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1.
PLoS Biol ; 19(12): e3001426, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34928952

RESUMO

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.


Assuntos
Código de Barras de DNA Taxonômico/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Malária/prevenção & controle , Acetobacteraceae/genética , Animais , Anopheles/genética , Anopheles/microbiologia , Antimaláricos/farmacologia , Inseticidas , Malária/parasitologia , Malária/transmissão , Mosquitos Vetores/microbiologia , RNA Ribossômico 16S/genética
2.
Proc Natl Acad Sci U S A ; 115(29): E6920-E6926, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29967151

RESUMO

Isoxazolines are oral insecticidal drugs currently licensed for ectoparasite control in companion animals. Here we propose their use in humans for the reduction of vector-borne disease incidence. Fluralaner and afoxolaner rapidly killed Anopheles, Aedes, and Culex mosquitoes and Phlebotomus sand flies after feeding on a drug-supplemented blood meal, with IC50 values ranging from 33 to 575 nM, and were fully active against strains with preexisting resistance to common insecticides. Based on allometric scaling of preclinical pharmacokinetics data, we predict that a single human median dose of 260 mg (IQR, 177-407 mg) for afoxolaner, or 410 mg (IQR, 278-648 mg) for fluralaner, could provide an insecticidal effect lasting 50-90 days against mosquitoes and Phlebotomus sand flies. Computational modeling showed that seasonal mass drug administration of such a single dose to a fraction of a regional population would dramatically reduce clinical cases of Zika and malaria in endemic settings. Isoxazolines therefore represent a promising new component of drug-based vector control.


Assuntos
Controle de Doenças Transmissíveis/métodos , Culicidae/crescimento & desenvolvimento , Inseticidas/farmacologia , Controle de Mosquitos/métodos , Mosquitos Vetores/crescimento & desenvolvimento , Psychodidae/crescimento & desenvolvimento , Animais , Humanos
3.
Cell Microbiol ; 19(1)2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27324409

RESUMO

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti-malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3-phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N-terminal targeting sequence to GFP and 3' tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site-directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3-phosphate acyltransferase in malaria parasites.


Assuntos
Apicoplastos/metabolismo , Ácidos Graxos/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Plasmodium berghei/metabolismo , Plasmodium falciparum/metabolismo , Apicoplastos/enzimologia , Bactérias/genética , Bactérias/metabolismo , Análise Mutacional de DNA , Técnicas de Inativação de Genes , Teste de Complementação Genética , Plasmodium berghei/enzimologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Transporte Proteico
4.
Proc Natl Acad Sci U S A ; 112(33): 10216-23, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-25831536

RESUMO

Mitochondrial ATP synthase is driven by chemiosmotic oxidation of pyruvate derived from glycolysis. Blood-stage malaria parasites eschew chemiosmosis, instead relying almost solely on glycolysis for their ATP generation, which begs the question of whether mitochondrial ATP synthase is necessary during the blood stage of the parasite life cycle. We knocked out the mitochondrial ATP synthase ß subunit gene in the rodent malaria parasite, Plasmodium berghei, ablating the protein that converts ADP to ATP. Disruption of the ß subunit gene of the ATP synthase only marginally reduced asexual blood-stage parasite growth but completely blocked mouse-to-mouse transmission via Anopheles stephensi mosquitoes. Parasites lacking the ß subunit gene of the ATP synthase generated viable gametes that fuse and form ookinetes but cannot progress beyond this stage. Ookinetes lacking the ß subunit gene of the ATP synthase had normal motility but were not viable in the mosquito midgut and never made oocysts or sporozoites, thereby abrogating transmission to naive mice via mosquito bite. We crossed the self-infertile ATP synthase ß subunit knockout parasites with a male-deficient, self-infertile strain of P. berghei, which restored fertility and production of oocysts and sporozoites, which demonstrates that mitochondrial ATP synthase is essential for ongoing viability through the female, mitochondrion-carrying line of sexual reproduction in P. berghei malaria. Perturbation of ATP synthase completely blocks transmission to the mosquito vector and could potentially be targeted for disease control.


Assuntos
Regulação Enzimológica da Expressão Gênica , Malária/parasitologia , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Plasmodium berghei/enzimologia , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Animais , Proteínas de Bactérias/metabolismo , Biologia Computacional , Cruzamentos Genéticos , Culicidae , Feminino , Glicólise , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Oocistos/enzimologia , Oxigênio/química , Fenótipo , Plasmodium berghei/patogenicidade , Esporozoítos/enzimologia , Transgenes
5.
Blood ; 125(3): 534-41, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25414439

RESUMO

Many red cell polymorphisms are a result of selective pressure by the malarial parasite. Here, we add another red cell disease to the panoply of erythrocytic changes that give rise to resistance to malaria. Erythrocytes from individuals with erythropoietic protoporphyria (EPP) have low levels of the final enzyme in the heme biosynthetic pathway, ferrochelatase. Cells from these patients are resistant to the growth of Plasmodium falciparum malarial parasites. This phenomenon is due to the absence of ferrochelatase and not an accumulation of substrate, as demonstrated by the normal growth of P falciparum parasites in the EPP phenocopy, X-linked dominant protoporphyria, which has elevated substrate, and normal ferrochelatase levels. This observation was replicated in a mouse strain with a hypomorphic mutation in the murine ferrochelatase gene. The parasite enzyme is not essential for parasite growth as Plasmodium berghei parasites carrying a complete deletion of the ferrochelatase gene grow normally in erythrocytes, which confirms previous studies. That ferrochelatase is essential to parasite growth was confirmed by showing that inhibition of ferrochelatase using the specific competitive inhibitor, N-methylprotoporphyrin, produced a potent growth inhibition effect against cultures of P falciparum. This raises the possibility of targeting human ferrochelatase in a host-directed antimalarial strategy.


Assuntos
Eritrócitos/parasitologia , Ferroquelatase/fisiologia , Malária Falciparum/prevenção & controle , Plasmodium berghei/crescimento & desenvolvimento , Protoporfiria Eritropoética/prevenção & controle , Animais , Eritrócitos/enzimologia , Feminino , Ferroquelatase/antagonistas & inibidores , Heme/metabolismo , Humanos , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Protoporfiria Eritropoética/enzimologia , Protoporfiria Eritropoética/parasitologia , Protoporfirinas/farmacologia
6.
Cell Microbiol ; 18(3): 399-412, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26347246

RESUMO

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium translocon of exported proteins (PTEX) and a cleavable Plasmodium export element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic Plasmodium berghei parasites deficient in EXP2, the putative pore-forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood-stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL-bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL-bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection.


Assuntos
Malária/parasitologia , Plasmodium berghei/patogenicidade , Proteínas de Protozoários/metabolismo , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/metabolismo , Interações Hospedeiro-Parasita , Fígado/parasitologia , Camundongos , Plasmodium berghei/genética , Transporte Proteico/genética , Proteínas de Protozoários/genética , Tetraciclinas/farmacologia
7.
PLoS Pathog ; 10(5): e1004135, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24854165

RESUMO

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunização Secundária/métodos , Estágios do Ciclo de Vida/imunologia , Malária/prevenção & controle , Plasmodium berghei/imunologia , Receptores de Antígenos de Linfócitos T/genética , Esporozoítos/imunologia , Transferência Adotiva , Animais , Anopheles , Sangue/parasitologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Células Cultivadas , Fígado/imunologia , Fígado/parasitologia , Malária/sangue , Malária/imunologia , Malária/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium chabaudi , Plasmodium yoelii , Receptores de Antígenos de Linfócitos T/imunologia
9.
Mol Microbiol ; 89(6): 1167-86, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23869529

RESUMO

Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX. Previously PTEX150, HSP101 and EXP2 have been shown to be bona fide members of PTEX. Here we validate that PTEX88 and TRX2 are also genuine members of PTEX and provide evidence that expression of PTEX components are also expressed in early gametocytes, mosquito and liver stages, consistent with observations that protein export is not restricted to asexual stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in Plasmodium berghei, in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth. In contrast, the putative thioredoxin-like protein TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro, and reduced capacity to cause severe disease in a cerebral malaria model. Thus, while not essential for parasite survival, TRX2 may help to optimize PTEX activity. Importantly, the generation of TRX2 knockout parasites that display altered phenotypes provides a much-needed tool to dissect PTEX function.


Assuntos
Parasitemia/parasitologia , Plasmodium berghei/enzimologia , Plasmodium berghei/patogenicidade , Tiorredoxinas/metabolismo , Fatores de Virulência/metabolismo , Animais , Modelos Animais de Doenças , Deleção de Genes , Malária Cerebral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Análise de Sobrevida , Tiorredoxinas/genética , Virulência , Fatores de Virulência/genética
10.
J Med Chem ; 64(17): 12582-12602, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34437804

RESUMO

A phenotypic high-throughput screen allowed discovery of quinazolinone-2-carboxamide derivatives as a novel antimalarial scaffold. Structure-activity relationship studies led to identification of a potent inhibitor 19f, 95-fold more potent than the original hit compound, active against laboratory-resistant strains of malaria. Profiling of 19f suggested a fast in vitro killing profile. In vivo activity in a murine model of human malaria in a dose-dependent manner constitutes a concomitant benefit.


Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Malária Falciparum/tratamento farmacológico , Quinazolinonas/farmacologia , Administração Oral , Animais , Humanos , Camundongos , Estrutura Molecular , Plasmodium falciparum/efeitos dos fármacos , Quinazolinonas/química , Relação Estrutura-Atividade
11.
ACS Infect Dis ; 7(6): 1680-1689, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33929818

RESUMO

Prolyl-tRNA synthetase (PRS) is a clinically validated antimalarial target. Screening of a set of PRS ATP-site binders, initially designed for human indications, led to identification of 1-(pyridin-4-yl)pyrrolidin-2-one derivatives representing a novel antimalarial scaffold. Evidence designates cytoplasmic PRS as the drug target. The frontrunner 1 and its active enantiomer 1-S exhibited low-double-digit nanomolar activity against resistant Plasmodium falciparum (Pf) laboratory strains and development of liver schizonts. No cross-resistance with strains resistant to other known antimalarials was noted. In addition, a similar level of growth inhibition was observed against clinical field isolates of Pf and P. vivax. The slow killing profile and the relative high propensity to develop resistance in vitro (minimum inoculum resistance of 8 × 105 parasites at a selection pressure of 3 × IC50) constitute unfavorable features for treatment of malaria. However, potent blood stage and antischizontal activity are compelling for causal prophylaxis which does not require fast onset of action. Achieving sufficient on-target selectivity appears to be particularly challenging and should be the primary focus during the next steps of optimization of this chemical series. Encouraging preliminary off-target profile and oral efficacy in a humanized murine model of Pf malaria allowed us to conclude that 1-(pyridin-4-yl)pyrrolidin-2-one derivatives represent a promising starting point for the identification of novel antimalarial prophylactic agents that selectively target Plasmodium PRS.


Assuntos
Aminoacil-tRNA Sintetases , Antimaláricos , Malária Falciparum , Malária , Animais , Antimaláricos/farmacologia , Humanos , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Camundongos , Plasmodium falciparum
12.
Front Cell Infect Microbiol ; 10: 591046, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33392104

RESUMO

Chimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing PfCSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting PvCSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum. We generated chimeric P. falciparum parasites expressing both PfCSP and PvCSP. These Pf-PvCSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed PfCSP and PvCSP on the sporozoite surface. Pf-PvCSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both PfCSP and PvCSP after immunization of mice. These results support the use of Pf-PvCSP sporozoites in studies optimizing vaccines targeting PvCSP.


Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária , Plasmodium falciparum , Plasmodium vivax , Animais , Anticorpos Antiprotozoários , Vacinas Antimaláricas/genética , Malária Falciparum/prevenção & controle , Camundongos , Plasmodium falciparum/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética
13.
Protist ; 160(1): 51-63, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19026596

RESUMO

The rodent malaria parasite Plasmodium berghei develops in hepatocytes within 48-52h from a single sporozoite into up to 20,000 daughter parasites, so-called merozoites. The cellular and molecular details of this extensive proliferation are still largely unknown. Here we have used a transgenic, RFP-expressing P. berghei parasite line and molecular imaging techniques including intravital microscopy to decipher various aspects of parasite development within the hepatocyte. In late schizont stages, MSP1 is expressed and incorporated into the parasite plasma membrane that finally forms the membrane of developing merozoites by continuous invagination steps. We provide first evidence for activation of a verapamil-sensitive Ca(2+) channel in the plasma membrane of liver stage parasites before invagination occurs. During merozoite formation, the permeability of the parasitophorous vacuole membrane changes considerably before it finally becomes completely disrupted, releasing merozoites into the host cell cytoplasm.


Assuntos
Membrana Celular/metabolismo , Malária/parasitologia , Plasmodium berghei/crescimento & desenvolvimento , Vacúolos/metabolismo , Animais , Canais de Cálcio/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Hepatócitos/parasitologia , Humanos , Fígado/parasitologia , Merozoítos/crescimento & desenvolvimento , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência/métodos , Ratos , Esporozoítos/crescimento & desenvolvimento , Verapamil
14.
Cell Microbiol ; 10(8): 1723-34, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18419771

RESUMO

Cysteine proteases mediate liberation of Plasmodium berghei merozoites from infected hepatocytes. In an attempt to identify the responsible parasite proteases, we screened the genome of P. berghei for cysteine protease-encoding genes. RT-PCR analyses revealed that transcription of four out of five P. berghei serine repeat antigen (PbSERA) genes was strongly upregulated in late liver stages briefly before the parasitophorous vacuole membrane ruptured to release merozoites into the host cell cytoplasm, suggesting a role of PbSERA proteases in these processes. In order to characterize PbSERA3 processing, we raised an antiserum against a non-conserved region of the protein and generated a transgenic P. berghei strain expressing a TAP-tagged PbSERA3 under the control of the endogenous promoter. Immunofluorescence assays revealed that PbSERA3 leaks into the host cell cytoplasm during merozoite development, where it might contribute to host cell death or activate host cell proteases that execute cell death. Importantly, processed PbSERA3 has been detected by Western blot analysis in cell extracts of schizont-infected cells and merozoite-infected detached hepatic cells.


Assuntos
Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Cisteína Endopeptidases/metabolismo , Fígado/parasitologia , Malária/imunologia , Plasmodium berghei/imunologia , Animais , Antígenos de Protozoários/análise , Linhagem Celular Tumoral , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Feminino , Malária/parasitologia , Organismos Geneticamente Modificados , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Ratos , Regulação para Cima , Vacúolos/parasitologia
15.
Genome Biol ; 20(1): 151, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370870

RESUMO

BACKGROUND: In multicellular organisms, alternative splicing is central to tissue differentiation and identity. Unicellular protists lack multicellular tissue but differentiate into variable cell types during their life cycles. The role of alternative splicing in transitions between cell types and establishing cellular identity is currently unknown in any unicellular organism. RESULTS: To test whether alternative splicing in unicellular protists plays a role in cellular differentiation, we conduct RNA-seq to compare splicing in female and male sexual stages to asexual intraerythrocytic stages in the rodent malaria parasite Plasmodium berghei. We find extensive changes in alternative splicing between stages and a role for alternative splicing in sexual differentiation. Previously, general gametocyte differentiation was shown to be modulated by specific transcription factors. Here, we show that alternative splicing establishes a subsequent layer of regulation, controlling genes relating to consequent sex-specific differentiation of gametocytes. CONCLUSIONS: We demonstrate that alternative splicing is reprogrammed during cellular differentiation of a unicellular protist. Disruption of an alternative splicing factor, PbSR-MG, perturbs sex-specific alternative splicing and decreases the ability of the parasites to differentiate into male gametes and oocysts, thereby reducing transmission between vertebrate and insect hosts. Our results reveal alternative splicing as an integral, stage-specific phenomenon in these protists and as a regulator of cellular differentiation that arose early in eukaryotic evolution.


Assuntos
Processamento Alternativo , Plasmodium berghei/genética , Animais , Células Germinativas/metabolismo , Estágios do Ciclo de Vida/genética , Camundongos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Transcrição Gênica
16.
Science ; 352(6283): 349-53, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27081071

RESUMO

Drug resistance compromises control of malaria. Here, we show that resistance to a commonly used antimalarial medication, atovaquone, is apparently unable to spread. Atovaquone pressure selects parasites with mutations in cytochrome b, a respiratory protein with low but essential activity in the mammalian blood phase of the parasite life cycle. Resistance mutations rescue parasites from the drug but later prove lethal in the mosquito phase, where parasites require full respiration. Unable to respire efficiently, resistant parasites fail to complete mosquito development, arresting their life cycle. Because cytochrome b is encoded by the maternally inherited parasite mitochondrion, even outcrossing with wild-type strains cannot facilitate spread of resistance. Lack of transmission suggests that resistance will be unable to spread in the field, greatly enhancing the utility of atovaquone in malaria control.


Assuntos
Anopheles/parasitologia , Antimaláricos/farmacologia , Atovaquona/farmacologia , Citocromos b/genética , Resistência a Medicamentos/genética , Malária/parasitologia , Mitocôndrias/genética , Plasmodium berghei/efeitos dos fármacos , Animais , Antimaláricos/uso terapêutico , Atovaquona/uso terapêutico , Linhagem Celular , Genes Mitocondriais/genética , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/genética , Malária/tratamento farmacológico , Malária/transmissão , Masculino , Camundongos , Mutação , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Seleção Genética
17.
Int J Parasitol ; 42(6): 519-27, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22406332

RESUMO

Vector-borne diseases constitute an enormous burden on public health across the world. However, despite the importance of interactions between infectious pathogens and their respective vector for disease transmission, the biology of the pathogen in the insect is often less well understood than the forms that cause human infections. Even with the global impact of Plasmodium parasites, the causative agents of malarial disease, no vaccine exists to prevent infection and resistance to all frontline drugs is emerging. Malaria parasite migration through the mosquito host constitutes a major population bottleneck of the lifecycle and therefore represents a powerful, although as yet relatively untapped, target for therapeutic intervention. The understanding of parasite-mosquito interactions has increased in recent years with developments in genome-wide approaches, genomics and proteomics. Each development has shed significant light on the biology of the malaria parasite during the mosquito phase of the lifecycle. Less well understood, however, is the process of midgut colonisation and oocyst formation, the precursor to parasite re-infection from the next mosquito bite. Here, we review the current understanding of cellular and molecular events underlying midgut colonisation centred on the role of the motile ookinete. Further insight into the major interactions between the parasite and the mosquito will help support the broader goal to identify targets for transmission-blocking therapies against malarial disease.


Assuntos
Culicidae/parasitologia , Interações Hospedeiro-Parasita , Plasmodium/crescimento & desenvolvimento , Animais , Entomologia/tendências , Trato Gastrointestinal/parasitologia , Genômica/métodos , Parasitologia/tendências , Plasmodium/patogenicidade , Proteômica/métodos
18.
Mol Biochem Parasitol ; 182(1-2): 93-6, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22138565

RESUMO

Malaria parasite motility relies on an internal parasite actomyosin motor that, when linked to the host cell substrate, propels motile zoites forward. Despite their key role in this process, attempts to visualize actin microfilaments (F-actin) during motility and under native microscopy conditions have not to date been successful. Towards facilitating their visualization we present here a Plasmodium berghei transgenic line in which a green fluorescent protein (GFP)-actin fusion is constitutively expressed through the lifecycle. Focused investigation of the largest motile form, the insect stage ookinete, demonstrates a large cytosolic pool of actin with no obvious F-actin structures. However, following treatment with the actin filament-stabilizing drug Jasplakinolide, we show evidence for concentration of F-actin dynamics in the parasite pellicle and at polar apices. These observations support current models for gliding motility and establish a cellular tool for further exploration of the diverse roles actin is thought to play throughout parasite development.


Assuntos
Actinas/química , Proteínas de Fluorescência Verde/química , Estágios do Ciclo de Vida , Plasmodium berghei/química , Actinas/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/química , Depsipeptídeos/farmacologia , Processamento de Imagem Assistida por Computador/métodos , Locomoção , Proteínas dos Microfilamentos/química , Microscopia Eletrônica , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/ultraestrutura , Proteínas de Protozoários/química
19.
PLoS One ; 7(2): e32188, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22389687

RESUMO

Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu.


Assuntos
Citoesqueleto de Actina/metabolismo , Malária/parasitologia , Proteínas de Protozoários/metabolismo , Citoesqueleto de Actina/química , Animais , Humanos , Estágios do Ciclo de Vida/fisiologia , Merozoítos/metabolismo , Camundongos , Plasmodium berghei/metabolismo , Plasmodium falciparum/metabolismo , Estrutura Secundária de Proteína , Esporozoítos/metabolismo
20.
J Exp Med ; 208(7): 1547-59, 2011 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-21690250

RESUMO

Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The apicoplast is indispensable and an attractive drug target. The apicoplast is home to a 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for the synthesis of isoprenoid precursors. This pathway is believed to be the most conserved function of the apicoplast, and fosmidomycin, a specific inhibitor of the pathway, is an effective antimalarial. Surprisingly, fosmidomycin has no effect on most other apicomplexans. Using Toxoplasma gondii, we establish that the pathway is essential in parasites that are highly fosmidomycin resistant. We define the molecular basis of resistance and susceptibility, experimentally testing various host and parasite contributions in T. gondii and Plasmodium. We demonstrate that in T. gondii the parasite plasma membrane is a critical barrier to drug uptake. In strong support of this hypothesis, we engineer de novo drug-sensitive T. gondii parasites by heterologous expression of a bacterial transporter protein. Mice infected with these transgenic parasites can now be cured from a lethal challenge with fosmidomycin. We propose that the varied extent of metabolite exchange between host and parasite is a crucial determinator of drug susceptibility and a predictor of future resistance.


Assuntos
Fosfomicina/análogos & derivados , Terpenos/metabolismo , Toxoplasma/efeitos dos fármacos , Toxoplasma/metabolismo , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Animais , Animais Geneticamente Modificados , Antiprotozoários/farmacologia , Permeabilidade da Membrana Celular , Resistência a Medicamentos/fisiologia , Fosfomicina/farmacologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Redes e Vias Metabólicas , Camundongos , Modelos Biológicos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Organelas/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Pentosefosfatos/metabolismo , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/metabolismo , Plasmodium berghei/patogenicidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxoplasma/genética , Toxoplasma/patogenicidade
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