RESUMEN
Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.
Asunto(s)
Vacunas contra la Malaria , Malaria , Animales , Ratones , Células T de Memoria , Malaria/prevención & control , Hígado , Plasmodium berghei/genética , Linfocitos T CD8-positivosRESUMEN
Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle. We discover 461 genes required for efficient parasite transmission to mosquitoes through the liver stage and back into the bloodstream of mice. We analyze the screen in the context of genomic, transcriptomic, and metabolomic data by building a thermodynamic model of P. berghei liver-stage metabolism, which shows a major reprogramming of parasite metabolism to achieve rapid growth in the liver. We identify seven metabolic subsystems that become essential at the liver stages compared with asexual blood stages: type II fatty acid synthesis and elongation (FAE), tricarboxylic acid, amino sugar, heme, lipoate, and shikimate metabolism. Selected predictions from the model are individually validated in single mutants to provide future targets for drug development.
Asunto(s)
Genoma de Protozoos , Estadios del Ciclo de Vida/genética , Hígado/metabolismo , Hígado/parasitología , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/genética , Alelos , Amino Azúcares/biosíntesis , Animales , Culicidae/parasitología , Eritrocitos/parasitología , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/metabolismo , Técnicas de Inactivación de Genes , Genotipo , Modelos Biológicos , Mutación/genética , Parásitos/genética , Parásitos/crecimiento & desarrollo , Fenotipo , Plasmodium berghei/metabolismo , Ploidias , ReproducciónRESUMEN
Hereditary xerocytosis is thought to be a rare genetic condition characterized by red blood cell (RBC) dehydration with mild hemolysis. RBC dehydration is linked to reduced Plasmodium infection in vitro; however, the role of RBC dehydration in protection against malaria in vivo is unknown. Most cases of hereditary xerocytosis are associated with gain-of-function mutations in PIEZO1, a mechanically activated ion channel. We engineered a mouse model of hereditary xerocytosis and show that Plasmodium infection fails to cause experimental cerebral malaria in these mice due to the action of Piezo1 in RBCs and in T cells. Remarkably, we identified a novel human gain-of-function PIEZO1 allele, E756del, present in a third of the African population. RBCs from individuals carrying this allele are dehydrated and display reduced Plasmodium infection in vitro. The existence of a gain-of-function PIEZO1 at such high frequencies is surprising and suggests an association with malaria resistance.
Asunto(s)
Anemia Hemolítica Congénita/patología , Población Negra/genética , Hidropesía Fetal/patología , Canales Iónicos/genética , Malaria/patología , Alelos , Anemia Hemolítica Congénita/genética , Animales , Deshidratación , Modelos Animales de Enfermedad , Eritrocitos/citología , Eritrocitos/metabolismo , Eliminación de Gen , Genotipo , Humanos , Hidropesía Fetal/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/deficiencia , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales Iónicos/química , Malaria/genética , Malaria/parasitología , Malaria/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/patogenicidad , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine.
Asunto(s)
Genoma de Protozoos , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/genética , Animales , Evolución Biológica , Femenino , Técnicas de Inactivación de Genes , Genes Esenciales , Interacciones Huésped-Parásitos , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos BALB C , Plasmodium berghei/metabolismo , Saccharomyces cerevisiae/genética , Toxoplasma/genética , Trypanosoma brucei brucei/genéticaRESUMEN
Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.
Asunto(s)
Lisofosfatidilcolinas/metabolismo , Malaria/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Animales , Femenino , Humanos , Malaria/inmunología , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/fisiología , ReproducciónRESUMEN
Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Malaria Cerebral/inmunología , Inflamación Neurogénica/inmunología , Factores de Transcripción/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Proteínas de Unión al ADN/genética , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Células HEK293 , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Malaria Cerebral/tratamiento farmacológico , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Terapia Molecular Dirigida , Glicoproteína Mielina-Oligodendrócito/inmunología , Inflamación Neurogénica/tratamiento farmacológico , Fragmentos de Péptidos/inmunología , Plasmodium berghei/inmunología , Factores de Transcripción/genética , Proteasas Ubiquitina-Específicas/genéticaRESUMEN
Malaria infection involves an obligatory, yet clinically silent liver stage1,2. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis3, but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing4 and single-molecule transcript imaging5 to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These 'abortive hepatocytes', which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte.
Asunto(s)
Regulación de la Expresión Génica , Hepatocitos , Hígado , Malaria , Parásitos , Plasmodium berghei , Análisis de la Célula Individual , Animales , Hepatocitos/citología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Hepatocitos/parasitología , Hígado/anatomía & histología , Hígado/citología , Hígado/inmunología , Hígado/parasitología , Malaria/genética , Malaria/inmunología , Malaria/parasitología , Parásitos/genética , Parásitos/inmunología , Parásitos/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/inmunología , Plasmodium berghei/metabolismo , Imagen Individual de Molécula , Análisis de Secuencia de ARN , Hierro/metabolismo , Ácidos Grasos/metabolismo , Transcripción Genética , Genes Protozoarios/genética , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunologíaRESUMEN
The malaria parasite life cycle alternates between two hosts: a vertebrate and the female Anopheles mosquito vector. Cell division, proliferation, and invasion are essential for parasite development, transmission, and survival. Most research has focused on Plasmodium development in the vertebrate, which causes disease; however, knowledge of malaria parasite development in the mosquito (the sexual and transmission stages) is now rapidly accumulating, gathered largely through investigation of the rodent malaria model, with Plasmodium berghei. In this review, we discuss the seminal genome-wide screens that have uncovered key regulators of cell proliferation, invasion, and transmission during Plasmodium sexual development. Our focus is on the roles of transcription factors, reversible protein phosphorylation, and molecular motors. We also emphasize the still-unanswered important questions around key pathways in cell division during the vector transmission stages and how they may be targeted in future studies.
Asunto(s)
Anopheles , Malaria , Parásitos , Animales , Anopheles/parasitología , Femenino , Malaria/parasitología , Mosquitos Vectores , Plasmodium berghei/genéticaRESUMEN
Cerebral malaria is a deadly complication of Plasmodium infection and involves blood brain barrier (BBB) disruption following infiltration of white blood cells. During experimental cerebral malaria (ECM), mice inoculated with Plasmodium berghei ANKA-infected red blood cells develop a fatal CM-like disease caused by CD8+ T cell-mediated pathology. We found that treatment with interleukin-15 complex (IL-15C) prevented ECM, whereas IL-2C treatment had no effect. IL-15C-expanded natural killer (NK) cells were necessary and sufficient for protection against ECM. IL-15C treatment also decreased CD8+ T cell activation in the brain and prevented BBB breakdown without influencing parasite load. IL-15C induced NK cells to express IL-10, which was required for IL-15C-mediated protection against ECM. Finally, we show that ALT-803, a modified human IL-15C, mediates similar induction of IL-10 in NK cells and protection against ECM. These data identify a regulatory role for cytokine-stimulated NK cells in the prevention of a pathogenic immune response.
Asunto(s)
Interleucina-10/inmunología , Interleucina-15/inmunología , Células Asesinas Naturales/inmunología , Malaria Cerebral/inmunología , Plasmodium berghei/inmunología , Proteínas/farmacología , Animales , Barrera Hematoencefálica/patología , Encéfalo/inmunología , Encéfalo/patología , Linfocitos T CD8-positivos/inmunología , Interleucina-10/biosíntesis , Activación de Linfocitos/inmunología , Malaria Cerebral/microbiología , Malaria Cerebral/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes de FusiónRESUMEN
Plasmodium sporozoites are the infective forms of the malaria parasite in the mosquito and vertebrate host. Gliding motility allows sporozoites to migrate and invade mosquito salivary glands and mammalian hosts. Motility and invasion are powered by an actin-myosin motor complex linked to the glideosome, which contains glideosome-associated proteins (GAPs), MyoA and the myosin A tail-interacting protein (MTIP). However, the role of several proteins involved in gliding motility remains unknown. We identified that the S14 gene is upregulated in sporozoite from transcriptome data of Plasmodium yoelii and further confirmed its transcription in P. berghei sporozoites using real-time PCR. C-terminal 3×HA-mCherry tagging revealed that S14 is expressed and localized on the inner membrane complex of the sporozoites. We disrupted S14 in P. berghei and demonstrated that it is essential for sporozoite gliding motility, and salivary gland and hepatocyte invasion. The gliding and invasion-deficient S14 knockout sporozoites showed normal expression and organization of inner membrane complex and surface proteins. Taken together, our data show that S14 plays a role in the function of the glideosome and is essential for malaria transmission.
Asunto(s)
Malaria , Plasmodium berghei , Proteínas Protozoarias , Esporozoítos , Esporozoítos/metabolismo , Plasmodium berghei/metabolismo , Plasmodium berghei/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Animales , Ratones , Malaria/parasitología , Glándulas Salivales/parasitología , Glándulas Salivales/metabolismo , Anopheles/parasitologíaRESUMEN
Leucine-rich repeat (LRR) proteins are commonly involved in innate immunity of animals and plants, including for pattern recognition of pathogen-derived elicitors. The Anopheles secreted LRR proteins APL1C and LRIM1 are required for malaria ookinete killing in conjunction with the complement-like TEP1 protein. However, the mechanism of parasite immune recognition by the mosquito remains unclear, although it is known that TEP1 lacks inherent binding specificity. Here, we find that APL1C and LRIM1 bind specifically to Plasmodium berghei ookinetes, even after depletion of TEP1 transcript and protein, consistent with a role for the LRR proteins in pathogen recognition. Moreover, APL1C does not bind to ookinetes of the human malaria parasite Plasmodium falciparum, and is not required for killing of this parasite, which correlates LRR binding specificity and immune protection. Most of the live P. berghei ookinetes that migrated into the extracellular space exposed to mosquito hemolymph, and almost all dead ookinetes, are bound by APL1C, thus associating LRR protein binding with parasite killing. We also find that APL1C binds to the surface of P. berghei sporozoites released from oocysts into the mosquito hemocoel and forms a potent barrier limiting salivary gland invasion and mosquito infectivity. Pathogen binding by APL1C provides the first functional explanation for the long-known requirement of APL1C for P. berghei ookinete killing in the mosquito midgut. We propose that secreted mosquito LRR proteins are required for pathogen discrimination and orientation of immune effector activity, potentially as functional counterparts of the immunoglobulin-based receptors used by vertebrates for antigen recognition.
Asunto(s)
Anopheles , Malaria , Animales , Humanos , Proteínas Repetidas Ricas en Leucina , Anopheles/parasitología , Esporozoítos/metabolismo , Proteínas/metabolismo , Plasmodium berghei/metabolismoRESUMEN
Plasmodium parasites cause malaria, a global health disease that is responsible for more than 200 million clinical cases and 600 000 deaths each year. Most deaths are caused by various complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Despite the very rapid and efficient killing of parasites with antimalarial drugs, 15% of patients with complicated malaria succumb. This stresses the importance of investigating resolution mechanisms that are involved in the recovery from these complications once the parasite is killed. To study the resolution of MA-ARDS, P. berghei NK65-infected C57BL/6 mice were treated with antimalarial drugs after onset of symptoms, resulting in 80% survival. Micro-computed tomography revealed alterations of the lungs upon infection, with an increase in total and non-aerated lung volume due to edema. Whole body plethysmography confirmed a drastically altered lung ventilation, which was restored during resolution. Single-cell RNA sequencing indicated an increased inflammatory state in the lungs upon infection, which was accompanied by a drastic decrease in endothelial cells, consistent with CD8+ T cell-mediated killing. During resolution, anti-inflammatory pathways were upregulated and proliferation of endothelial cells was observed. MultiNicheNet interactome analysis identified important changes in the ligand-receptor interactions during disease resolution that warrant further exploration in order to develop new therapeutic strategies. In conclusion, our study provides insights in pro-resolving pathways that limit inflammation and promote endothelial cell proliferation in experimental MA-ARDS. This information may be useful for the design of adjunctive treatments to enhance resolution after Plasmodium parasite killing by antimalarial drugs.
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Antimaláricos , Malaria , Síndrome de Dificultad Respiratoria , Humanos , Animales , Ratones , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Células Endoteliales/metabolismo , Microtomografía por Rayos X/efectos adversos , Ratones Endogámicos C57BL , Síndrome de Dificultad Respiratoria/etiología , Síndrome de Dificultad Respiratoria/metabolismo , Malaria/parasitología , Análisis de Secuencia de ARN , Plasmodium bergheiRESUMEN
Immune response (Ir) genes, originally proposed by Baruj Benacerraf to explain differential antigen-specific responses in animal models, have become synonymous with the major histocompatibility complex (MHC). We discovered a non-MHC-linked Ir gene in a T cell receptor (TCR) locus that was required for CD8+ T cell responses to the Plasmodium berghei GAP5040-48 epitope in mice expressing the MHC class I allele H-2Db. GAP5040-48-specific CD8+ T cell responses emerged from a very large pool of naive Vß8.1+ precursors, which dictated susceptibility to cerebral malaria and conferred protection against recombinant Listeria monocytogenes infection. Structural analysis of a prototypical Vß8.1+ TCR-H-2Db-GAP5040-48 ternary complex revealed that germline-encoded complementarity-determining region 1ß residues present exclusively in the Vß8.1 segment mediated essential interactions with the GAP5040-48 peptide. Collectively, these findings demonstrated that Vß8.1 functioned as an Ir gene that was indispensable for immune reactivity against the malaria GAP5040-48 epitope.
Asunto(s)
Antígeno de Histocompatibilidad H-2D/genética , Plasmodium berghei/inmunología , Proteínas Protozoarias/inmunología , Receptores de Antígenos de Linfocitos T/genética , Animales , Linfocitos T CD8-positivos/inmunología , Regiones Determinantes de Complementariedad , Epítopos , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fragmentos de Péptidos/inmunologíaRESUMEN
Antibodies against the NANP repeat of circumsporozoite protein (CSP), the major surface antigen of Plasmodium falciparum (Pf) sporozoites, can protect from malaria in animal models but protective humoral immunity is difficult to induce in humans. Here we cloned and characterized rare affinity-matured human NANP-reactive memory B cell antibodies elicited by natural Pf exposure that potently inhibited parasite transmission and development in vivo. We unveiled the molecular details of antibody binding to two distinct protective epitopes within the NANP repeat. NANP repeat recognition was largely mediated by germline encoded and immunoglobulin (Ig) heavy-chain complementarity determining region 3 (HCDR3) residues, whereas affinity maturation contributed predominantly to stabilizing the antigen-binding site conformation. Combined, our findings illustrate the power of exploring human anti-CSP antibody responses to develop tools for malaria control in the mammalian and the mosquito vector and provide a molecular basis for the structure-based design of next-generation CSP malaria vaccines.
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Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Inmunidad Humoral , Cadenas Pesadas de Inmunoglobulina/inmunología , Malaria Falciparum/prevención & control , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/química , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Linfocitos B/inmunología , Linfocitos B/parasitología , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Femenino , Expresión Génica , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/química , Memoria Inmunológica , Malaria/inmunología , Malaria/parasitología , Malaria/prevención & control , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Masculino , Ratones , Modelos Moleculares , Plasmodium berghei/inmunología , Plasmodium falciparum/inmunología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esporozoítos/química , Esporozoítos/inmunologíaRESUMEN
Malaria, which results from infection with Plasmodium parasites, remains a major public health problem. Although humans do not develop long-lived, sterilizing immunity, protection against symptomatic disease develops after repeated exposure to Plasmodium parasites and correlates with the acquisition of humoral immunity. Despite the established role Abs play in protection from malaria disease, dysregulated inflammation is thought to contribute to the suboptimal immune response to Plasmodium infection. Plasmodium berghei ANKA (PbA) infection results in a fatal severe malaria disease in mice. We previously demonstrated that treatment of mice with IL-15 complex (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) induces IL-10 expression in NK cells, which protects mice from PbA-induced death. Using a novel MHC class II tetramer to identify PbA-specific CD4+ T cells, in this study we demonstrate that IL-15C treatment enhances T follicular helper (Tfh) differentiation and modulates cytokine production by CD4+ T cells. Moreover, genetic deletion of NK cell-derived IL-10 or IL-10R expression on T cells prevents IL-15C-induced Tfh differentiation. Additionally, IL-15C treatment results in increased anti-PbA IgG Ab levels and improves survival following reinfection. Overall, these data demonstrate that IL-15C treatment, via its induction of IL-10 from NK cells, modulates the dysregulated inflammation during Plasmodium infection to promote Tfh differentiation and Ab generation, correlating with improved survival from reinfection. These findings will facilitate improved control of malaria infection and protection from disease by informing therapeutic strategies and vaccine design.
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Malaria , Plasmodium , Ratones , Humanos , Animales , Interleucina-10/metabolismo , Interleucina-15/metabolismo , Formación de Anticuerpos , Reinfección , Linfocitos T CD4-Positivos , Linfocitos T Colaboradores-Inductores , Inflamación/metabolismo , Ratones Endogámicos C57BL , Plasmodium bergheiRESUMEN
Sickle human hemoglobin (Hb) confers a survival advantage to individuals living in endemic areas of malaria, the disease caused by Plasmodium infection. As demonstrated hereby, mice expressing sickle Hb do not succumb to experimental cerebral malaria (ECM). This protective effect is exerted irrespectively of parasite load, revealing that sickle Hb confers host tolerance to Plasmodium infection. Sickle Hb induces the expression of heme oxygenase-1 (HO-1) in hematopoietic cells, via a mechanism involving the transcription factor NF-E2-related factor 2 (Nrf2). Carbon monoxide (CO), a byproduct of heme catabolism by HO-1, prevents further accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of ECM. Moreover, sickle Hb inhibits activation and/or expansion of pathogenic CD8(+) T cells recognizing antigens expressed by Plasmodium, an immunoregulatory effect that does not involve Nrf2 and/or HO-1. Our findings provide insight into molecular mechanisms via which sickle Hb confers host tolerance to severe forms of malaria.
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Hemoglobina Falciforme/inmunología , Malaria/inmunología , Plasmodium berghei , Animales , Linfocitos T CD8-positivos/inmunología , Monóxido de Carbono/metabolismo , Quimiocinas/metabolismo , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Hemo-Oxigenasa 1/metabolismo , Interacciones Huésped-Patógeno , Humanos , Malaria/fisiopatología , Malaria Cerebral/inmunología , Malaria Cerebral/fisiopatología , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
Gametocyte development of the Plasmodium parasite is a key step for transmission of the parasite. Male and female gametocytes are produced from a subpopulation of asexual blood-stage parasites, but the mechanisms that regulate the differentiation of sexual stages are still under investigation. In this study, we investigated the role of PbARID, a putative subunit of a SWI/SNF chromatin remodeling complex, in transcriptional regulation during the gametocyte development of P. berghei. PbARID expression starts in early gametocytes before the manifestation of male and female-specific features, and disruption of its gene results in the complete loss of gametocytes with detectable male features and the production of abnormal female gametocytes. ChIP-seq analysis of PbARID showed that it forms a complex with gSNF2, an ATPase subunit of the SWI/SNF chromatin remodeling complex, associating with the male cis-regulatory element, TGTCT. Further ChIP-seq of PbARID in gsnf2-knockout parasites revealed an association of PbARID with another cis-regulatory element, TGCACA. RIME and DNA-binding assays suggested that HDP1 is the transcription factor that recruits PbARID to the TGCACA motif. Our results indicated that PbARID could function in two chromatin remodeling events and paly essential roles in both male and female gametocyte development.
Asunto(s)
Ensamble y Desensamble de Cromatina , Plasmodium berghei , Proteínas Protozoarias , Factores de Transcripción , Animales , Femenino , Masculino , Ratones , Ensamble y Desensamble de Cromatina/genética , Plasmodium berghei/genética , Plasmodium berghei/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Genotipo , Análisis de Secuencia de ARN , Cromatina/genética , Cromatina/metabolismo , Secuencia de Aminoácidos , Análisis de Secuencia de Proteína , Filogenia , Transcriptoma , Genoma de ProtozoosRESUMEN
Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins, which are channels facilitating the movement of water and other solutes across membranes. We identify an aquaporin in malaria parasites and demonstrate that it is important for completion of Plasmodium development in the mosquito vector. Disruption of AQP2 in the human parasite Plasmodium falciparum and the rodent parasite Plasmodium berghei blocks sporozoite production inside oocysts established on mosquito midguts, greatly limiting parasite infection of salivary glands and transmission to a new host. In vivo epitope tagging of AQP2 in P. berghei, combined with immunofluorescence assays, reveals that the protein is localized in vesicle-like organelles found in the cytoplasm of gametocytes, ookinetes, and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endomembrane system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely resembles intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature among known aquaporins. This in conjunction with the importance of AQP2 for malaria transmission suggests that AQP2 may be a fruitful target of antimalarial interventions.
Asunto(s)
Acuaporina 2 , Mosquitos Vectores , Proteínas Protozoarias , Animales , Malaria , Mosquitos Vectores/parasitología , Filogenia , Plasmodium berghei/metabolismo , Proteínas Protozoarias/metabolismo , Esporozoítos/metabolismoRESUMEN
Plasmodium sporozoites are the highly motile and invasive forms of the malaria parasite transmitted by mosquitoes. Sporozoites form within oocysts at the midgut wall of the mosquito, egress from oocysts and enter salivary glands prior to transmission. The GPI-anchored major surface protein, the circumsporozoite protein (CSP) is important for Plasmodium sporozoite formation, egress, migration and invasion. To visualize CSP, we previously generated full-length versions of CSP internally tagged with the green fluorescent protein, GFP. However, while these allowed for imaging of sporogony in oocysts, sporozoites failed to egress. Here, we explore different strategies to overcome this block in egress and obtain salivary gland resident sporozoites that express CSP-GFP. Replacing the N-terminal and repeat region with GFP did not allow sporozoite formation. Lowering expression of CSP-GFP at the endogenous locus allowed sporozoite formation but did not overcome egress block. Crossing of CSP-GFP expressing parasites that are blocked in egress with wild-type parasites yielded a small fraction of parasites that entered salivary glands and expressed various levels of CSP-GFP. Expressing CSP-GFP constructs from a silent chromosome region from promoters that are active only post salivary gland invasion yielded normal numbers of fluorescent salivary gland sporozoites, albeit with low levels of fluorescence. We also show that lowering CSP expression by 50% allowed egress from oocysts but not salivary gland entry. In conclusion, Plasmodium berghei parasites with normal CSP expression tolerate a certain level of CSP-GFP without disruption of oocyst egress and salivary gland invasion.
Asunto(s)
Anopheles , Esporozoítos , Animales , Esporozoítos/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Anopheles/parasitología , Oocistos , Plasmodium berghei/genética , Plasmodium berghei/metabolismoRESUMEN
Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver-stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre-erythrocytic immunity and hence its utility as a GAP.