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1.
PLoS Pathog ; 16(8): e1008131, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866196

RESUMO

Invasion of hepatocytes by Plasmodium sporozoites initiates the pre-erythrocytic step of a malaria infection. Subsequent development of the parasite within hepatocytes and exit from them is essential for starting the disease-causing erythrocytic cycle. Identification of signaling pathways that operate in pre-erythrocytic stages provides insight into a critical step of infection and potential targets for chemoprotection from malaria. We demonstrate that P. berghei homologs of Calcium Dependent Protein Kinase 1 (CDPK1), CDPK4 and CDPK5 play overlapping but distinct roles in sporozoite invasion and parasite egress from hepatocytes. All three kinases are expressed in sporozoites. All three are required for optimal motility of sporozoites and consequently their invasion of hepatocytes. Increased cGMP can compensate for the functional loss of CDPK1 and CDPK5 during sporozoite invasion but cannot overcome loss of CDPK4. CDPK1 and CDPK5 expression is downregulated after sporozoite invasion. CDPK5 reappears in a subset of late stage liver stages and is present in all merosomes. Chemical inhibition of CDPK4 and depletion of CDPK5 in liver stages implicate these kinases in the formation and/or release of merosomes from mature liver stages. Furthermore, depletion of CDPK5 in merosomes significantly delays initiation of the erythrocytic cycle without affecting infectivity of hepatic merozoites. These data suggest that CDPK5 may be required for the rupture of merosomes. Our work provides evidence that sporozoite invasion requires CDPK1 and CDPK5, and suggests that CDPK5 participates in the release of hepatic merozoites.


Assuntos
Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Malária/epidemiologia , Merozoítos/enzimologia , Plasmodium berghei/enzimologia , Proteínas Quinases/biossíntese , Proteínas de Protozoários/biossíntese , Esporozoítos/enzimologia , Animais , Eritrócitos/enzimologia , Eritrócitos/parasitologia , Feminino , Células Hep G2 , Humanos , Fígado/enzimologia , Fígado/parasitologia , Malária/patologia , Camundongos
2.
Trends Parasitol ; 36(11): 914-926, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32958385

RESUMO

Research on Plasmodium parasites has driven breakthroughs in reducing malaria morbidity and mortality. Experimental analysis of in vivo/ex vivo versus in vitro samples serve unique roles in Plasmodium research. However, these distinctly different environments lead to discordant biology between parasites in host circulation and those under laboratory cultivation. Here, we review how in vitro factors, such as nutrient levels and physical forces, differ from those in the human host and the resulting implications for parasite growth, survival, and virulence. Additionally, we discuss the current utility of direct-from-host methodologies, which avoid the potentially confounding effects of in vitro cultivation. Finally, we make the case for methodological improvements that will drive research progress of physiologically relevant phenotypes.


Assuntos
Interações Hospedeiro-Parasita , Malária/parasitologia , Plasmodium/fisiologia , Plasmodium/patogenicidade , Animais , Células Cultivadas , Eritrócitos/parasitologia , Humanos , Técnicas In Vitro
3.
Nature ; 585(7826): 579-583, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939086

RESUMO

Malaria has had a major effect on the human genome, with many protective polymorphisms-such as the sickle-cell trait-having been selected to high frequencies in malaria-endemic regions1,2. The blood group variant Dantu provides 74% protection against all forms of severe malaria in homozygous individuals3-5, a similar degree of protection to that afforded by the sickle-cell trait and considerably greater than that offered by the best malaria vaccine. Until now, however, the protective mechanism has been unknown. Here we demonstrate the effect of Dantu on the ability of the merozoite form of the malaria parasite Plasmodium falciparum to invade red blood cells (RBCs). We find that Dantu is associated with extensive changes to the repertoire of proteins found on the RBC surface, but, unexpectedly, inhibition of invasion does not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we find a strong link between RBC tension and merozoite invasion, and identify a tension threshold above which invasion rarely occurs, even in non-Dantu RBCs. Dantu RBCs have higher average tension than non-Dantu RBCs, meaning that a greater proportion resist invasion. These findings provide both an explanation for the protective effect of Dantu, and fresh insight into why the efficiency of P. falciparum invasion might vary across the heterogenous populations of RBCs found both within and between individuals.


Assuntos
Antígenos de Grupos Sanguíneos/genética , Eritrócitos/citologia , Eritrócitos/parasitologia , Malária Falciparum/patologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/metabolismo , Polimorfismo Genético , Antígenos de Grupos Sanguíneos/classificação , Antígenos de Grupos Sanguíneos/metabolismo , Criança , Eritrócitos/metabolismo , Eritrócitos/patologia , Feminino , Genótipo , Humanos , Quênia , Ligantes , Masculino , Merozoítos/metabolismo , Merozoítos/patogenicidade , Microscopia de Vídeo , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/patogenicidade
4.
Nat Commun ; 11(1): 3825, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732874

RESUMO

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.


Assuntos
Lipídeos , Malária Falciparum/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/parasitologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Transporte Proteico , Vacúolos/metabolismo , Vacúolos/parasitologia
5.
J Vis Exp ; (162)2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32831311

RESUMO

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.


Assuntos
Anticorpos Antiprotozoários/análise , Bioensaio/métodos , Citometria de Fluxo/métodos , Parasitos/imunologia , Fagocitose , Plasmodium falciparum/imunologia , Animais , Antígenos de Protozoários/imunologia , Eritrócitos/parasitologia , Feminino , Humanos , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Proteínas Opsonizantes/metabolismo , Gravidez , Receptores Fc/metabolismo , Reprodutibilidade dos Testes , Células THP-1
6.
Exp Parasitol ; 218: 107969, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32858043

RESUMO

Invasion of human red blood cells (RBCs) by Plasmodium parasites is a crucial yet poorly characterised phenotype. Two-color flow cytometry (2cFCM) promises to be a very sensitive and high throughput method for phenotyping parasite invasion. However, current protocols require high (~1.0%) parasitemia for assay set-up and need to be adapted for low parasitemia samples, which are becoming increasingly common in low transmission settings. Background fluorescence from nuclei-containing uninfected RBCs and high autologous reinvasion rates (merozoite invasion of donor uninfected RBCs present at 50% assay volume) are some of the limitations to the method's sensitivity to enumerate low parasitemia (<0.5%) with nucleic acid-based stains. Here, we describe modifications for plating unlabeled donor to labeled target RBCs per assay well and for gating parasitemia, that produces accurate quantifications of low reinvasion parasitemia. Plasmodium falciparum 3D7, Dd2 and field isolates at various low and high parasitemia (0.05%-2.0%) were used to set-up SyBr Green 1-based 2cFCM invasion assays. Target RBCs were labeled with CTFR proliferation dye. We show that this dye combination allowed for efficient parasite invasion into target RBCs and that a 1:3 ratio of unlabeled to labeled RBCs per assay greatly skewed autologous reinvasion (p < 0.001). Accuracy of quantifying reinvasion was limited to an assay parasitemia of 0.02% with minimal background interference. Invasion inhibition by enzymatic treatments increased averagely by 10% (p<0.05) across the entire parasitemia range. The effect was greater for samples with <0.5% parasitemia. Overall, a more sensitive method for phenotyping invasion of low P. falciparum parasitemia is described.


Assuntos
Citometria de Fluxo/métodos , Malária Falciparum/parasitologia , Parasitemia/parasitologia , Plasmodium falciparum/isolamento & purificação , Rastreamento de Células/métodos , Corantes , Eritrócitos/parasitologia , Humanos , Fenótipo , Plasmodium falciparum/classificação , Plasmodium falciparum/fisiologia , Recidiva , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
7.
Nat Commun ; 11(1): 4015, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782246

RESUMO

Intracellular pathogens mobilize host signaling pathways of their host cell to promote their own survival. Evidence is emerging that signal transduction elements are activated in a-nucleated erythrocytes in response to infection with malaria parasites, but the extent of this phenomenon remains unknown. Here, we fill this knowledge gap through a comprehensive and dynamic assessment of host erythrocyte signaling during infection with Plasmodium falciparum. We used arrays of 878 antibodies directed against human signaling proteins to interrogate the activation status of host erythrocyte phospho-signaling pathways at three blood stages of parasite asexual development. This analysis reveals a dynamic modulation of many host signalling proteins across parasite development. Here we focus on the hepatocyte growth factor receptor (c-MET) and the MAP kinase pathway component B-Raf, providing a proof of concept that human signaling kinases identified as activated by malaria infection represent attractive targets for antimalarial intervention.


Assuntos
Antimaláricos/farmacologia , Eritrócitos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Concentração Inibidora 50 , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Fosforilação/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Plasmodium falciparum/fisiologia , Análise Serial de Proteínas , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
PLoS Pathog ; 16(8): e1008230, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797076

RESUMO

Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.


Assuntos
Eritrócitos/imunologia , Armadilhas Extracelulares/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Malária/imunologia , Neutrófilos/imunologia , Plasmodium/imunologia , Receptores CXCR4/metabolismo , Animais , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/parasitologia , Humanos , Malária/metabolismo , Malária/parasitologia , Malária/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/parasitologia , Parasitemia/imunologia , Parasitemia/metabolismo , Parasitemia/parasitologia , Parasitemia/patologia
9.
PLoS One ; 15(7): e0236375, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726331

RESUMO

BACKGROUND: Malaria in pregnancy causes maternal, fetal and neonatal morbidity and mortality, and maternal innate immune responses are implicated in pathogenesis of these complications. The effects of malaria exposure and obstetric and demographic factors on the early maternal immune response are poorly understood. METHODS: Peripheral blood mononuclear cell responses to Plasmodium falciparum-infected erythrocytes and phytohemagglutinin were compared between pregnant women from Papua New Guinea (malaria-exposed) with and without current malaria infection and from Australia (unexposed). Elicited levels of inflammatory cytokines at 48 h and 24 h (interferon γ, IFN-γ only) and the cellular sources of IFN-γ were analysed. RESULTS: Among Papua New Guinean women, microscopic malaria at enrolment did not alter peripheral blood mononuclear cell responses. Compared to samples from Australia, cells from Papua New Guinean women secreted more inflammatory cytokines tumor necrosis factor-α, interleukin 1ß, interleukin 6 and IFN-γ; p<0.001 for all assays, and more natural killer cells produced IFN-γ in response to infected erythrocytes and phytohemagglutinin. In both populations, cytokine responses were not affected by gravidity, except that in the Papua New Guinean cohort multigravid women had higher IFN-γ secretion at 24 h (p = 0.029) and an increased proportion of IFN-γ+ Vδ2 γδ T cells (p = 0.003). Cytokine levels elicited by a pregnancy malaria-specific CSA binding parasite line, CS2, were broadly similar to those elicited by CD36-binding line P6A1. CONCLUSIONS: Geographic location and, to some extent, gravidity influence maternal innate immunity to malaria.


Assuntos
Imunidade Inata/genética , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Complicações Parasitárias na Gravidez/imunologia , Adolescente , Adulto , Austrália/epidemiologia , Antígenos CD36/genética , Eritrócitos/imunologia , Eritrócitos/parasitologia , Eritrócitos/patologia , Feminino , Número de Gestações/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-6/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/parasitologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Leucócitos Mononucleares/patologia , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Pessoa de Meia-Idade , Papua Nova Guiné/epidemiologia , Plasmodium falciparum/patogenicidade , Gravidez , Complicações Parasitárias na Gravidez/parasitologia , Complicações Parasitárias na Gravidez/patologia , Linfócitos T/imunologia , Linfócitos T/parasitologia , Adulto Jovem
10.
Nat Commun ; 11(1): 3532, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669539

RESUMO

Asexual proliferation of the Plasmodium parasites that cause malaria follows a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. By phosphoproteomic and chemical-genetic analysis, we isolate two functional targets of PfPP1 for egress: a HECT E3 protein-ubiquitin ligase; and GCα, a fusion protein composed of a guanylyl cyclase and a phospholipid transporter domain. We hypothesize that PfPP1 regulates lipid sensing by GCα and find that phosphatidylcholine stimulates PfPP1-dependent egress. PfPP1 acts as a key regulator that integrates multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells.


Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/enzimologia , Proteína Fosfatase 1/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Proliferação de Células , GMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Fosfatidilcolinas/química , Domínios Proteicos , Proteoma , Ubiquitina-Proteína Ligases/metabolismo
11.
PLoS One ; 15(7): e0235798, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673324

RESUMO

During the course of the asexual erythrocytic stage of development, Plasmodium spp. parasites undergo a series of morphological changes and induce alterations in the host cell. At the end of this stage, the parasites egress from the infected cell, after which the progeny invade a new host cell. These processes are rapid and occur in a time-dependent manner. Of particular importance, egress and invasion of erythrocytes by the parasite are difficult to capture in an unsynchronized culture, or even a culture that has been synchronized within a window of one to several hours. Therefore, precise synchronization of parasite cultures is of paramount importance for the investigation of these processes. Here we describe a method for synchronizing Plasmodium falciparum and Plasmodium knowlesi asexual blood stage parasites with ML10, a highly specific inhibitor of the cGMP-dependent protein kinase (PKG) that arrests parasite growth approximately 15 minutes prior to egress. This inhibitor allows parasite cultures to be synchronized so that all parasites are within a window of development of several minutes, with a simple wash step. Furthermore, we show that parasites remain viable for several hours after becoming arrested by the compound and that ML10 has advantages, owing to its high specificity and low EC50, over the previously used PKG inhibitor Compound 2. Here, we demonstrate that ML10 is an invaluable tool for the study of Plasmodium spp. asexual blood stage biology and for the routine synchronization of P. falciparum and P. knowlesi cultures.


Assuntos
Eritrócitos/parasitologia , Malária/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium knowlesi/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium knowlesi/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Fatores de Tempo
12.
PLoS Pathog ; 16(6): e1008485, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32589689

RESUMO

Ozonide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel), are synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a "multi-omics" workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of ozonide-treated P. falciparum infected red blood cells revealed a rapid depletion of short Hb-derived peptides followed by subsequent alterations in lipid and nucleotide metabolism, while untargeted peptidomics showed accumulation of longer Hb-derived peptides. Quantitative proteomics and ABPP assays demonstrated that Hb-digesting proteases were increased in abundance and activity following treatment, respectively. Ozonide-induced depletion of short Hb-derived peptides was less extensive in a drug-treated K13-mutant artemisinin resistant parasite line (Cam3.IIR539T) than in the drug-treated isogenic sensitive strain (Cam3.IIrev), further confirming the association between ozonide activity and Hb catabolism. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in ozonide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short ozonide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate ozonide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage ozonide-induced damage.


Assuntos
Adamantano/análogos & derivados , Antimaláricos/farmacologia , Eritrócitos , Hemoglobinas/metabolismo , Compostos Heterocíclicos com 1 Anel/farmacologia , Peróxidos/farmacologia , Plasmodium falciparum/metabolismo , Compostos de Espiro/farmacologia , Adamantano/farmacologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Hemoglobinas/genética , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Plasmodium falciparum/genética , Proteômica
13.
Parasitol Res ; 119(8): 2659-2666, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32529297

RESUMO

Blood samples from 72 Ameiva ameiva lizards from Central Amazonian upland forests were collected, and thin smears of 40 (55.5%) animals were positive for gamonts of Hepatozoon with a mean level of intensity of infection of 14 parasites/2000 blood erythrocytes (0.73%). The gametocytes were found attached with host cells' nuclei, and their dimensions were 14.28 ± 1.05 µm in length and 4.50 ± 0.80 µm in width. Phylogenetic analyses of the 18S rRNA gene showed that the new sequences obtained from A. ameiva constitute a monophyletic sister clade to the Hepatozoon spp. from Brazilian snakes. Based on morphological features and new molecular data, we redescribe this hemogregarine as Hepatozoon ameivae. This study also provides the first molecular characterization of a Hepatozoon species from a Brazilian lizard.


Assuntos
Coccidiose/veterinária , Eucoccidiida/classificação , Lagartos/parasitologia , Animais , Brasil , Coccidiose/parasitologia , Eritrócitos/parasitologia , Eucoccidiida/citologia , Eucoccidiida/genética , Eucoccidiida/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Carga Parasitária , Filogenia , RNA Ribossômico 18S/genética
14.
Parasitol Res ; 119(8): 2631-2640, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32556500

RESUMO

The genus Plasmodium (Plasmodiidae) ranks among the most widespread intracellular protozoan parasites affecting a wide range of mammals, birds, and reptiles. Little information is available about lizard malaria parasites in South America, and the pathological features of the resulting parasitoses remain unknown or poorly understood. To partially fill in these gaps, we conducted blood smear analysis, molecular detection, and phylogenetic and pathological investigations in lizards inhabiting an Atlantic Forest fragment in Paraiba, Brazil. From 104 striped forest whiptails (Kentropyx calcarata) screened for the presence of haemosporidian parasites, 67 (64.4%) were positive. Four of five Amazon lava lizards (Strobilurus torquatus) we collected from this same area were also positive. A total of 27 forest whiptails were infected with a new genetic lineage of Plasmodium kentropyxi and other Plasmodium lineages were also detected. Histopathological analysis in infected forest whiptails revealed systemic intraerythrocytic Plasmodium stages, mainly gametocytes, in the liver, lung, and heart. Also, the liver of infected lizards had mild to moderate levels of Kupffer cell and melanomacrophage hypertrophy/hyperplasia with sinusoid leukocytosis. Overall, our findings suggest that an endemic Plasmodium species causes histological alterations that are not related to major pathological processes in striped forest whiptails.


Assuntos
Lagartos/parasitologia , Plasmodium/genética , Plasmodium/patogenicidade , Infecções Protozoárias em Animais/parasitologia , Animais , Brasil , Eritrócitos/parasitologia , Florestas , Fígado/parasitologia , Fígado/patologia , Filogenia , Plasmodium/classificação , Infecções Protozoárias em Animais/patologia
15.
Science ; 368(6492): 746-753, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32409471

RESUMO

Malarial rhythmic fevers are the consequence of the synchronous bursting of red blood cells (RBCs) on completion of the malaria parasite asexual cell cycle. Here, we hypothesized that an intrinsic clock in the parasite Plasmodium chabaudi underlies the 24-hour-based rhythms of RBC bursting in mice. We show that parasite rhythms are flexible and lengthen to match the rhythms of hosts with long circadian periods. We also show that malaria rhythms persist even when host food intake is evenly spread across 24 hours, suggesting that host feeding cues are not required for synchrony. Moreover, we find that the parasite population remains synchronous and rhythmic even in an arrhythmic clock mutant host. Thus, we propose that parasite rhythms are generated by the parasite, possibly to anticipate its circadian environment.


Assuntos
Ritmo Circadiano/fisiologia , Febre/fisiopatologia , Febre/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Malária/fisiopatologia , Malária/parasitologia , Plasmodium chabaudi/fisiologia , Animais , Proteínas CLOCK/genética , Ritmo Circadiano/genética , Sinais (Psicologia) , Escuridão , Ingestão de Alimentos , Eritrócitos/parasitologia , Comportamento Alimentar , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Camundongos , Camundongos Mutantes , Plasmodium chabaudi/genética , Transcrição Genética
16.
Science ; 368(6492): 754-759, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32409472

RESUMO

The blood stage of the infection of the malaria parasite Plasmodium falciparum exhibits a 48-hour developmental cycle that culminates in the synchronous release of parasites from red blood cells, which triggers 48-hour fever cycles in the host. This cycle could be driven extrinsically by host circadian processes or by a parasite-intrinsic oscillator. To distinguish between these hypotheses, we examine the P. falciparum cycle in an in vitro culture system and show that the parasite has molecular signatures associated with circadian and cell cycle oscillators. Each of the four strains examined has a different period, which indicates strain-intrinsic period control. Finally, we demonstrate that parasites have low cell-to-cell variance in cycle period, on par with a circadian oscillator. We conclude that an intrinsic oscillator maintains Plasmodium's rhythmic life cycle.


Assuntos
Relógios Circadianos/fisiologia , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Estágios do Ciclo de Vida , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Relógios Circadianos/genética , Expressão Gênica , Genes de Protozoários/fisiologia , Interações Hospedeiro-Parasita/genética , Camundongos , Plasmodium falciparum/genética , Transcriptoma
17.
PLoS One ; 15(5): e0232183, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379764

RESUMO

Due to the lack of efficiency to control malaria elicited by sub-unit vaccine preparations, vaccination with live-attenuated Plasmodium parasite as reported 70 years ago with irradiated sporozoites regained recently a significant interest. The complex life cycle of the parasite and the different stages of development between mammal host and anopheles do not help to propose an easy vaccine strategy. In order to achieve a complete long-lasting protection against Plasmodium infection and disease, we considered a genetically attenuated blood stage parasite in the hmgb2 gene coding for the high-mobility-group-box 2 (HMGB2). This Plasmodium protein belongs to the HMGB family and hold as the mammal proteins, a double life since it acts first as a nuclear factor involved in chromatin remodelling and transcription regulation and second, when secreted as an active pro-inflammatory alarmin protein. Even though the number of reports on whole living attenuated blood stage parasites is limited when compared to attenuated sporozoites, the results reported with Plasmodium KO parasites are very encouraging. In this report, we present a novel strategy based on pre-immunization with Δhmgb2PbNK65 parasitized red blood cells that confer long-lasting protection in a murine experimental cerebral malaria model against two highly pathogenic homologous and heterologous parasites.


Assuntos
Proteína HMGB2/genética , Malária Cerebral/prevenção & controle , Plasmodium berghei/genética , Animais , Anopheles/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteção Cruzada/imunologia , Modelos Animais de Doenças , Eritrócitos/parasitologia , Feminino , Proteína HMGB2/metabolismo , Imunização/métodos , Vacinas Antimaláricas/imunologia , Malária Cerebral/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium berghei/patogenicidade , Esporozoítos/genética , Vacinação/métodos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia
18.
Trends Parasitol ; 36(6): 512-519, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32360314

RESUMO

Trager and Jensen established a method for culturing Plasmodium falciparum, a breakthrough for malaria research worldwide. Since then, multiple attempts to establish Plasmodium vivax in continuous culture have failed. Unlike P. falciparum, which can invade all aged erythrocytes, P. vivax is restricted to reticulocytes. Thus, a constant supply of reticulocytes is considered critical for continuous P. vivax growth in vitro. A critical question remains why P. vivax selectively invades reticulocytes? What do reticulocytes offer to P. vivax that is not present in mature erythrocytes? One possibility is protection from oxidative stress by glucose-6-phosphate dehydrogenase (G6PD). Here, we also suggest supplements to the media and procedures that may reduce oxidative stress and, as a result, establish a system for the continuous culture of P. vivax.


Assuntos
Técnicas de Cultura/normas , Estágios do Ciclo de Vida/fisiologia , Plasmodium vivax/crescimento & desenvolvimento , Reticulócitos/parasitologia , Técnicas de Cultura/tendências , Eritrócitos/enzimologia , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Estresse Oxidativo , Reticulócitos/enzimologia
19.
Parasitol Res ; 119(7): 2227-2235, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32435898

RESUMO

Babesiosis among humans is on the rise in North America. Current diagnostic assays for the screening of babesiosis require blood collection by venipuncture, which is an invasive method. Urine on the other hand is a desirable biospecimen for biomarker analysis of Babesia microti infections because it can be collected periodically and non-invasively. Our group uses a new class of biomarker harvesting nanocage technology, which, when combined with mass spectrometry (MS), can determine the presence of parasite proteins shed in different bodily fluids of mammalian hosts, including urine. Using the hamster model of babesiosis, our nanoparticle-MS approach identified several B. microti proteins in erythrocytes, plasma, and urine samples. Surface and secreted antigens previously shown to elicit host immune responses against the parasite were particularly abundant in erythrocytes and plasma compared to other proteins. Two of these antigens, BmSA1 and BMR1_03g00947, showed different localization patterns by immunofluorescence of infected erythrocytes. Hamster urine samples from parasitemic animals harbored lower numbers of B. microti proteins compared to erythrocytes and plasma, with glycolytic enzymes, cytoskeletal components, and chaperones being the most frequently detected proteins. By applying novel nanoparticle-MS methods, a high level of analytical sensitivity can be achieved to detect multiple B. microti proteins in blood and urine. This is generally difficult to obtain with other techniques due to the masking of parasite biomarkers by the complex biomolecular matrix of bodily fluids from the host.


Assuntos
Babesia microti/isolamento & purificação , Babesiose/diagnóstico , Eritrócitos/parasitologia , Proteínas de Protozoários/metabolismo , Animais , Babesia microti/metabolismo , Babesiose/sangue , Babesiose/urina , Biomarcadores/sangue , Biomarcadores/metabolismo , Biomarcadores/urina , Cricetinae , Espectrometria de Massas , Proteômica , Proteínas de Protozoários/sangue , Proteínas de Protozoários/urina , Sensibilidade e Especificidade
20.
Nature ; 582(7810): 104-108, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32427965

RESUMO

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.


Assuntos
Apoptose/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Parasitos/imunologia , Plasmodium falciparum/citologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Aotidae/imunologia , Aotidae/parasitologia , Caspases/metabolismo , Criança , Estudos de Coortes , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Ativação Enzimática , Eritrócitos/parasitologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Quênia , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Masculino , Camundongos , Parasitos/citologia , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Tanzânia , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/imunologia , Vacúolos/imunologia
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