RESUMO
Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.
Assuntos
Antiprotozoários , Naftoquinonas , Parasitos , Theileria annulata , Theileriose , Carrapatos , Animais , Bovinos , Theileriose/tratamento farmacológico , Theileriose/parasitologia , Theileria annulata/genética , Citocromos b/genética , Isoleucina/farmacologia , Metionina/farmacologia , Antiprotozoários/farmacologia , Mutação , Racemetionina/farmacologia , Antiparasitários/farmacologia , Carrapatos/parasitologiaRESUMO
Despite several initiatives to subside the global malaria burden, the spread of artemisinin-resistant parasites poses a big threat to malaria elimination. Mutations in PfKelch13 are predictive of ART resistance, whose underpinning molecular mechanism remains obscure. Recently, endocytosis and stress response pathways such as the ubiquitin-proteasome machinery have been linked to artemisinin resistance. With Plasmodium, however, ambiguity persists regarding a role in ART resistance for another cellular stress defence mechanism called autophagy. Therefore, we investigated whether, in the absence of ART treatment, basal autophagy is augmented in PfK13-R539T mutant ART-resistant parasites and analyzed whether PfK13-R539T endowed mutant parasites with an ability to utilize autophagy as a pro-survival strategy. We report that in the absence of any ART treatment, PfK13-R539T mutant parasites exhibit increased basal autophagy compared to PfK13-WT parasites and respond aggressively through changes in autophagic flux. A clear cytoprotective role of autophagy in parasite resistance mechanism is evident by the observation that a suppression of PI3-Kinase (PI3K) activity (a master autophagy regulator) rendered difficulty in the survival of PfK13-R539T ART-resistant parasites. In conclusion, we now show that higher PI3P levels reported for mutant PfKelch13 backgrounds led to increased basal autophagy that acts as a pro-survival response to ART treatment. Our results highlight PfPI3K as a druggable target with the potential to re-sensitize ART-resistant parasites and identify autophagy as a pro-survival function that modulates ART-resistant parasite growth.
RESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that can play critical roles in regulating various cellular processes, including during many parasitic infections. Here, we report a regulatory role for miR-34c-3p in cAMP-independent regulation of host cell protein kinase A (PKA) activity in Theileria annulata-infected bovine leukocytes. We identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit) as a novel miR-34c-3p target gene and demonstrate how infection-induced upregulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity. As a result, the disseminating tumorlike phenotype of T. annulata-transformed macrophages is enhanced. Finally, we extend our observations to Plasmodium falciparum-parasitized red blood cells, where infection-induced augmentation in miR-34c-3p levels led to a drop in the amount of prkar2b mRNA and increased PKA activity. Collectively, our findings represent a novel cAMP-independent way of regulating host cell PKA activity in infections by Theileria and Plasmodium parasites. IMPORTANCE Small microRNA levels are altered in many diseases, including those caused by parasites. Here, we describe how infection by two important animal and human parasites, Theileria annulata and Plasmodium falciparum, induce changes in infected host cell miR-34c-3p levels to regulate host cell PKA kinase activity by targeting mammalian prkar2b. Infection-induced changes in miR-34c-3p levels provide a novel epigenetic mechanism for regulating host cell PKA activity independent of fluxes in cAMP to both aggravate tumor dissemination and improve parasite fitness.
Assuntos
MicroRNAs , Theileria annulata , Humanos , Bovinos , Animais , Theileria annulata/genética , Theileria annulata/metabolismo , MicroRNAs/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Mamíferos , Subunidade RIIbeta da Proteína Quinase Dependente de AMP CíclicoRESUMO
A fungal metabolite, FR235222, specifically inhibits a histone deacetylase of the apicomplexan parasite Toxoplasma gondii and TgHDAC3 has emerged as a key factor regulating developmental stage transition in this species. Here, we exploited FR235222 to ask if changes in histone acetylation regulate developmental stage transition of Theileria annulata, another apicomplexan species. We found that FR235222 treatment of T. annulata-infected transformed leukocytes induced a proliferation arrest. The blockade in proliferation was due to drug-induced conversion of intracellular schizonts to merozoites that lack the ability to maintain host leukocyte cell division. Induction of merogony by FR235222 leads to an increase in expression of merozoite-marker (rhoptry) proteins. RNA-seq of FR235222-treated T. annulata-infected B cells identified deregulated expression of 468 parasite genes including a number encoding parasite ApiAP2 transcription factors. Thus, similar to T. gondii, FR235222 inhibits T. annulata HDAC (TaHDAC1) activity and places parasite histone acetylation as a major regulatory event of the transition from schizonts to merozoites.
Assuntos
Theileria annulata , Theileria , Animais , Histona Desacetilase 1/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Merozoítos/metabolismo , Esquizontes/metabolismo , Theileria/metabolismoRESUMO
We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites.
RESUMO
Theileria are tick-transmitted parasites that cause often fatal leuko-proliferative diseases in cattle called tropical theileriosis (T. annulata) and East Coast fever (T. parva). However, upon treatment with anti-theilerial drug-transformed leukocytes die of apoptosis indicating that Theileria-induced transformation is reversible making infected leukocytes a powerful example of how intracellular parasites interact with their hosts. Theileria-transformed leukocytes disseminate throughout infected cattle causing a cancer-like disease and here, we discuss how cytokines, noncoding RNAs and oncometabolites can contribute to the transformed phenotype and disease pathology.
Assuntos
Doenças dos Bovinos/fisiopatologia , Leucócitos/parasitologia , Theileria/fisiologia , Theileriose/fisiopatologia , Animais , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Citocinas/genética , Citocinas/imunologia , Interações Hospedeiro-Parasita , Leucócitos/imunologia , Theileria/genética , Theileriose/genética , Theileriose/imunologia , Theileriose/parasitologiaRESUMO
Theileria parasites are classified in the phylum Apicomplexa that includes several genera of medical and veterinary importance such as Plasmodium, Babesia, Toxoplasma and Cryptosporidium. These protozoans have evolved subtle ways to reshape their intracellular niche for their own benefit and Theileria is no exception. This tick transmitted microorganism is unique among all eukaryotes in that its intracellular schizont stage is able to transform its mammalian host leukocytes into an immortalised highly disseminating cell that phenocopies tumour cells. Here, we describe what is known about secreted Theileria-encoded host cell manipulators.
Assuntos
Apicomplexa , Leucócitos , Theileria , Animais , Antígenos de Protozoários , Apicomplexa/imunologia , Apicomplexa/metabolismo , Transformação Celular Neoplásica , Interações Hospedeiro-Parasita/imunologia , Interações Hospedeiro-Parasita/fisiologia , Leucócitos/parasitologia , Leucócitos/patologia , Mamíferos/parasitologia , Theileria/imunologia , Theileria/metabolismoRESUMO
Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex. Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasites.IMPORTANCE Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion. Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites.
Assuntos
Proteínas 14-3-3/metabolismo , Eritrócitos/parasitologia , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Proteínas 14-3-3/genética , Humanos , Merozoítos/fisiologia , Fosforilação , Plasmodium falciparum/genética , Proteômica , Proteínas de Protozoários/genéticaRESUMO
Theileria annulata is a tick-transmitted apicomplexan parasite that infects and transforms bovine leukocytes into disseminating tumours that cause a disease called tropical theileriosis. Using comparative transcriptomics we identified genes transcriptionally perturbed during Theileria-induced leukocyte transformation. Dataset comparisons highlighted a small set of genes associated with Theileria-transformed leukocyte dissemination. The roles of Granzyme A (GZMA) and RAS guanyl-releasing protein 1 (RASGRP1) were verified by CRISPR/Cas9-mediated knockdown. Knocking down expression of GZMA and RASGRP1 in attenuated macrophages led to a regain in their dissemination in Rag2/γC mice confirming their role as dissemination suppressors in vivo. We further evaluated the roles of GZMA and RASGRP1 in human B lymphomas by comparing the transcriptome of 934 human cancer cell lines to that of Theileria-transformed bovine host cells. We confirmed dampened dissemination potential of human B lymphomas that overexpress GZMA and RASGRP1. Our results provide evidence that GZMA and RASGRP1 have a novel tumour suppressor function in both T. annulata-infected bovine host leukocytes and in human B lymphomas.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Supressores de Tumor/fisiologia , Granzimas/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Leucócitos/parasitologia , Linfoma de Células B/genética , Macrófagos/parasitologia , Theileria annulata/genética , Animais , Bovinos , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Linfoma de Células B/parasitologia , Camundongos , Theileria annulata/patogenicidadeRESUMO
Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.
Assuntos
Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Ubiquitina/genéticaRESUMO
Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells.
Assuntos
Vesículas Transportadoras/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Linhagem Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Parasitos/metabolismo , Transporte Proteico , Proteínas de Protozoários , Toxoplasma/metabolismo , Toxoplasmose/metabolismo , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
Intracellular pathogens have evolved intricate mechanisms to subvert host cell signaling pathways and ensure their own propagation. A lineage of the protozoan parasite genus Theileria infects bovine leukocytes and induces their uncontrolled proliferation causing a leukemia-like disease. Given the importance of E2F transcription factors in mammalian cell cycle regulation, we investigated the role of E2F signaling in Theileria-induced host cell proliferation. Using comparative genomics and surface plasmon resonance, we identified parasite-derived peptides that have the sequence-specific ability to increase E2F signaling by binding E2F negative regulator Retinoblastoma-1 (RB). Using these peptides as a tool to probe host E2F signaling, we show that the disruption of RB complexes ex vivo leads to activation of E2F-driven transcription and increased leukocyte proliferation in an infection-dependent manner. This result is consistent with existing models and, together, they support a critical role of E2F signaling for Theileria-induced host cell proliferation, and its potential direct manipulation by one or more parasite proteins.
Assuntos
Fatores de Transcrição E2F/metabolismo , Leucócitos/citologia , Leucócitos/parasitologia , Transdução de Sinais , Theileria/fisiologia , Linhagem Celular , Proliferação de Células , Fator de Transcrição E2F1/metabolismoRESUMO
We study the effect of different chemical moieties on the rigidity of red blood cells (RBCs) induced by Plasmodium falciparum infection, and the bystander effect previously found. The infected cells are obtained from a culture of parasite-infected RBCs grown in the laboratory. The rigidity of RBCs is measured by looking at the Brownian fluctuations of individual cells in an optical-tweezers trap. The results point towards increased intracellular cyclic adenosine monophosphate (cAMP) levels as being responsible for the increase in rigidity.
Assuntos
Eritrócitos , Malária Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Efeito Espectador , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Eritrócitos/patologia , Humanos , Pinças ÓpticasRESUMO
Plasmodium sp. are obligate intracellular parasites that derive most of their nutrients from their host meaning the metabolic circuitry of both are intricately linked. We employed untargeted, global mass spectrometry to identify metabolites present in the culture supernatants of P. falciparum-infected red blood cells synchronized at ring, trophozoite and schizont developmental stages. This revealed a temporal regulation in release of a distinct set of metabolites compared with supernatants of non-infected red blood cells. Of the distinct metabolites we identified pipecolic acid to be abundantly present in parasite lysate, infected red blood cells and infected culture supernatant. Further, we performed targeted metabolomics to quantify pipecolic acid concentrations in both the supernatants of red blood cells infected with P. falciparum, as well as in the plasma and infected RBCs of P. berghei-infected mice. Measurable and significant hyperpipecolatemia suggest that pipecolic acid has the potential to be a diagnostic marker for malaria.
Assuntos
Eritrócitos , Malária Falciparum/sangue , Plasmodium berghei/metabolismo , Plasmodium falciparum/metabolismo , Animais , Biomarcadores/sangue , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , CamundongosRESUMO
Theileria annulata is a protozoan parasite that infects and transforms bovine macrophages causing a myeloid-leukaemia-like disease called tropical theileriosis. TGF-ß2 is highly expressed in many cancer cells and is significantly increased in Theileria-transformed macrophages, as are levels of Reactive Oxygen Species (ROS), notably H2O2. Here, we describe the interplay between TGF-ß2 and ROS in cellular transformation. We show that TGF-ß2 drives expression of catalase to reduce the amount of H2O2 produced by T. annulata-transformed bovine macrophages, as well as by human lung (A549) and colon cancer (HT-29) cell lines. Theileria-transformed macrophages attenuated for dissemination express less catalase and produce more H2O2, but regain both virulent migratory and matrigel traversal phenotypes when stimulated either with TGF-ß2, or catalase to reduce H2O2 output. Increased H2O2 output therefore, underpins the aggressive dissemination phenotype of diverse tumour cell types, but in contrast, too much H2O2 can dampen dissemination.
Assuntos
Adenocarcinoma de Pulmão/secundário , Catalase/metabolismo , Neoplasias do Colo/secundário , Peróxido de Hidrogênio/metabolismo , Macrófagos/imunologia , Fator de Crescimento Transformador beta2/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Animais , Catalase/genética , Bovinos , Neoplasias do Colo/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Oxidantes/metabolismo , Oxirredução , Fenótipo , Theileria annulata/isolamento & purificação , Theileriose/parasitologia , Fator de Crescimento Transformador beta2/genética , Células Tumorais CultivadasRESUMO
Constitutive c-Jun N-terminal kinase (JNK) activity characterizes bovine T and B cells infected with Theileria parva, and B cells and macrophages infected with Theileria annulata. Here, we show that T. annulata infection of macrophages manipulates JNK activation by recruiting JNK2 and not JNK1 to the parasite surface, whereas JNK1 is found predominantly in the host cell nucleus. At the parasite's surface, JNK2 forms a complex with p104, a GPI-(GlycosylPhosphatidylInositol)-anchor T. annulata plasma membrane protein. Sequestration of JNK2 depended on Protein Kinase-A (PKA)-mediated phosphorylation of a JNK-binding motif common to T. parva and a cell penetrating peptide harbouring the conserved p104 JNK-binding motif competitively ablated binding, whereupon liberated JNK2 became ubiquitinated and degraded. Cytosolic sequestration of JNK2 suppressed small mitochondrial ARF-mediated autophagy, whereas it sustained nuclear JNK1 levels, c-Jun phosphorylation, and matrigel traversal. Therefore, T. annulata sequestration of JNK2 contributes to both survival and dissemination of Theileria-transformed macrophages.
Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Macrófagos/parasitologia , Proteínas de Membrana/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Proteínas de Protozoários/metabolismo , Theileria annulata/crescimento & desenvolvimento , Animais , Macrófagos/imunologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Modelos Teóricos , Ligação Proteica , Theileria annulata/metabolismo , Theileriose/parasitologia , Theileriose/patologiaRESUMO
Theileria annulata is an obligate intracellular protozoan parasite of the phylum Apicomplexa. Theileria sporozoites invade bovine leukocytes and develop into a multinucleate syncytial macroschizont that causes uncontrolled proliferation and dissemination of infected and transformed leukocytes. Activator protein 1 (AP-1) is a transcription factor driving expression of genes involved in proliferation and dissemination and is therefore a key player in Theileria-induced leukocytes transformation. Ta9 possesses a signal peptide allowing it to be secreted into the infected leukocyte cytosol and be presented to CD8 T cells in the context of MHC class I. First, we confirmed that Ta9 is secreted into the infected leukocyte cytosol, and then we generated truncated versions of GFP-tagged Ta9 and tested their ability to activate AP-1 in non-infected HEK293T human kidney embryo cells. The ability to activate AP-1-driven transcription was found to reside in the C-terminal 100 amino acids of Ta9 distant to the N-terminally located epitopes recognised by CD8+ T cells. Secreted Ta9 has therefore, not only the ability to stimulate CD8+ T cells, but also the potential to activate AP-1-driven transcription and contribute to T. annulata-induced leukocyte transformation.
Assuntos
Sinais Direcionadores de Proteínas/genética , Infecções Protozoárias em Animais/genética , Proteínas de Protozoários/genética , Theileria annulata/genética , Fator de Transcrição AP-1/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Bovinos , Proliferação de Células/genética , Epitopos/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Células HEK293 , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Infecções Protozoárias em Animais/imunologia , Infecções Protozoárias em Animais/parasitologia , Linfócitos T/imunologia , Theileria annulata/patogenicidadeRESUMO
Theileria annulata is an apicomplexan parasite that infects and transforms bovine macrophages that disseminate throughout the animal causing a leukaemia-like disease called tropical theileriosis. Using deep RNAseq of T. annulata-infected B cells and macrophages we identify a set of microRNAs induced by infection, whose expression diminishes upon loss of the hyper-disseminating phenotype of virulent transformed macrophages. We describe how infection-induced upregulation of miR-126-5p ablates JIP-2 expression to release cytosolic JNK to translocate to the nucleus and trans-activate AP-1-driven transcription of mmp9 to promote tumour dissemination. In non-disseminating attenuated macrophages miR-126-5p levels drop, JIP-2 levels increase, JNK1 is retained in the cytosol leading to decreased c-Jun phosphorylation and dampened AP-1-driven mmp9 transcription. We show that variation in miR-126-5p levels depends on the tyrosine phosphorylation status of AGO2 that is regulated by Grb2-recruitment of PTP1B. In attenuated macrophages Grb2 levels drop resulting in less PTP1B recruitment, greater AGO2 phosphorylation, less miR-126-5p associated with AGO2 and a consequent rise in JIP-2 levels. Changes in miR-126-5p levels therefore, underpin both the virulent hyper-dissemination and the attenuated dissemination of T. annulata-infected macrophages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , MAP Quinase Quinase 4/metabolismo , Macrófagos/microbiologia , MicroRNAs/genética , Theileriose/microbiologia , Fator de Transcrição AP-1/metabolismo , Virulência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Bovinos , Células Cultivadas , MAP Quinase Quinase 4/genética , Macrófagos/metabolismo , Theileria annulata/patogenicidade , Theileriose/genética , Theileriose/metabolismo , Fator de Transcrição AP-1/genéticaRESUMO
One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKA is an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Theileria/patogenicidade , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/metabolismo , Hexoquinase/química , Hexoquinase/metabolismo , Humanos , Leucócitos/citologia , Leucócitos/metabolismo , Leucócitos/parasitologia , Fosforilação Oxidativa , Mapas de Interação de ProteínasRESUMO
Apicomplexan parasites are responsible for a number of important human pathologies. Obviously, as Eukaryotes they share a number of cellular features and pathways with their respective host cells. One of them is autophagy, a process involved in the degradation of the cell's own components. These intracellular parasites nonetheless seem to present a number of original features compared to their very evolutionarily distant host cells. In mammals and other metazoans, autophagy has been identified as an important contributor to the defence against microbial pathogens. Thus, host autophagy also likely plays a key role in the control of apicomplexan parasites, although its potential manipulation and subversion by intracellular parasites creates a complex interplay in the regulation of host and parasite autophagy. In this mini-review, we summarise current knowledge on autophagy in both parasites and their host cells, in the context of infection by three Apicomplexa: Plasmodium, Toxoplasma, and Theileria.