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1.
PLoS Biol ; 19(12): e3001483, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34879056

RESUMEN

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.


Asunto(s)
Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/genética , Animales , Dominio Catalítico , Línea Celular , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Eritrocitos/parasitología , Humanos , Malaria , Malaria Falciparum/parasitología , Merozoítos , Parásitos/metabolismo , Proteínas Protozoarias/metabolismo
2.
Cell Microbiol ; 22(2): e13123, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31652487

RESUMEN

A hallmark of the biology of Plasmodium falciparum blood stage parasites is their extensive host cell remodelling, facilitated by parasite proteins that are exported into the erythrocyte. Although this area has received extensive attention, only a few exported parasite proteins have been analysed in detail, and much of this remodelling process remains unknown, particularly for gametocyte development. Recent advances to induce high rates of sexual commitment enable the production of large numbers of gametocytes. We used this approach to study the Plasmodium helical interspersed subtelomeric (PHIST) protein GEXP02, which is expressed during sexual development. We show by immunofluorescence that GEXP02 is exported to the gametocyte-infected host cell periphery. Co-immunoprecipitation revealed potential interactions between GEXP02 and components of the erythrocyte cytoskeleton as well as other exported parasite proteins. This indicates that GEXP02 targets the erythrocyte cytoskeleton and is likely involved in its remodelling. GEXP02 knock-out parasites show no obvious phenotype during gametocyte maturation, transmission through mosquitoes, and hepatocyte infection, suggesting auxiliary or redundant functions for this protein. In summary, we performed a detailed cellular and biochemical analysis of a sexual stage-specific exported parasite protein using a novel experimental approach that is broadly applicable to study the biology of P. falciparum gametocytes.


Asunto(s)
Membrana Eritrocítica/metabolismo , Células Germinativas/citología , Malaria Falciparum/parasitología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/fisiología , Interacciones Huésped-Parásitos , Humanos
3.
FASEB J ; 33(12): 14611-14624, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31690116

RESUMEN

Plasmodium falciparum is the most lethal of human-infective malaria parasites. A hallmark of P. falciparum malaria is extensive remodeling of host erythrocytes by the parasite, which facilitates the development of virulence properties such as host cell adhesion to the endothelial lining of the microvasculature. Host remodeling is mediated by a large complement of parasite proteins exported to the erythrocyte; among them is a single heat shock protein (Hsp)70-class protein chaperone, P. falciparum Hsp70-x (PfHsp70-x). PfHsp70-x was previously shown to assist the development of virulent cytoadherence characteristics. Here, we show that PfHsp70-x also supports parasite growth under elevated temperature conditions that simulate febrile episodes, especially at the beginning of the parasite life cycle when most of host cell remodeling takes place. Biochemical and biophysical analyses of PfHsp70-x, including crystallographic structures of its catalytic domain and the J-domain of its stimulatory Hsp40 cochaperone, suggest that PfHsp70-x is highly similar to human Hsp70 chaperones endogenous to the erythrocyte. Nevertheless, our results indicate that selective inhibition of PfHsp70-x function using small molecules may be possible and highlight specific sites of its catalytic domain as potentially of high interest. We discuss the likely roles of PfHsp70-x and human chaperones in P. falciparum biology and how specific inhibitors may assist us in disentangling their relative contributions.-Day, J., Passecker, A., Beck, H.-P., Vakonakis, I. The Plasmodium falciparum Hsp70-x chaperone assists the heat stress response of the malaria parasite.


Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas HSP70 de Choque Térmico/química , Dominios Proteicos , Proteínas Protozoarias/química
4.
ACS Infect Dis ; 10(4): 1286-1297, 2024 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-38556981

RESUMEN

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.


Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Malaria , Humanos , Antimaláricos/farmacología , Plasmodium falciparum , Malaria/tratamiento farmacológico , Malaria Falciparum/parasitología , Antagonistas del Ácido Fólico/farmacología , Galactósidos/farmacología , Galactósidos/uso terapéutico
5.
Elife ; 122023 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-37934560

RESUMEN

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.


Asunto(s)
Antimaláricos , Malaria Falciparum , Humanos , Tacrolimus , Antibacterianos , Sistemas de Liberación de Medicamentos , Homeostasis , Proteínas de Unión a Tacrolimus
6.
Front Cell Infect Microbiol ; 12: 802341, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35223540

RESUMEN

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.


Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Parásitos , Animales , Antimaláricos/farmacología , Humanos , Malaria/parasitología , Malaria Falciparum/parasitología , Preparaciones Farmacéuticas , Plasmodium falciparum
7.
mSphere ; 6(1)2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33536327

RESUMEN

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.


Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Silenciador del Gen , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Variación Antigénica , Sistemas CRISPR-Cas , Línea Celular , Homólogo de la Proteína Chromobox 5 , Eritrocitos/parasitología , Regulación de la Expresión Génica , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo
8.
Commun Biol ; 4(1): 336, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712726

RESUMEN

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.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Eritrocitos/parasitología , Gametogénesis , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Antimaláricos/farmacología , Sistemas CRISPR-Cas , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/genética , Dominio Catalítico , Edición Génica , Humanos , Estadios del Ciclo de Vida , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Protozoarias/genética , Reproducción Asexuada
9.
Nat Commun ; 12(1): 4806, 2021 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-34376675

RESUMEN

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.


Asunto(s)
Sistemas CRISPR-Cas , Malaria Falciparum/sangre , Plasmodium falciparum/genética , Esporas Protozoarias/genética , Animales , Anopheles/parasitología , Células Cultivadas , Eritrocitos/parasitología , Hepatocitos/citología , Hepatocitos/parasitología , Interacciones Huésped-Parásitos , Humanos , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Microscopía Fluorescente , Mosquitos Vectores/parasitología , Plasmodium falciparum/fisiología , Esporas Protozoarias/fisiología , Esporozoítos/genética , Esporozoítos/fisiología
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