RESUMO
Plasmodium sporozoites are extracellular forms introduced during mosquito bite that selectively invade mammalian hepatocytes. Sporozoites are delimited by a cell membrane that is linked to the underlying acto-myosin molecular motor. While membrane proteins with roles in motility and invasion have been well studied, very little is known about proteins that maintain the sporozoite shape. We demonstrate that in Plasmodium berghei (Pb) a conserved hypothetical gene, PBANKA_1422900 specifies sporozoite structural integrity maintenance protein (SIMP) required for maintaining the sporozoite shape and motility. Sporozoites lacking SIMP exhibited loss of regular shape, extensive membrane blebbing at multiple foci, and membrane detachment. The mutant sporozoites failed to infect hepatocytes, though the altered shape did not affect the organization of cytoskeleton or inner membrane complex (IMC). Interestingly, the components of IMC failed to extend under the membrane blebs likely suggesting that SIMP may assist in anchoring the membrane to IMC. Endogenous C-terminal HA tagging localized SIMP to membrane and revealed the C-terminus of the protein to be extracellular. Since SIMP is highly conserved among Plasmodium species, these findings have important implications for utilizing it as a novel sporozoite-specific vaccine candidate.
Assuntos
Proteínas de Protozoários , Esporozoítos , Animais , Dipeptídeos , Hepatócitos/metabolismo , Mamíferos/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Proteínas de Protozoários/metabolismo , Esporozoítos/metabolismoRESUMO
The interplay between ATP generating and utilizing pathways in a cell is responsible for maintaining cellular ATP/energy homeostasis that is reflected by Adenylate Energy Charge (AEC) ratio. Adenylate kinase (AK), that catalyzes inter-conversion of ADP, ATP and AMP, plays a major role in maintaining AEC and is regulated by cellular AMP levels. Hence, the enzymes AMP deaminase (AMPD) and nucleotidases, which catabolize AMP, indirectly regulate AK activity and in-turn affect AEC. Here, we present the first report on AMPD from Plasmodium, the causative agent of malaria. The recombinant enzyme expressed in Saccharomyces cerevisiae was studied using functional complementation assay and residues vital for enzyme activity have been identified. Similarities and differences between Plasmodium falciparum AMPD (PfAMPD) and its homologs from yeast, Arabidopsis and humans are also discussed. The AMPD gene was deleted in the murine malaria parasite P. berghei and was found to be dispensable during all stages of the parasite life cycle. However, when episomal expression was attempted, viable parasites were not obtained, suggesting that perturbing AMP homeostasis by over-expressing AMPD might be lethal. As AMPD is known to be allosterically modulated by ATP, GTP and phosphate, allosteric activators of PfAMPD could be developed as anti-parasitic agents.
Assuntos
AMP Desaminase/química , AMP Desaminase/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , AMP Desaminase/genética , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Catálise , Humanos , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genéticaRESUMO
SUMOylation is a reversible post translational modification of proteins that regulates protein stabilization, nucleocytoplasmic transport, and protein-protein interactions. Several viruses and bacteria modulate host SUMOylation machinery for efficient infection. Plasmodium sporozoites are infective forms of malaria parasite that invade mammalian hepatocytes and transforms into exoerythrocytic forms (EEFs). Here, we show that during EEF development, the distribution of SUMOylated proteins in host cell nuclei was significantly reduced and expression of the SUMOylation enzymes was downregulated. Plasmodium EEFs destabilized the host cytoplasmic protein SMAD4 by inhibiting its SUMOylation. SUMO1 overexpression was detrimental to EEF growth, and insufficiency of the only conjugating enzyme Ubc9/E2 promoted EEF growth. The expression of genes involved in suppression of host cell defense pathways during infection was reversed during SUMO1 overexpression, as revealed by transcriptomic analysis. The inhibition of host cell SUMOylation was also observed during Toxoplasma infection. We provide a hitherto unknown mechanism of regulating host gene expression by Apicomplexan parasites through altering host SUMOylation.
Assuntos
Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Proteína SUMO-1/biossíntese , Sumoilação/fisiologia , Toxoplasma/genética , Toxoplasma/metabolismo , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Plasmodium berghei/citologia , Plasmodium berghei/crescimento & desenvolvimento , Interferência de RNA , RNA Interferente Pequeno/genética , Coelhos , Proteína Smad4/metabolismo , Esporozoítos/citologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
BACKGROUND: Emerging resistance of the malaria parasite Plasmodium to current therapies underscores the critical importance of exploring novel strategies for disease eradication. Plasmodium species are obligate intracellular protozoan parasites. They rely on an unusual form of substrate-dependent motility for their migration on and across host-cell membranes and for host cell invasion. This peculiar motility mechanism is driven by the 'glideosome', an actin-myosin associated, macromolecular complex anchored to the inner membrane complex of the parasite. Myosin A, actin, aldolase, and thrombospondin-related anonymous protein (TRAP) constitute the molecular core of the glideosome in the sporozoite, the mosquito stage that brings the infection into mammals. METHODS: Virtual library screening of a large compound library against the PfAldolase-TRAP complex was used to identify candidate compounds that stabilize and prevent the disassembly of the glideosome. The mechanism of these compounds was confirmed by biochemical, biophysical and parasitological methods. RESULTS: A novel inhibitory effect on the parasite was achieved by stabilizing a protein-protein interaction within the glideosome components. Compound 24 disrupts the gliding and invasive capabilities of Plasmodium parasites in in vitro parasite assays. A high-resolution, ternary X-ray crystal structure of PfAldolase-TRAP in complex with compound 24 confirms the mode of interaction and serves as a platform for future ligand optimization. CONCLUSION: This proof-of-concept study presents a novel approach to anti-malarial drug discovery and design. By strengthening a protein-protein interaction within the parasite, an avenue towards inhibiting a previously "undruggable" target is revealed and the motility motor responsible for successful invasion of host cells is rendered inactive. This study provides new insights into the malaria parasite cell invasion machinery and convincingly demonstrates that liver cell invasion is dramatically reduced by 95 % in the presence of the small molecule stabilizer compound 24.
Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/química , Proteínas de Protozoários/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Frutose-Bifosfato Aldolase/química , Hepatócitos/efeitos dos fármacos , Humanos , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Complexos Multiproteicos/efeitos dos fármacos , Plasmodium falciparum/química , Estabilidade Proteica/efeitos dos fármacos , Proteínas de Protozoários/química , Coelhos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/toxicidade , Ressonância de Plasmônio de SuperfícieRESUMO
Malaria parasites must respond to stresses and environmental signals to perpetuate efficiently during their multistage development in diverse environments. To gain insights into the parasite's stress response mechanisms, we investigated a conserved Plasmodium protein, which we have named plasmoDJ1 on the basis of the presence of a putative cysteine protease motif of the DJ-1/PfpI superfamily, for its activities, potential to respond to stresses and role in parasite development. PlasmoDJ1 is expressed in all intraerythrocytic stages and ookinetes. Its expression was increased 7-9-fold upon heat shock and oxidative stress due to H2O2 and artemisinin; its expression in a stress-sensitive Escherichia coli mutant conferred tolerance against oxidative stress, indicating that plasmoDJ1 has the potential to sense and/or protect from stresses. Recombinant plasmoDJ1 efficiently neutralized H2O2, facilitated renaturation of denatured citrate synthase and showed protease activity, indicating that plasmoDJ1 is a multi-activity protein. Mutation of the catalytic cysteine residue, but not other residues, reduced H2O2-neutralization activity by ~90% and significantly decreased chaperone and protease activities, indicating that these activities are intrinsic to plasmoDJ1. The plasmoDJ1 gene knockout in Plasmodium berghei ANKA attenuated virulence and reduced oocyst production, suggesting a major role for plasmoDJ1 in parasite development, which probably depends on its multiple activities.
Assuntos
Oocistos/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/patogenicidade , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Adaptação Fisiológica , Sequência de Aminoácidos , Animais , Antimaláricos/farmacologia , Artemisininas/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Deleção de Genes , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Oocistos/crescimento & desenvolvimento , Estresse Oxidativo , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência , VirulênciaRESUMO
Malaria parasites must respond to stresses and environmental signals to perpetuate efficiently during their multistage development in diverse environments. To gain insights into the parasite's stress response mechanisms, we investigated a conserved Plasmodium protein, which we have named plasmoDJ1 on the basis of the presence of a putative cysteine protease motif of the DJ-1/PfpI superfamily, for its activities, potential to respond to stresses and role in parasite development. PlasmoDJ1 is expressed in all intraerythrocytic stages and ookinetes. Its expression was increased 7-9-fold upon heat shock and oxidative stress due to H2O2 and artemisinin; its expression in a stress-sensitive Escherichia coli mutant conferred tolerance against oxidative stress, indicating that plasmoDJ1 has the potential to sense and/or protect from stresses. Recombinant plasmoDJ1 efficiently neutralized H2O2, facilitated renaturation of denatured citrate synthase and showed protease activity, indicating that plasmoDJ1 is a multi-activity protein. Mutation of the catalytic cysteine residue, but not other residues, reduced H2O2-neutralization activity by ~90% and significantly decreased chaperone and protease activities, indicating that these activities are intrinsic to plasmoDJ1. The plasmoDJ1 gene knockout in Plasmodium berghei ANKA attenuated virulence and reduced oocyst production, suggesting a major role for plasmoDJ1 in parasite development, which probably depends on its multiple activities.
Assuntos
Cisteína Endopeptidases/genética , Oocistos/enzimologia , Plasmodium berghei/enzimologia , Plasmodium berghei/patogenicidade , Plasmodium falciparum/enzimologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Artemisininas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Família Multigênica , Mutação , Oocistos/efeitos dos fármacos , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/fisiologia , Ratos , Ratos Wistar , Virulência/efeitos dos fármacos , Virulência/genéticaRESUMO
Theileriosis is a tick-borne disease caused by Theileria annulata, an intracellular parasite that belongs to the phylum Apicomplexa. The infective forms of the parasite to cattle are sporozoites that are introduced into the host when the infected ticks take a blood meal. The sporozoites selectively invade bovine B cells, macrophages, or monocytes, leading to their cellular transformation. The parasite factors involved in the host cell transformation are not well explored. In pursuit of this, we revisited the probable secretome of the parasite and, following a stringent downscaling criterion, have identified Theileria prohibitin (TaPHB-1) as one of factors secreted into the host cells. Interestingly, in infected cells, TaPHB-1 localized both on the parasite surface and in the host cytoplasm, and independent approaches such as coimmunoprecipitation, yeast two-hybrid screening (Y2H), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed RuvB-like AAA ATPase 1 (RUVBL-1) as one of its interacting partners. Further, the T. annulata infection does not affect the localization of bovine prohibitin. Mitigating the expression of bovine RUVBL-1 precluded the translocation of TaPHB-1 in the host cell cytoplasm without affecting the host cell viability. Taken together, we report for the first time a unique interaction of TaPHB-1 with bovine RUVBL-1 that is likely needed to cause cancer-like hallmarks during theileriosis. IMPORTANCE Theileria annulata is an apicomplexan parasite that causes tropical theileriosis in cattle. It is the only eukaryotic pathogen able to cause cellular transformation of host cells yielding a cancer-like phenotype. The parasite factors responsible for the transformation of the host cell are largely unknown. This study demonstrates for the first time the partial role of Theileria prohibitin (TaPHB-1) in maintaining the transformed state of the host cell and its interaction with RuvB-like AAA ATPase 1 (RUVBL-1) in a T. annulata-infected bovine cell line. Interestingly, the knockdown of bovine RUVBL-1 rendered the parasites metabolically inactive, implying that the identified interaction is critical for parasite survival. This study contributes to our understanding the Theileria-host interactions and offers scope for novel therapeutic interventions to control theileriosis.
Assuntos
Theileria annulata , Theileriose , Bovinos , Animais , Theileriose/parasitologia , Adenosina Trifosfatases/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Cromatografia Líquida , Proibitinas , Espectrometria de Massas em Tandem , Theileria annulata/genética , Theileria annulata/metabolismoRESUMO
DNA damage inducible 1 protein (DDI1) is involved in a variety of cellular processes including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of a RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is important for parasite survival, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating the potential of DDI1 knock-down parasites as a whole organism vaccine. Plasmodium falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA-damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. As DDI1 knock-down parasites confer protective immunity and it could be a target of HIV protease inhibitors, Plasmodium DDI1 is a potential therapeutic target for malaria control.
Assuntos
Antimaláricos , Inibidores da Protease de HIV , Plasmodium , Proteínas de Saccharomyces cerevisiae , Animais , Humanos , Camundongos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Dano ao DNA , Plasmodium/genética , DNA , Cromatina , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genéticaRESUMO
Malaria infection starts when mosquitoes inject sporozoites into the skin. The parasites enter the blood stream and make their way to the liver where they develop into the exo-erythrocytic forms (EEFs). Immunization with irradiated sporozoites (IrSp) leads to robust protection against malaria infection in rodents, monkeys and humans by eliciting antibodies to circumsporozoite protein (CS) that inhibit sporozoite infectivity, and T cells that destroy the EEFs. To study the role of non-CS antigens in protection, we produced CS transgenic mice that were tolerant to CS T-cell epitopes. Here we show that in the absence of T-cell-dependent immune responses to CS, protection induced by immunization with two doses of IrSp was greatly reduced. Thus, although hundreds of other Plasmodium genes are expressed in sporozoites and EEFs, CS is a dominant protective antigen. Nevertheless, sterile immunity could be obtained by immunization of CS transgenics with three doses of IrSp.
Assuntos
Antígenos de Protozoários/imunologia , Epitopos Imunodominantes/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Esporozoítos/efeitos da radiação , Animais , Vacinas Antimaláricas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esporozoítos/química , Linfócitos T Citotóxicos/imunologiaRESUMO
The interplay between yHSP90α (Hsp82) and Rad51 has been implicated in the DNA double-strand break repair (DSB) pathway in yeast. Here we report that nuclear translocation of yHSP90α and its recruitment to the DSB end are essential for homologous recombination (HR)-mediated DNA repair in yeast. The HsHSP90α possesses an amino-terminal extension which is phosphorylated upon DNA damage. We find that the absence of the amino-terminal extension in yHSP90α does not compromise its nuclear import, and the nonphosphorylatable-mutant HsHSP90αT7A could be imported to the yeast nucleus upon DNA damage. Interestingly, the flexible charged-linker (CL) domains of both yHSP90α and HsHSP90α play a critical role during their nuclear translocation. The conformational restricted CL mutant yHSP90α∆(211-259), but not a shorter deletion version yHSP90α∆(211-242), fails to reach the nucleus. As the CL domain of yHSP90α is critical for its interaction with Aha1, we investigated whether Aha1 promotes the nuclear import of yHSP90α. We found that the nuclear import of yHSP90α is severely affected in ∆aha1 strain. Moreover, Aha1 is accumulated in the nucleus during DNA damage. Hence Aha1 may serve as a potential target for inhibiting nuclear function of yHSP90α. The increased sensitivity of ∆aha1 strain to genotoxic agents strengthens this notion.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Quebras de DNA de Cadeia Dupla , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Dano ao DNA , Reparo do DNA , Rad51 Recombinase/metabolismo , Proteínas de Choque Térmico HSP90/metabolismoRESUMO
The homologous recombination (HR) pathway has been implicated as the predominant mechanism for the repair of chromosomal DNA double-strand breaks (DSBs) of the malarial parasite. Although the extrachromosomal mitochondrial genome of this parasite experiences a greater number of DSBs due to its close proximity to the electron transport chain, nothing is known about the proteins involved in the repair of the mitochondrial genome. We investigated the involvement of nucleus-encoded HR proteins in the repair of the mitochondrial genome, as this genome does not code for any DNA repair proteins. Here, we provide evidence that the nucleus-encoded "recombinosome" of the parasite is also involved in mitochondrial genome repair. First, two crucial HR proteins, namely, Plasmodium falciparum Rad51 (PfRad51) and P. falciparum Bloom helicase (PfBlm) are located in the mitochondria. They are recruited to the mitochondrial genome at the schizont stage, a stage that is prone to DSBs due to exposure to various endogenous and physiologic DNA-damaging agents. Second, the recruitment of these two proteins to the damaged mitochondrial genome coincides with the DNA repair kinetics. Moreover, both the proteins exit the mitochondrial DNA (mtDNA) once the genome is repaired. Most importantly, the specific chemical inhibitors of PfRad51 and PfBlm block the repair of UV-induced DSBs of the mitochondrial genome. Additionally, overexpression of these two proteins resulted in a kinetically faster repair. Given the essentiality of the mitochondrial genome, blocking its repair by inhibiting the HR pathway could offer a novel strategy for curbing malaria. IMPORTANCE The impact of malaria on global public health and the world economy continues to surge despite decades of vaccine research and drug development efforts. An alarming rise in resistance toward all the commercially available antimalarial drugs and the lack of an effective malaria vaccine brings us to the urge to identify novel intervention strategies for curbing malaria. Here, we uncover the molecular mechanism behind the repair of the most deleterious form of DNA lesions on the parasitic mitochondrial genome. Given that the single-copy mitochondrion is an indispensable organelle of the malaria parasite, we propose that targeting the mitochondrial DNA repair pathways should be exploited as a potential malaria control strategy. The establishment of the parasitic homologous recombination machinery as the predominant repair mechanism of the mitochondrial DNA double-strand breaks underscores the importance of this pathway as a novel druggable target.
Assuntos
Antimaláricos/farmacologia , Genoma Mitocondrial/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Rad51 Recombinase/antagonistas & inibidores , RecQ Helicases/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Recombinação Homóloga , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismoRESUMO
Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.
Assuntos
Adenina/análogos & derivados , Antimaláricos/farmacologia , Cicloexilaminas/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Malária/parasitologia , Plasmodium falciparum/enzimologia , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas de Protozoários/antagonistas & inibidores , Adenina/química , Adenina/farmacologia , Adenina/uso terapêutico , Animais , Antimaláricos/química , Antimaláricos/uso terapêutico , Células CHO , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Cicloexilaminas/química , Cicloexilaminas/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária/tratamento farmacológico , Malária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Movimento/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Testes de Sensibilidade Parasitária , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas Quinases/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
In Plasmodium, protein kinases govern key biological processes of the parasite life cycle involved in the establishment of infection, dissemination and sexual reproduction. The rodent malaria model P lasmodium berghei encodes for 66 putative eukaryotic protein kinases (ePKs) as identified through modelling domain signatures and are highly conserved in Plasmodium falciparum We report here the functional characterisation of a putative serine-threonine kinase P BANKA_0311400 identified in this kinome analysis and designate it as Pbstk2 To elucidate its role, we knocked out Pbstk2 locus and performed a detailed phenotypic analysis at different life cycle stages. The Pbstk2 knockout (KO) was not compromised in asexual blood stage propagation, transmission and development in the mosquito vector. The Pbstk2 KO produced viable salivary gland sporozoites that successfully transformed into exo-erythrocytic forms (EEFs) and were morphologically indistinguishable from wild-type GFP (WT GFP) with regard to size and shape until 48â h. An intravenous dose of 1×103 Pbstk2 KO sporozoites in C57BL/6 mice failed to establish blood stage infection and a higher dose of 5X103 showed a 2-3â day delay in prepatency as compared to WT GFP parasites. Consistent with such an observation, analysis of in vitro EEF development at 62â h revealed that the hepatic merozoite numbers were reduced to nearly 40% as compared to WT GFP and showed meagre expression of MSP1. Our studies provide evidence for the role of PbSTK2 in late liver stage development and for the successful establishment of a timely blood stage infection.
RESUMO
Plasmodium sporozoites are infective forms of the parasite to mammalian hepatocytes. Sporozoite surface or secreted proteins likely play an important role in recognition, invasion and successful establishment of hepatocyte infection. By approaches of reverse genetics, we report the functional analysis of two Plasmodium berghei (Pb) sporozoite specific genes- PbS10 and PbS23/SSP3 that encode for proteins with a putative signal peptide. The expression of both genes was high in oocyst and salivary gland sporozoite stages as compared to other life cycle stages and PbS23/SSP3 protein was detected in salivary gland sporozoites. Both mutants were indistinguishable to wild-type parasites with regard to asexual growth in RBC, ability to complete sexual reproduction and form sporozoites in vector host. While the sporozoite stage of both mutants were able to glide and invade hepatocytes normally in vitro and in vivo, PbS10 mutants suffered growth attenuation at an early stage while PbS23/SSP3 mutants manifested defect during late exo-erythrocytic form maturation. Interestingly, both mutants gave rare breakthrough infections, suggesting that while both were critical for liver stage development, their depletion did not completely abrogate blood stage infection. These findings have important implications for weakening sporozoites by multiple gene attenuation towards the generation of a safe whole organism vaccine.
Assuntos
Malária/parasitologia , Plasmodium berghei/metabolismo , Proteínas de Protozoários/metabolismo , Esporozoítos/crescimento & desenvolvimento , Animais , Eritrócitos/parasitologia , Feminino , Humanos , Estágios do Ciclo de Vida , Camundongos , Camundongos Endogâmicos C57BL , Oocistos/genética , Oocistos/crescimento & desenvolvimento , Oocistos/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Especificidade da Espécie , Esporozoítos/genética , Esporozoítos/metabolismoRESUMO
The irradiated-sporozoite vaccine elicits sterile immunity against Plasmodium parasites in experimental rodent hosts and human volunteers. Based on rodent malaria models, it has been proposed that CD8+ T cells are the key protective effector mechanism required in sporozoite-induced immunity. To investigate the role of class II-restricted immunity in protective immunity, we immunized beta2-microglobulin knockout (beta2M-/-) mice with irradiated Plasmodium yoelii or P. berghei sporozoites. Sterile immunity was obtained in the CD8+-T-cell-deficient mice immunized with either P. berghei or P. yoelii sporozoites. beta2M-/- mice with the BALB/c (H-2d) genetic background as well as those with the C57BL (H-2b) genetic background were protected. Effector mechanisms included CD4+ T cells, mediated in part through the production of gamma interferon, and neutralizing antibodies that targeted the extracellular sporozoites. We conclude that in the absence of class I-restricted CD8+ T cells, sporozoite-induced protective immunity can be effectively mediated by class II-restricted immune effector mechanisms. These results support efforts to develop subunit vaccines that effectively elicit high levels of antibody and CD4+ T cells to target Plasmodium pre-erythrocytic stages.
Assuntos
Malária/prevenção & controle , Plasmodium berghei/imunologia , Plasmodium yoelii/imunologia , Esporozoítos/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Linfócitos T CD4-Positivos/imunologia , Interferon gama/imunologia , Fígado/parasitologia , Depleção Linfocítica , Malária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Testes de Neutralização , Plasmodium berghei/efeitos da radiação , Plasmodium yoelii/efeitos da radiação , Microglobulina beta-2/deficiênciaRESUMO
In Plasmodium, the shikimate pathway is a potential target for malaria chemotherapy owing to its absence in the mammalian host. Chorismate, the end product of this pathway, serves as a precursor for aromatic amino acids, Para-aminobenzoic acid and ubiquinone, and is synthesised by Chorismate synthase (CS). Therefore, it follows that the Cs locus may be refractory to genetic manipulation. By utilising a conditional mutagenesis system of yeast Flp/FRT, we demonstrate an unexpectedly dispensable role of CS in Plasmodium. Our studies reiterate the need to establish an obligate reliance on Plasmodium metabolic enzymes through genetic approaches before their selection as drug targets.
Assuntos
Ácido Corísmico/metabolismo , Malária/parasitologia , Mosquitos Vetores/parasitologia , Fósforo-Oxigênio Liases/metabolismo , Plasmodium berghei/crescimento & desenvolvimento , Ácido Chiquímico/metabolismo , Sequência de Aminoácidos , Animais , Anopheles/parasitologia , Feminino , Técnicas de Inativação de Genes , Células Hep G2 , Humanos , Fígado/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/genética , Filogenia , Plasmodium berghei/enzimologia , Plasmodium berghei/genéticaRESUMO
Malaria sporozoites migrate through several cells prior to a productive invasion that involves the formation of a parasitophorous vacuole (PV) where sporozoites undergo transformation into Exo-erythorcytic forms (EEFs). The precise mechanism leading to sporozoite activation for invasion is unknown, but prior traversal of host cells is required. During cell migration sporozoites are exposed to large shifts in K(+) concentration. We report here that incubation of sporozoites to the intracellular K(+) concentration enhances 8-10 times the infectivity of Plasmodium berghei and 4-5 times the infectivity of Plasmodium yoelli sporozoites for a hepatocyte cell line, while simultaneously decreasing cell passage activity. The K(+) enhancing effect was time and concentration dependent, and was significantly decreased by K(+) channel inhibitors. Potassium-treated P. berghei sporozoites also showed enhanced numbers of EEFs in non-permissive cell lines. Treated sporozoites had reduced infectivity for mice, but infectivity was enhanced upon Kupffer cell depletion. Transcriptional analysis of K(+) treated and control sporozoites revealed a high degree of correlation in their levels of gene expression, indicating that the observed phenotypic changes are not due to radical changes in gene transcription. Only seven genes were upregulated by more than two-fold in K(+) treated sporozoites. The highest level was noted in PP2C, a phosphatase known to dephosphorylate the AKT potassium channel in plants.
Assuntos
Plasmodium berghei/patogenicidade , Plasmodium yoelii/patogenicidade , Potássio/farmacologia , Esporozoítos/efeitos dos fármacos , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Hepatócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Células de Kupffer/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium yoelii/efeitos dos fármacos , Plasmodium yoelii/crescimento & desenvolvimento , Inoculações Seriadas , Esporozoítos/crescimento & desenvolvimento , Esporozoítos/fisiologiaRESUMO
Plasmodium aspartic proteases, termed plasmepsins (PMs) play many critical roles such as haemoglobin degradation, cleavage of PEXEL proteins and sporozoite development in the parasite life cycle. Most of the plasmepsins are well characterized, however the role of PM VIII in Plasmodium remains unknown. Here, we elucidate the functions of PM VIII (PBANKA_132910) in the rodent malaria parasite Plasmodium berghei (Pb). By targeted gene deletion, we show that PbPM VIII is critical for sporozoite egress from an oocyst and gliding motility, which is a prerequisite for the invasion of salivary glands and subsequent transmission to the vertebrate host.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Plasmodium berghei/fisiologia , Proteínas de Protozoários/metabolismo , Animais , Anopheles/parasitologia , Ácido Aspártico Endopeptidases/genética , Culicidae/parasitologia , Modelos Animais de Doenças , Feminino , Células Hep G2 , Humanos , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Movimento/fisiologia , Oocistos/enzimologia , Oocistos/fisiologia , Fenótipo , Plasmodium berghei/enzimologia , Proteínas de Protozoários/genética , Glândulas Salivares/parasitologia , Esporozoítos/enzimologia , Esporozoítos/fisiologiaRESUMO
Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus.
Assuntos
Sequência Conservada , Eritrócitos/parasitologia , Proteínas de Membrana/metabolismo , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Esporozoítos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anopheles/parasitologia , Eritrócitos/metabolismo , Feminino , Dosagem de Genes , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde , Células Hep G2 , Humanos , Imunidade , Imunização , Estágios do Ciclo de Vida , Fígado/parasitologia , Malária/imunologia , Malária/parasitologia , Malária/transmissão , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Fenótipo , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glândulas Salivares/metabolismo , Especificidade da Espécie , Esporozoítos/crescimento & desenvolvimentoRESUMO
UNLABELLED: Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity. IMPORTANCE: Malaria is responsible for more mortality than any other parasitic disease. Resistance to antimalarial medicines is a recurring problem; new drugs are urgently needed. A key to the parasite's successful intracellular development in the liver is the metabolic changes necessary to convert the parasite from a sporozoite to a replication-competent, metabolically active trophozoite form. Our study reinforces the burgeoning concept that organellar changes during parasite differentiation are mediated by an autophagy-like process. We have identified ATG8 in Plasmodium liver forms as an important effector that controls the development and fate of organelles, e.g., the clearance of micronemes that are required for hepatocyte invasion and the expansion of the apicoplast that produces many metabolites indispensable for parasite replication. Given the unconventional properties and the importance of ATG8 for parasite development in hepatocytes, targeting the parasite's autophagic pathway may represent a novel approach to control malarial infections.