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1.
mSphere ; 9(6): e0011124, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38809064

RESUMO

Asexual replication in the apicomplexan Sarcocystis neurona involves two main developmental stages: the motile extracellular merozoite and the sessile intracellular schizont. Merozoites invade host cells and transform into schizonts that undergo replication via endopolygeny to form multiple (64) daughter merozoites that are invasive to new host cells. Given that the capabilities of the merozoite vary significantly from the schizont, the patterns of transcript levels throughout the asexual lifecycle were determined and compared in this study. RNA-Seq data were generated from extracellular merozoites and four intracellular schizont development time points. Of the 6,938 genes annotated in the S. neurona genome, 6,784 were identified in the transcriptome. Of these, 4,111 genes exhibited significant differential expression between the merozoite and at least one schizont development time point. Transcript levels were significantly higher for 2,338 genes in the merozoite and 1,773 genes in the schizont stages. Included in this list were genes encoding the secretory pathogenesis determinants (SPDs), which encompass the surface antigen and SAG-related sequence (SAG/SRS) and the secretory organelle proteins of the invasive zoite stage (micronemes, rhoptries, and dense granules). As anticipated, many of the S. neurona SPD gene transcripts were abundant in merozoites. However, several SPD transcripts were elevated in intracellular schizonts, suggesting roles unrelated to host cell invasion and the initial establishment of the intracellular niche. The hypothetical genes that are potentially unique to the genus Sarcocystis are of particular interest. Their conserved expression patterns are instructive for future investigations into the possible functions of these putative Sarcocystis-unique genes. IMPORTANCE: The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona, putative Sarcocystis-unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis-unique transcripts will provide insights into the interesting biology of this parasite genus.


Assuntos
Merozoítos , Sarcocystis , Sarcocystis/genética , Sarcocystis/crescimento & desenvolvimento , Merozoítos/crescimento & desenvolvimento , Esquizontes/genética , Esquizontes/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transcriptoma , Perfilação da Expressão Gênica , Reprodução Assexuada/genética , Animais , Sarcocistose/parasitologia , Sarcocistose/veterinária , Estágios do Ciclo de Vida/genética
2.
Parasitol Int ; 85: 102420, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34265466

RESUMO

Malaria is a haemato-protozoan disease which causes thousands of deaths every year. Due to the alarming increase of drug resistant strains of P. falciparum, malaria is now becoming more deadly. Helicases are the most important components of the cellular machinery without which cells are unable to survive. The importance of helicases has been proven in variety of organisms. In this study we have reported detailed biochemical characterization of human homologue of DDX3X from Plasmodium falciparum (PfDDX3X). Our study revealed that PfDDX3X is ATP- dependent DNA helicase whereas in human host it is ATP-dependent RNA helicase. We show that N-terminal is essential for its activity and it is present in nucleus and cytoplasm in intraerythrocytic developmental stages of P. falciparum 3D7 strain. Also, it is highly expressed in the schizont stage of P. falciparum 3D7strain. The present study suggests that a protein can perform different functions in different systems. The present study will help to understand the basic biology of malaria parasite P. falciparum.


Assuntos
DNA Helicases/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , DNA Helicases/química , DNA Helicases/metabolismo , Malária Falciparum/metabolismo , Filogenia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Esquizontes/enzimologia , Esquizontes/genética , Esquizontes/crescimento & desenvolvimento , Esquizontes/metabolismo , Alinhamento de Sequência
3.
Parasitol Int ; 84: 102403, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34119684

RESUMO

The transcription factor (TF) AP2-G is essential for gametocytogenesis in the malaria parasite; however, it remains unclear if AP2-G determines commitment to sexual stage development fate in the schizont stage, or whether AP2-G directly initiates sexual stage differentiation and development beginning in the late-trophozoite stage. In this study, we addressed this issue by investigating the expression profile of AP2-G and determining genome-wide target genes in Plasmodium berghei. Fluorescence microscopy showed that AP2-G expression was first observed in the parasite 12 h after erythrocyte invasion and peaked at 18 h when sexual features were first manifested in early gametocytes. Expression of AP2-G decreased with manifestation of sex-specific features. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed at peak AP2-G expression and identified over 1000 binding sites in the genome. The main binding motif of the TF predicted from the binding sites was GTACNY. Predicted targets contained a number of genes related to protein biogenesis, suggesting that AP2-G plays a role in establishing a cellular basis required for sexual differentiation. AP2-G binding sites also existed upstream of gametocyte-specific TFs, namely AP2-G2, AP2-FG, and AP2-G itself. Furthermore, the target contained two AP2 TF-related genes. Disruption of these genes resulted in the arrest of ookinete development. These results suggest another role of AP2-G: activating a transcriptional cascade to promote conversion into early gametocytes. Taken together, AP2-G is involved not in establishing sexual commitment of schizonts, but rather in triggering the initiation of differentiation and the early development of gametocytes in the late trophozoite stage.


Assuntos
Malária/metabolismo , Plasmodium berghei/fisiologia , Proteínas de Protozoários/metabolismo , Esquizontes/fisiologia , Animais , Gametogênese , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/crescimento & desenvolvimento , Ratos , Ratos Wistar , Esquizontes/crescimento & desenvolvimento
4.
Turkiye Parazitol Derg ; 44(4): 226-231, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33269565

RESUMO

OBJECTIVE: Plasmodium falciparum is a protozoan parasite that causes many deaths worldwide. It's cultivation in an in vitro culture setting contributes significantly to scientific studies. However, there are no laboratories in Turkey that cultivate P. falciparum in vitro. Hence, the purpose of this study was to cultivate P. falciparum in vitro. METHODS: Five P. falciparum strains were used in our study and were kept frozen in liquid nitrogen tanks. These parasite strains were then thawed in a 37 °C water bath and transferred to the Albumax-complete medium that was previously prepared. After that, the petri dishes were placed in the chamber. For 30 seconds, a special gas mixture containing 5% CO2, 5% O2 and 90% N2 was added into the chamber which was placed in a 37 °C oven and left for incubation for 2 days. At the end of the incubation period, thin smear preparations were prepared from the medium, stained with Giemsa and examined using an immersion lens. RESULTS: Examination of the smears revealed that trophozoite and schizont forms of all P. falciparum isolates were present at a rate of 2% in in vitro culture medium. CONCLUSION: As a result of our study, the in vitro culture of P. falciparum was successfully developed. With this, several projects such as biological and chemical characteristics, pathogenicity, phenotypic and molecular-level drug sensitivities and parasite vaccination studies can be carried out more easily in our country.


Assuntos
Plasmodium falciparum/crescimento & desenvolvimento , Animais , Meios de Cultura , Humanos , Técnicas In Vitro , Plasmodium falciparum/isolamento & purificação , Esquizontes/crescimento & desenvolvimento , Trofozoítos/crescimento & desenvolvimento , Turquia
5.
Artigo em Inglês | MEDLINE | ID: mdl-32660993

RESUMO

Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 µM) and hypnozoites (IC50, 29.24 µM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Ivermectina/farmacologia , Fígado/efeitos dos fármacos , Malária/tratamento farmacológico , Plasmodium cynomolgi/efeitos dos fármacos , Animais , Antimaláricos/sangue , Antimaláricos/farmacocinética , Disponibilidade Biológica , Cloroquina/sangue , Cloroquina/farmacocinética , Esquema de Medicação , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/parasitologia , Ivermectina/sangue , Ivermectina/farmacocinética , Fígado/parasitologia , Macaca mulatta , Malária/parasitologia , Masculino , Parasitemia/tratamento farmacológico , Plasmodium cynomolgi/crescimento & desenvolvimento , Plasmodium cynomolgi/patogenicidade , Cultura Primária de Células , Esquizontes/efeitos dos fármacos , Esquizontes/crescimento & desenvolvimento
6.
Malar J ; 18(1): 148, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023359

RESUMO

BACKGROUND: Long-term in vitro culture of blood stage Plasmodium parasites invariably leads to asynchronous parasite development. The most often used technique to synchronize Plasmodium falciparum culture is sorbitol treatment, which differentially induces osmotic lysis of trophozoite- and schizont-infected red blood cells due to presence of the new permeation pathways in the membranes of these cells. However, sorbitol treatment does not work well when used to synchronize the culture-adapted Plasmodium knowlesi A1-H.1 line. METHODS: A number of common solutes were tested in lieu of sorbitol for synchronization of P. knowlesi A1-H.1 ring stage. RESULTS: Guanidine hydrochloride was found to selectively lyse trophozoite- and schizont-infected red blood cells, yielding highly synchronous and viable rings. CONCLUSIONS: A method for synchronization of P. knowlesi in human red blood cells was developed. Requiring only common laboratory reagents, this method is simple and should be applicable to most laboratory settings.


Assuntos
Eritrócitos/efeitos dos fármacos , Guanidina/farmacologia , Parasitologia/métodos , Plasmodium knowlesi/efeitos dos fármacos , Plasmodium knowlesi/crescimento & desenvolvimento , Eritrócitos/parasitologia , Humanos , Malária/parasitologia , Esquizontes/crescimento & desenvolvimento , Sorbitol/farmacologia
7.
Exp Parasitol ; 201: 34-41, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31034815

RESUMO

Ovine Eimeria spp. infections cause increased mortality, reduced welfare and substantial economic losses, and anticocccidials are important for their control. Recent reports of anticoccidial resistance against ovine Eimeria spp. necessitate the development of in vitro methods for the detection of reduced anticoccidial efficacy, especially since the in vivo methods are both expensive, time consuming and requires the use of otherwise healthy animals. The aim of the present study was therefore to approach a preliminary standardization of in vitro assays for evaluation of the efficacy of the most commonly used anticoccidials in ruminants. For this purpose, apart from the evaluation of inhibition of oocyst sporulation, most effort was concentrated on assessment of the capacity of the different anticoccidials to inhibit both the invasion and further development (up to the first schizogony) of E. ninakohlyakimovae sporozoites in bovine colonic epithelial cells (BCEC). For this purpose, infected cultures were monitored 1, 8 and 15 days post infection to determine the infection rate, number of immature schizonts and number, size and appearance of mature schizonts, respectively. No clear inhibitory effect was found with any of the anticoccidial formulations tested, and we could not identify why there were no measurable effects from the different anticoccidials. Despite the lack of positive results, further investigations should be encouraged, as this could decrease the need for animal experiments and could be used in the initial assessment of anticoccidial efficacy of new drugs.


Assuntos
Coccidiose/veterinária , Coccidiostáticos/farmacologia , Eimeria/efeitos dos fármacos , Doenças das Cabras/parasitologia , Animais , Bovinos , Células Cultivadas , Coccidiose/tratamento farmacológico , Coccidiose/parasitologia , Colo/citologia , Colo/parasitologia , Decoquinato/farmacologia , Resistência a Medicamentos , Eimeria/crescimento & desenvolvimento , Eimeria/isolamento & purificação , Células Epiteliais/parasitologia , Fezes/parasitologia , Doenças das Cabras/tratamento farmacológico , Cabras , Mucosa Intestinal/citologia , Mucosa Intestinal/parasitologia , Nitrilas/farmacologia , Oocistos/isolamento & purificação , Esquizontes/efeitos dos fármacos , Esquizontes/crescimento & desenvolvimento , Esporozoítos/isolamento & purificação , Sulfonamidas/farmacologia , Triazinas/farmacologia
9.
PLoS Biol ; 17(2): e3000154, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30794532

RESUMO

Cyclic nucleotide signalling is a major regulator of malaria parasite differentiation. Phosphodiesterase (PDE) enzymes are known to control cyclic GMP (cGMP) levels in the parasite, but the mechanisms by which cyclic AMP (cAMP) is regulated remain enigmatic. Here, we demonstrate that Plasmodium falciparum phosphodiesterase ß (PDEß) hydrolyses both cAMP and cGMP and is essential for blood stage viability. Conditional gene disruption causes a profound reduction in invasion of erythrocytes and rapid death of those merozoites that invade. We show that this dual phenotype results from elevated cAMP levels and hyperactivation of the cAMP-dependent protein kinase (PKA). Phosphoproteomic analysis of PDEß-null parasites reveals a >2-fold increase in phosphorylation at over 200 phosphosites, more than half of which conform to a PKA substrate consensus sequence. We conclude that PDEß plays a critical role in governing correct temporal activation of PKA required for erythrocyte invasion, whilst suppressing untimely PKA activation during early intra-erythrocytic development.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Transdução de Sinais/genética , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Eritrócitos/parasitologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hidrólise , Merozoítos/enzimologia , Merozoítos/genética , Merozoítos/crescimento & desenvolvimento , Fosfoproteínas/classificação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteoma/classificação , Proteoma/genética , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Esquizontes/enzimologia , Esquizontes/genética , Esquizontes/crescimento & desenvolvimento , Fatores de Tempo
10.
Nat Commun ; 9(1): 1837, 2018 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-29743474

RESUMO

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.


Assuntos
Antimaláricos/administração & dosagem , Malária Falciparum/tratamento farmacológico , Malária Vivax/tratamento farmacológico , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium vivax/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Hepatócitos/parasitologia , Humanos , Fígado/parasitologia , Malária Falciparum/parasitologia , Malária Vivax/parasitologia , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium vivax/efeitos dos fármacos , Esquizontes/efeitos dos fármacos , Esquizontes/crescimento & desenvolvimento , Esporozoítos/efeitos dos fármacos , Esporozoítos/crescimento & desenvolvimento
11.
J Aquat Anim Health ; 30(2): 95-102, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29698999

RESUMO

Other than the initial infectious cell, schizonts are the only stage of the parasite Ichthyophonus sp. that has been identified in the tissues of a living host, and they are known to initiate new infections when ingested by a suitable host. However, after feeding Ichthyophonus-infected tissue to Rainbow Trout Oncorhynchus mykiss, we observed that once infection was initiated, some schizonts proceeded to develop into several other morphologic forms indistinguishable from those previously described from recently deceased hosts, decomposing infected corpses, and in vitro culture. It appeared that not all schizonts participated in the infection process; some initiated infection, as expected, while others passed into the intestines, where they morphed into multiple cell types (e.g., schizonts, some with partially digested or ruptured capsules, ameboid plasmodia, merozoites, hyphenated cells, and empty capsules). Some of these cells were viable when cultured, but none was infectious to naïve Rainbow Trout when administered by gavage. We posit that (1) not all tissue schizonts are programmed to perform the same function or (2) not all respond similarly to their environment. After consumption by a piscivore, those schizonts that do not initiate an infection do not die but rather metamorphose into different cell types as they transit the gastrointestinal tract and are ultimately released back into the aquatic environment through defecation. The fate of these cells after exiting the host is presently unknown, but they likely represent a segment of the Ichthyophonus life cycle.


Assuntos
Doenças dos Peixes/parasitologia , Infecções por Mesomycetozoea/parasitologia , Mesomycetozoea/crescimento & desenvolvimento , Oncorhynchus mykiss , Animais , Doenças dos Peixes/transmissão , Trato Gastrointestinal/parasitologia , Estágios do Ciclo de Vida , Infecções por Mesomycetozoea/transmissão , Metamorfose Biológica , Esquizontes/crescimento & desenvolvimento
12.
Elife ; 62017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29215331

RESUMO

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.


Assuntos
Perfilação da Expressão Gênica , Fígado/parasitologia , Macaca mulatta/parasitologia , Plasmodium cynomolgi/crescimento & desenvolvimento , Plasmodium cynomolgi/genética , Esquizontes/crescimento & desenvolvimento , Esquizontes/genética , Animais , Feminino , Masculino
13.
Malar J ; 16(1): 392, 2017 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-28964258

RESUMO

BACKGROUND: While intensive Plasmodium falciparum multidrug resistance surveillance continues in Cambodia, relatively little is known about Plasmodium vivax drug resistance in Cambodia or elsewhere. To investigate P. vivax anti-malarial susceptibility in Cambodia, 76 fresh P. vivax isolates collected from Oddar Meanchey (northern Cambodia) in 2013-2015 were assessed for ex vivo drug susceptibility using the microscopy-based schizont maturation test (SMT) and a Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. P. vivax multidrug resistance gene 1 (pvmdr1) mutations, and copy number were analysed in a subset of isolates. RESULTS: Ex vivo testing was interpretable in 80% of isolates using the pLDH-ELISA, but only 25% with the SMT. Plasmodium vivax drug susceptibility by pLDH-ELISA was directly compared with 58 P. falciparum isolates collected from the same locations in 2013-4, tested by histidine-rich protein-2 ELISA. Median pLDH-ELISA IC50 of P. vivax isolates was significantly lower for dihydroartemisinin (3.4 vs 6.3 nM), artesunate (3.2 vs 5.7 nM), and chloroquine (22.1 vs 103.8 nM) than P. falciparum but higher for mefloquine (92 vs 66 nM). There were not significant differences for lumefantrine or doxycycline. Both P. vivax and P. falciparum had comparable median piperaquine IC50 (106.5 vs 123.8 nM), but some P. falciparum isolates were able to grow in much higher concentrations above the normal standard range used, attaining up to 100-fold greater IC50s than P. vivax. A high percentage of P. vivax isolates had pvmdr1 Y976F (78%) and F1076L (83%) mutations but none had pvmdr1 amplification. CONCLUSION: The findings of high P. vivax IC50 to mefloquine and piperaquine, but not chloroquine, suggest significant drug pressure from drugs used to treat multidrug resistant P. falciparum in Cambodia. Plasmodium vivax isolates are frequently exposed to mefloquine and piperaquine due to mixed infections and the long elimination half-life of these drugs. Difficulty distinguishing infection due to relapsing hypnozoites versus blood-stage recrudescence complicates clinical detection of P. vivax resistance, while well-validated molecular markers of chloroquine resistance remain elusive. The pLDH assay may be a useful adjunctive tool for monitoring for emerging drug resistance, though more thorough validation is needed. Given high grade clinical chloroquine resistance observed recently in neighbouring countries, low chloroquine IC50 values seen here should not be interpreted as susceptibility in the absence of clinical data. Incorporating pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax will help to further validate this field-expedient method.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Ensaio de Imunoadsorção Enzimática/métodos , Microscopia/métodos , Plasmodium vivax/efeitos dos fármacos , Camboja , Variações do Número de Cópias de DNA , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Esquizontes/crescimento & desenvolvimento
14.
Malar J ; 16(1): 366, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28899381

RESUMO

BACKGROUND: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. RESULTS: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. CONCLUSIONS: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.


Assuntos
Antimaláricos/farmacologia , Carbolinas/farmacologia , NADP/análogos & derivados , Piperazinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Transdução de Sinais , Eritrócitos/parasitologia , Humanos , NADP/fisiologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/fisiologia , Esquizontes/efeitos dos fármacos , Esquizontes/crescimento & desenvolvimento , Esquizontes/fisiologia
15.
Malar J ; 16(1): 305, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28764716

RESUMO

BACKGROUND: Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. METHODS: HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector® electroporation technology. RESULTS: Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. CONCLUSION: An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8-18 days.


Assuntos
Merozoítos/fisiologia , Microrganismos Geneticamente Modificados/fisiologia , Plasmodium berghei/fisiologia , Animais , Técnicas de Cultura de Células , Fígado , Merozoítos/genética , Merozoítos/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/crescimento & desenvolvimento , Plasmodium berghei/genética , Esquizontes/genética , Esquizontes/crescimento & desenvolvimento , Esquizontes/fisiologia , Transfecção
16.
Parasitol Int ; 66(2): 106-111, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28033565

RESUMO

Raptors serve as the definitive host for several Sarcocystis species. The complete life cycles of only a few of these Sarcocystis species that use birds of prey as definitive hosts have been described. In the present study, Sarcocystis species sporocysts were obtained from the intestine of a Cooper's hawk (Accipiter cooperii) and were used to infect cell cultures of African green monkey kidney cells to isolate a continuous culture and describe asexual stages of the parasite. Two clones of the parasite were obtained by limiting dilution. Asexual stages were used to obtain DNA for molecular classification and identification. PCR amplification and sequencing were done at three nuclear ribosomal DNA loci; 18S rRNA, 28S rRNA, and ITS-1, and the mitochondrial cytochrome c oxidase subunit 1 (cox1) locus. Examination of clonal isolates of the parasite indicated a single species related to S. columbae (termed Sarcocystis sp. ex Accipiter cooperii) was present in the Cooper's hawk. Our results document for the first time Sarcocystis sp. ex A. cooperii occurs naturally in an unknown intermediate host in North America and that Cooper's hawks (A. cooperii) are a natural definitive host.


Assuntos
Doenças das Aves/parasitologia , Falcões/parasitologia , Sarcocystis/genética , Sarcocystis/isolamento & purificação , Sarcocistose/veterinária , Animais , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Ciclo-Oxigenase 1/genética , DNA Ribossômico/genética , Microscopia Eletrônica de Transmissão , Oocistos/ultraestrutura , Filogenia , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico/genética , Reprodução Assexuada/fisiologia , Sarcocystis/classificação , Sarcocystis/crescimento & desenvolvimento , Sarcocistose/parasitologia , Esquizontes/crescimento & desenvolvimento , Esquizontes/ultraestrutura , Análise de Sequência de DNA
17.
Indian J Med Microbiol ; 34(4): 509-512, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27934832

RESUMO

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Cloroquina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Esquizontes/efeitos dos fármacos , Esquizontes/crescimento & desenvolvimento , Trofozoítos/efeitos dos fármacos , Trofozoítos/crescimento & desenvolvimento
18.
PLoS One ; 11(10): e0165358, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27780272

RESUMO

The growth phenotype of asexual blood stage malaria parasites can influence their virulence and also their ability to survive and achieve transmission to the next host, but there are few methods available to characterise parasite growth parameters in detail. We developed a new assay to measure growth rates at different starting parasitaemias in a 96-well format and applied it to characterise the growth of Plasmodium falciparum lines 3D7-A and 3D7-B, previously shown to have different invasion rates and to use different invasion pathways. Using this simple and accurate assay we found that 3D7-B is more sensitive to high initial parasitaemia than 3D7-A. This result indicates that different parasite lines show variation in their levels of density-dependent growth inhibition. We also developed a new assay to compare the duration of the asexual blood cycle between different parasite lines. The assay is based on the tight synchronisation of cultures to a 1 h parasite age window and the subsequent monitoring of schizont bursting and formation of new rings by flow cytometry. Using this assay we observed differences in the duration of the asexual blood cycle between parasite lines 3D7 and HB3. These two new assays will be useful to characterise variation in growth-related parameters and to identify growth phenotypes associated with the targeted deletion of specific genes or with particular genomic, transcriptomic or proteomic patterns. Furthermore, the identification of density-dependent growth inhibition as an intrinsic parasite property that varies between parasite lines expands the repertoire of measurable growth-related phenotypic traits that have the potential to influence the outcome of a malarial blood infection.


Assuntos
Parasitemia/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Eritrócitos/parasitologia , Citometria de Fluxo , Genômica , Humanos , Estágios do Ciclo de Vida , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Merozoítos/fisiologia , Análise em Microsséries , Parasitemia/diagnóstico , Fenótipo , Plasmodium falciparum/fisiologia , Proteômica , Esquizontes/crescimento & desenvolvimento
19.
Parasitol Int ; 65(6 Pt A): 715-727, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27425600

RESUMO

The asexual erythrocytic cycle of the protozoan parasite Plasmodium falciparum is responsible for the pathogenesis of malaria and causes the overwhelming majority of malaria deaths. Rapidly increasing parasitaemia during this 48hour cycle threatens the survival of the human host and the parasite prior to transmission of the slow-maturing sexual stages to the mosquito host. The parasite may utilise regulated cell death (RCD) to control the burden of infection on the host and thus aid its own survival and transmission. The occurrence of RCD in P. falciparum remains a controversial topic. We provide strong evidence for the occurrence of an apoptosis-like phenotype of RCD in P. falciparum under conditions of high parasite density. P. falciparum was maintained in vitro and stressed by allowing growth to an unrestricted peak parasitaemia. Cell death markers, including morphological changes, DNA fragmentation, mitochondrial polarisation and phosphatidylserine externalisation were used to characterise parasite death at the time of peak parasitaemia and 24h later. At peak parasitaemia, mitochondrial depolarisation was observed, together with phosphatidylserine externalisation in both parasitised- and neighbouring non-infected erythrocytes. DNA fragmentation coincided with a decline in parasitaemia. Fewer merozoites were observed in mature schizonts at peak parasitaemia. Growth recovery to near-peak parasitaemia was noted within two intraerythrocytic cycles. The combination and chronological order of the biochemical markers of cell death suggest the occurrence of an apoptosis-like phenotype. The identification of a RCD pathway in P. falciparum may provide novel drug targets, particularly if the pathway differs from the host machinery.


Assuntos
Morte Celular/fisiologia , Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Parasitemia/patologia , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Culicidae/parasitologia , Fragmentação do DNA , Humanos , Merozoítos/metabolismo , Mitocôndrias/fisiologia , Fosfatidilserinas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Densidade Demográfica , Esquizontes/crescimento & desenvolvimento
20.
Sci Rep ; 6: 26993, 2016 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-27244695

RESUMO

The Plasmodium vivax reticulocyte-binding protein (RBP) family was identified based on the annotation of adhesive ligands in the P. vivax genome. Reticulocyte-specific interactions with the PvRBPs (PvRBP1 and PvRBP2) were previously reported. Plasmodium falciparum reticulocyte-binding protein homologue 4 (PfRh4, a homologue of PvRBP1) was observed to possess erythrocyte-binding activity via complement receptor 1 on the erythrocyte surface. However, the reticulocyte-binding mechanisms of P. vivax are unclear because of the large molecular mass of PvRBP1 (>326 kDa) and the difficulty associated with in vitro cultivation. In the present study, 34 kDa of PvRBP1a (PlasmoDB ID: PVX_098585) and 32 kDa of PvRBP1b (PVX_098582) were selected from a 30 kDa fragment of PfRh4 for reticulocyte-specific binding activity analysis. Both PvRBP1a and PvRBP1b were found to be localized at the microneme in the mature schizont-stage parasites. Naturally acquired immune responses against PvRBP1a-34 and PvRBP1b-32 were observed lower than PvDBP-RII. The reticulocyte-specific binding activities of PvRBP1a-34 and PvRBP1b-32 were significantly higher than normocyte binding activity and were significantly reduced by chymotrypsin treatment. PvRBP1a and 1b, bind to reticulocytes and that this suggests that these ligands may have an important role in P. vivax merozoite invasion.


Assuntos
Eritrócitos/metabolismo , Proteínas de Membrana/genética , Plasmodium falciparum/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Reticulócitos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Eritrócitos/parasitologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Malária Falciparum/parasitologia , Malária Vivax/parasitologia , Proteínas de Membrana/metabolismo , Merozoítos/genética , Merozoítos/crescimento & desenvolvimento , Merozoítos/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Plasmodium vivax/crescimento & desenvolvimento , Plasmodium vivax/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reticulócitos/parasitologia , Esquizontes/genética , Esquizontes/crescimento & desenvolvimento , Esquizontes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
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