 (PfHP1) that plays a crucial role in the epigenetic regulation of various survival-related processes. PfHP1 is essential for parasite proliferation and the heritable silencing of genes linked to antigenic variation, host cell invasion, and sexual conversion. Here, we employed CRISPR/Cas9-mediated genome editing combined with the DiCre/loxP system to investigate how the PfHP1 chromodomain (CD), hinge domain, and chromoshadow domain (CSD) contribute to overall PfHP1 function. We show that the 76 C-terminal residues are responsible for targeting PfHP1 to the nucleus. Furthermore, we reveal that each of the three functional domains of PfHP1 are required for heterochromatin formation, gene silencing, and mitotic parasite proliferation. Finally, we discovered that the hinge domain and CSD of HP1 are functionally conserved between P. falciparum and P. berghei, a related malaria parasite infecting rodents. In summary, our study provides new insights into PfHP1 function and offers a tool for further studies on epigenetic regulation and life cycle decision in malaria parasites.IMPORTANCE Malaria is caused by unicellular Plasmodium species parasites that repeatedly invade and replicate inside red blood cells. Some blood-stage parasites exit the cell cycle and differentiate into gametocytes that are essential for malaria transmission via the mosquito vector. Epigenetic control mechanisms allow the parasites to alter the expression of surface antigens and to balance the switch between parasite multiplication and gametocyte production. These processes are crucial to establish chronic infection and optimize parasite transmission. Here, we performed a mutational analysis of heterochromatin protein 1 (HP1) in P. falciparum We demonstrate that all three domains of this protein are indispensable for the proper function of HP1 in parasite multiplication, heterochromatin formation, and gene silencing. Moreover, expression of chimeric proteins revealed the functional conservation of HP1 proteins between different Plasmodium species. These results provide new insight into the function and evolution of HP1 as an essential epigenetic regulator of parasite survival.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Inativação Gênica , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Variação Antigênica , Sistemas CRISPR-Cas , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Eritrócitos/parasitologia , Regulação da Expressão Gênica , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Malaria is a life-threatening disease, caused by Apicomplexan parasites of the Plasmodium genus. The Anopheles mosquito is necessary for the sexual replication of these parasites and for their transmission to vertebrate hosts, including humans. Imaging of the parasite within the insect vector has been attempted using multiple microscopy methods, most of which are hampered by the presence of the light scattering opaque cuticle of the mosquito. So far, most imaging of the Plasmodium mosquito stages depended on either sectioning or surgical dissection of important anatomical sites, such as the midgut and the salivary glands. Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) enable imaging fields of view in the centimeter scale whilst providing micrometer resolution. In this paper, we compare different optical clearing protocols and present reconstructions of the whole body of Plasmodium-infected, optically cleared Anopheles stephensi mosquitoes and their midguts. The 3D-reconstructions from OPT imaging show detailed features of the mosquito anatomy and enable overall localization of parasites in midguts. Additionally, LSFM imaging of mosquito midguts shows detailed distribution of oocysts in extracted midguts. This work was submitted as a pre-print to bioRxiv, available at https://www.biorxiv.org/content/10.1101/682054v2.
Assuntos
Anopheles/fisiologia , Imageamento Tridimensional , Insetos Vetores/fisiologia , Microscopia de Fluorescência , Plasmodium/fisiologia , Tomografia Óptica , Animais , Anopheles/parasitologiaRESUMO
A recent report by Jennison et al. reveals an important role for plasmepsin V (PMV), an aspartyl protease, in the development of malaria transmission stages. The authors showed that PMV activity is critical for protein export in these stages and that specific PMV inhibitors block parasite transmission to mosquitoes.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Malária/parasitologia , Malária/transmissão , Plasmodium/enzimologia , Animais , Antimaláricos/farmacologia , Carbamatos/farmacologia , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/fisiologia , Malária/prevenção & controle , Oligopeptídeos/farmacologia , Plasmodium/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacosRESUMO
Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. However, in the malaria parasite Plasmodium falciparum both the function of HP1 phosphorylation and the identity of the protein kinases targeting HP1 are still elusive. In order to functionally analyze phosphorylation of P. falciparum HP1 (PfHP1), we first mapped PfHP1 phosphorylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of native PfHP1, which identified motifs from which potential kinases could be predicted; in particular, several phosphorylated residues were embedded in motifs rich in acidic residues, reminiscent of targets for P. falciparum casein kinase 2 (PfCK2). Secondly, we tested recombinant PfCK2 and a number of additional protein kinases for their ability to phosphorylate PfHP1 in in vitro kinase assays. These experiments validated our prediction that PfHP1 acts as a substrate for PfCK2. Furthermore, LC-MS/MS analysis showed that PfCK2 phosphorylates three clustered serine residues in an acidic motif within the central hinge region of PfHP1. To study the role of PfHP1 phosphorylation in live parasites we used CRISPR/Cas9-mediated genome editing to generate a number of conditional PfHP1 phosphomutants based on the DiCre/LoxP system. Our studies revealed that neither PfCK2-dependent phosphorylation of PfHP1, nor phosphorylation of the hinge domain in general, affect PfHP1's ability to localize to heterochromatin, and that PfHP1 phosphorylation in this region is dispensable for the proliferation of P. falciparum blood stage parasites.
Assuntos
Caseína Quinase II/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Malária Falciparum/parasitologia , Plasmodium falciparum/isolamento & purificação , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Caseína Quinase II/genética , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Humanos , Malária Falciparum/metabolismo , Mutação , Fosforilação , Proteínas de Protozoários/genéticaRESUMO
The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the Plasmodium falciparum protein kinase PfCLK3, which we used in combination with chemogenetics to validate PfCLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for PfCLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of PfCLK3 mediated rapid killing of asexual liver- and blood-stage P. falciparum and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against P. berghei and P. knowlesi Hence, our data establish PfCLK3 as a target for drugs, with the potential to offer a cure-to be prophylactic and transmission blocking in malaria.
Assuntos
Antimaláricos/farmacologia , Terapia de Alvo Molecular , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antimaláricos/química , Antimaláricos/isolamento & purificação , Antimaláricos/uso terapêutico , Gametogênese/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Inibidores de Proteínas Quinases/isolamento & purificação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas de Protozoários/genética , Splicing de RNA/genética , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Intravital microscopy allows the visualisation of how pathogens interact with host cells and tissues in living animals in real time. This method has enabled key advances in our understanding of host-parasite interactions under physiological conditions. A combination of genetics, microscopy techniques, and image analysis have recently facilitated the understanding of biological phenomena in living animals at cellular and subcellular resolution. In this review, we summarise findings achieved by intravital microscopy of the skin and adipose tissues upon infection with various parasites, and we present a view into possible future applications of this method.
Assuntos
Tecido Adiposo/diagnóstico por imagem , Tecido Adiposo/parasitologia , Interações Hospedeiro-Parasita , Microscopia Intravital/métodos , Pele/diagnóstico por imagem , Pele/parasitologia , Tecido Adiposo/citologia , Tecido Adiposo/patologia , Animais , Microscopia Intravital/tendências , Leishmania/metabolismo , Leishmania/patogenicidade , Plasmodium/metabolismo , Plasmodium/patogenicidade , Schistosoma/metabolismo , Schistosoma/patogenicidade , Pele/citologia , Pele/patologia , Trypanosoma/metabolismo , Trypanosoma/patogenicidadeRESUMO
Background: Malaria parasites go through major transitions during their complex life cycle, yet the underlying differentiation pathways remain obscure. Here we apply single cell transcriptomics to unravel the program inducing sexual differentiation in Plasmodium falciparum. Parasites have to make this essential life-cycle decision in preparation for human-to-mosquito transmission. Methods: By combining transcriptional profiling with quantitative imaging and genetics, we defined a transcriptional signature in sexually committed cells. Results: We found this transcriptional signature to be distinct from general changes in parasite metabolism that can be observed in response to commitment-inducing conditions. Conclusions: This proof-of-concept study provides a template to capture transcriptional diversity in parasite populations containing complex mixtures of different life-cycle stages and developmental programs, with important implications for our understanding of parasite biology and the ongoing malaria elimination campaign.
RESUMO
Transmission of Plasmodium parasites to the mosquito requires the formation and development of gametocytes. Studies in infected humans have shown that only the most mature forms of Plasmodium falciparum gametocytes are present in circulation, whereas immature forms accumulate in the hematopoietic environment of the bone marrow. We used the rodent model Plasmodium berghei to study gametocyte behavior through time under physiological conditions. Intravital microscopy demonstrated preferential homing of early gametocyte forms across the intact vascular barrier of the bone marrow and the spleen early during infection and subsequent development in the extravascular environment. During the acute phase of infection, we observed vascular leakage resulting in further parasite accumulation in this environment. Mature gametocytes showed high deformability and were found entering and exiting the intact vascular barrier. We suggest that extravascular gametocyte localization and mobility are essential for gametocytogenesis and transmission of Plasmodium to the mosquito.
Assuntos
Medula Óssea/parasitologia , Malária/patologia , Malária/parasitologia , Plasmodium/fisiologia , Migração Transendotelial e Transepitelial , Animais , Modelos Animais de Doenças , Interações Hospedeiro-Parasita , Humanos , Camundongos , Imagem Molecular , Sistema Fagocitário Mononuclear/parasitologiaRESUMO
Malaria is caused by Plasmodium parasites that proliferate in the bloodstream. During each replication cycle, some parasites differentiate into gametocytes, the only forms able to infect the mosquito vector and transmit malaria. Sexual commitment is triggered by activation of AP2-G, the master transcriptional regulator of gametocytogenesis. Heterochromatin protein 1 (HP1)-dependent silencing of ap2-g prevents sexual conversion in proliferating parasites. In this study, we identified Plasmodium falciparum gametocyte development 1 (GDV1) as an upstream activator of sexual commitment. We found that GDV1 targeted heterochromatin and triggered HP1 eviction, thus derepressing ap2-g Expression of GDV1 was responsive to environmental triggers of sexual conversion and controlled via a gdv1 antisense RNA. Hence, GDV1 appears to act as an effector protein that induces sexual differentiation by antagonizing HP1-dependent gene silencing.