RESUMO
The parasite Cryptosporidium is responsible for diarrheal disease in young children causing death, malnutrition, and growth delay. Cryptosporidium invades enterocytes where it develops in a unique intracellular niche. Infected cells exhibit profound changes in morphology, physiology, and transcriptional activity. How the parasite effects these changes is poorly understood. We explored the localization of highly polymorphic proteins and found members of the Cryptosporidium parvum MEDLE protein family to be translocated into the cytosol of infected cells. All intracellular life stages engage in this export, which occurs after completion of invasion. Mutational studies defined an N-terminal host-targeting motif and demonstrated proteolytic processing at a specific leucine residue. Direct expression of MEDLE2 in mammalian cells triggered an ER stress response, which was also observed during infection. Taken together, our studies reveal the presence of a Cryptosporidium secretion system capable of delivering parasite proteins into the infected enterocyte.
Assuntos
Criptosporidiose/parasitologia , Cryptosporidium parvum/fisiologia , Citosol/parasitologia , Interações Hospedeiro-Parasita , Proteínas de Protozoários/fisiologia , Animais , CamundongosRESUMO
With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.
Assuntos
Antiprotozoários/uso terapêutico , Chalcona/metabolismo , Chalcona/farmacologia , Citosol/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Peroxidases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/farmacologia , Células Cultivadas , Chalcona/administração & dosagem , Chalcona/análogos & derivados , Citosol/enzimologia , Citosol/parasitologia , Descoberta de Drogas , Humanos , Leishmania/classificação , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Toxoplasma gondii is an intracellular protozoan parasite that has the remarkable ability to infect and replicate in neutrophils, immune cells with an arsenal of antimicrobial effector mechanisms. We report that T. gondii infection extends the life span of primary human peripheral blood neutrophils by delaying spontaneous apoptosis, serum starvation-induced apoptosis, and tumor necrosis alpha (TNF-α)-mediated apoptosis. T. gondii blockade of apoptosis was associated with an inhibition of processing and activation of the apoptotic caspases caspase-8 and -3, decreased phosphatidylserine exposure on the plasma membrane, and reduced cell death. We performed a global transcriptome analysis of T. gondii-infected peripheral blood neutrophils using RNA sequencing (RNA-Seq) and identified gene expression changes associated with DNA replication and DNA repair pathways, which in mature neutrophils are indicative of changes in regulators of cell survival. Consistent with the RNA-Seq data, T. gondii infection upregulated transcript and protein expression of PCNA, which is found in the cytosol of human neutrophils, where it functions as a key inhibitor of apoptotic pro-caspases. Infection of neutrophils resulted in increased interaction of PCNA with pro-caspase-3. Inhibition of this interaction with an AlkB homologue 2 PCNA-interacting motif (APIM) peptide reversed the infection-induced delay in cell death. Taken together, these findings indicate a novel strategy by which T. gondii manipulates cell life span in primary human neutrophils, which may allow the parasite to maintain an intracellular replicative niche and avoid immune clearance.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that can cause life-threatening disease in immunocompromised individuals and in the developing fetus. Interestingly, T. gondii has evolved strategies to successfully manipulate the host immune system to establish a productive infection and evade host defense mechanisms. Although it is well documented that neutrophils are mobilized during acute T. gondii infection and infiltrate the site of infection, these cells can also be actively infected by T. gondii and serve as a replicative niche for the parasite. However, there is a limited understanding of the molecular processes occurring within T. gondii-infected neutrophils. This study reveals that T. gondii extends the life span of human neutrophils by inducing the expression of PCNA, which prevents activation of apoptotic caspases, thus delaying apoptosis. This strategy may allow the parasite to preserve its replicative intracellular niche.
Assuntos
Apoptose/imunologia , Caspase 8/metabolismo , Caspases/metabolismo , Citosol/metabolismo , Neutrófilos/parasitologia , Antígeno Nuclear de Célula em Proliferação/genética , Toxoplasma/imunologia , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspases/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Citosol/enzimologia , Citosol/parasitologia , Perfilação da Expressão Gênica , Humanos , Neutrófilos/enzimologia , Neutrófilos/fisiologia , Análise de Sequência de RNA , Regulação para CimaRESUMO
Many intracellular pathogens, including the protozoan parasite Toxoplasma gondii, live inside a vacuole that resides in the host cytosol. Vacuolar residence provides these pathogens with a defined niche for replication and protection from detection by host cytosolic pattern recognition receptors. However, the limiting membrane of the vacuole, which constitutes the host-pathogen interface, is also a barrier for pathogen effectors to reach the host cytosol and for the acquisition of host-derived nutrients. This review provides an update on the specialized secretion and trafficking systems used by Toxoplasma to overcome the barrier of the parasitophorous vacuole membrane and thereby allow the delivery of proteins into the host cell and the acquisition of host-derived nutrients.
Assuntos
Citosol/metabolismo , Interações Hospedeiro-Parasita , Nutrientes/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Citosol/parasitologia , Humanos , Redes e Vias Metabólicas , Transporte Proteico , Toxoplasma/patogenicidade , Vacúolos/parasitologia , Fatores de Virulência/metabolismoRESUMO
During development in human erythrocytes, the malaria parasite Plasmodium falciparum internalizes a large part of the cellular content of the host cell. The internalized cytosol, consisting largely of hemoglobin, is transported to the parasite's food vacuole where it is degraded, providing nutrients and space for growth. This host cell cytosol uptake (HCCU) is crucial for parasite survival but the parasite proteins mediating this process remain obscure. Here, we identify P. falciparum VPS45 as an essential factor in HCCU. Conditional inactivation of PfVPS45 led to an accumulation of host cell cytosol-filled vesicles within the parasite and inhibited the delivery of hemoglobin to the parasite's digestive vacuole, resulting in arrested parasite growth. A proportion of these HCCU vesicle intermediates was positive for phosphatidylinositol 3-phosphate, suggesting endosomal characteristics. Thus PfVPS45 provides insight into the elusive machinery of the ingestion pathway in a parasite that contains an endolysosomal system heavily repurposed for protein secretion.
Assuntos
Citosol/parasitologia , Eritrócitos/parasitologia , Hemoglobinas/metabolismo , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/metabolismo , Animais , Transporte Biológico , Citosol/metabolismo , Complexo de Golgi/metabolismo , Interações Hospedeiro-Parasita , Humanos , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/patogenicidade , Transporte Proteico , Proteínas de Protozoários/genética , Vacúolos/metabolismo , Vacúolos/parasitologia , Vacúolos/ultraestruturaRESUMO
Studying redox metabolism in malaria parasites is of great interest for understanding parasite biology, parasite-host interactions, and mechanisms of drug action. Genetically encoded fluorescent redox sensors have recently been described as powerful tools for determining the glutathione-dependent redox potential in living parasites. In the present study, we genomically integrated and expressed the ratiometric redox sensors hGrx1-roGFP2 (human glutaredoxin 1 fused to reduction-oxidation sensitive green fluorescent protein) and sfroGFP2 (superfolder roGFP2) in the cytosol of NF54- attB blood-stage Plasmodium falciparum parasites. Both sensors were evaluated in vitro and in cell culture with regard to their fluorescence properties and reactivity. As genomic integration allows for the stable expression of redox sensors in parasites, we systematically compared single live-cell imaging with plate reader detection. For these comparisons, short-term effects of redox-active compounds were analyzed along with mid- and long-term effects of selected antimalarial agents. Of note, the single components of the redox probes themselves did not influence the redox balance of the parasites. Our analyses revealed comparable results for both the hGrx1-roGFP2 and sfroGFP2 probes, with sfroGFP2 exhibiting a more pronounced fluorescence intensity in cellulo. Accordingly, the sfroGFP2 probe was employed to monitor the fluorescence signals throughout the parasites' asexual life cycle. Through the use of stable genomic integration, we demonstrate a means of overcoming the limitations of transient transfection, allowing more detailed in-cell studies as well as high-throughput analyses using plate reader-based approaches.
Assuntos
Corantes Fluorescentes , Glutarredoxinas/análise , Interações Hospedeiro-Parasita , Plasmodium falciparum/metabolismo , Antimaláricos/farmacologia , Citosol/efeitos dos fármacos , Citosol/parasitologia , Fluorescência , Proteínas de Fluorescência Verde/análise , Humanos , Oxirredução , Plasmodium falciparum/efeitos dos fármacos , Proteínas Recombinantes/análise , TransfecçãoRESUMO
Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for Leishmania donovani leucyl-tRNA synthetase (LdLRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival in vitro The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that Leishmania donovani recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages in vitro as well as in vivo in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LdLRS. We provide preliminary chemical validation of LdLRS as a drug target by showing that AN2690 is an inhibitor both of L. donovani LRS and of L. donovani cell growth.
Assuntos
Aminoacil-tRNA Sintetases/genética , Compostos de Boro/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leishmania donovani/efeitos dos fármacos , Parasitos/efeitos dos fármacos , Animais , Linhagem Celular , Citosol/parasitologia , Feminino , Deleção de Genes , Heterozigoto , Leishmania donovani/genética , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/parasitologia , Parasitos/genética , Proteínas de Protozoários/genéticaRESUMO
Recent years have witnessed a great gain in knowledge regarding parasite-host cell interactions during Plasmodium liver stage development. It is now an accepted fact that a large percentage of sporozoites invading hepatocytes fail to form infectious merozoites. There appears to be a delicate balance between parasite survival and elimination and we now start to understand why this is so. Plasmodium liver stage parasites replicate within the parasitophorous vacuole (PV), formed during invasion by invagination of the host cell plasma membrane. The main interface between the parasite and hepatocyte is the parasitophorous vacuole membrane (PVM) that surrounds the PV. Recently, it was shown that autophagy marker proteins decorate the PVM of Plasmodium liver stage parasites and eliminate a proportion of them by an autophagy-like mechanism. Successfully developing Plasmodium berghei parasites are initially also labeled but in the course of development, they are able to control this host defense mechanism by shedding PVM material into the tubovesicular network (TVN), an extension of the PVM that releases vesicles into the host cell cytoplasm. Better understanding of the molecular events at the PVM/TVN during parasite elimination could be the basis of new antimalarial measures.
Assuntos
Citosol/imunologia , Citosol/parasitologia , Interações Hospedeiro-Parasita/imunologia , Fígado/imunologia , Fígado/parasitologia , Plasmodium/crescimento & desenvolvimento , Plasmodium/imunologia , Animais , Hepatócitos/imunologia , Hepatócitos/parasitologia , Humanos , Estágios do Ciclo de VidaRESUMO
The intracellular Toxoplasma gondii parasite replicates within a parasitophorous vacuole (PV). T. gondii secretes proteins that remain soluble in the PV space, are inserted into PV membranes or are exported beyond the PV boundary. In addition to supporting T. gondii growth, these proteins can be processed and presented by MHC I for CD8+ T-cell recognition. Yet it is unclear whether membrane binding influences the processing pathways employed and if topology of membrane antigens impacts their MHC I presentation. Here we report that the MHC I pathways of soluble and membrane-bound antigens differ in their requirement for host ER recruitment. In contrast to the soluble SAG1-OVA model antigen, we find that presentation of the membrane-bound GRA6 is independent from the SNARE Sec22b, a key molecule for transfer of host endoplasmic reticulum components onto the PV. Using parasites modified to secrete a transmembrane antigen with opposite orientations, we further show that MHC I presentation is highly favored when the C-terminal epitope is exposed to the host cell cytosol, which corresponds to GRA6 natural orientation. Our data suggest that the biochemical properties of antigens released by intracellular pathogens critically guide their processing pathway and are valuable parameters to consider for vaccination strategies.
Assuntos
Apresentação de Antígeno , Antígenos de Protozoários/imunologia , Antígenos de Histocompatibilidade Classe I , Proteínas de Protozoários/imunologia , Proteínas R-SNARE/metabolismo , Toxoplasma/imunologia , Animais , Antígenos de Protozoários/química , Linfócitos T CD8-Positivos/imunologia , Citosol/imunologia , Citosol/parasitologia , Células Dendríticas/imunologia , Epitopos Imunodominantes , Camundongos , Proteínas de Protozoários/química , Toxoplasma/química , Vacúolos/imunologiaRESUMO
Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.
Assuntos
Arginase/metabolismo , Leishmania donovani/metabolismo , Animais , Células Cultivadas , Citosol/metabolismo , Citosol/parasitologia , Feminino , Leishmania infantum/metabolismo , Leishmania infantum/parasitologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Microcorpos/metabolismo , Microcorpos/parasitologia , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Putrescina/metabolismoRESUMO
Nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD) performs essential functions in cell metabolism and energy production due to its redox properties. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) enzyme catalyses the key step in the biosynthesis of NAD+. Previously, the enzyme NMNAT was identified in Trypanosoma cruzi (TcNMNAT), a pathogenic agent with epidemiological importance in Latin America. To continue with the functional characterisation of this enzyme, its subcellular location and its possible post-translational modifications were examined in this study. For this, polyclonal antibodies were generated in mice, with soluble and denatured recombinant protein being used to detect the parasite’s NMNAT. Immunodetection assays were performed on whole extracts of T. cruzi, and an approximation of its intracellular location was determined using confocal microscopy on wild and transgenic parasites, which revealed the cytosol distribution patterns. This localisation occurs according to the needs of the dinucleotides that exist in this compartment. Additionally, a bioinformatics study was performed as a first approach to establish the post-translational modifications of the enzyme. Possible phosphorylation events were experimentally analysed by western blot, highlighting TcNMNAT as a potential target for serine kinases.
Assuntos
Animais , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Citosol/parasitologia , Interações Hospedeiro-Parasita , Camundongos , Camundongos Endogâmicos BALB C , Nicotinamida-Nucleotídeo Adenililtransferase/isolamento & purificação , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas de Protozoários/isolamento & purificaçãoRESUMO
Nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD) performs essential functions in cell metabolism and energy production due to its redox properties. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) enzyme catalyses the key step in the biosynthesis of NAD+. Previously, the enzyme NMNAT was identified in Trypanosoma cruzi (TcNMNAT), a pathogenic agent with epidemiological importance in Latin America. To continue with the functional characterisation of this enzyme, its subcellular location and its possible post-translational modifications were examined in this study. For this, polyclonal antibodies were generated in mice, with soluble and denatured recombinant protein being used to detect the parasite's NMNAT. Immunodetection assays were performed on whole extracts of T. cruzi, and an approximation of its intracellular location was determined using confocal microscopy on wild and transgenic parasites, which revealed the cytosol distribution patterns. This localisation occurs according to the needs of the dinucleotides that exist in this compartment. Additionally, a bioinformatics study was performed as a first approach to establish the post-translational modifications of the enzyme. Possible phosphorylation events were experimentally analysed by western blot, highlighting TcNMNAT as a potential target for serine kinases.
Assuntos
Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Citosol/parasitologia , Interações Hospedeiro-Parasita , Camundongos , Camundongos Endogâmicos BALB C , Nicotinamida-Nucleotídeo Adenililtransferase/isolamento & purificação , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas de Protozoários/isolamento & purificaçãoRESUMO
Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.
Assuntos
Echinococcus granulosus/metabolismo , Proteínas de Helminto/isolamento & purificação , Histonas/isolamento & purificação , Processamento de Proteína Pós-Traducional , Proteoma/isolamento & purificação , Proteômica/métodos , Acetilação , Sequência de Aminoácidos , Animais , Bovinos , Núcleo Celular/química , Núcleo Celular/parasitologia , Cromatografia Líquida , Citosol/química , Citosol/parasitologia , Equinococose/parasitologia , Equinococose/patologia , Echinococcus granulosus/genética , Echinococcus granulosus/crescimento & desenvolvimento , Células Epiteliais/química , Células Epiteliais/parasitologia , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Histonas/genética , Histonas/metabolismo , Estágios do Ciclo de Vida/genética , Pulmão/química , Pulmão/parasitologia , Metionina/química , Metionina/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Proteoma/genética , Proteoma/metabolismo , Proteômica/instrumentação , Espectrometria de Massas em TandemRESUMO
Survival and virulence of the human malaria parasite Plasmodium falciparum during the blood stage of infection critically depend on extensive host cell refurbishments mediated through export of numerous parasite proteins into the host cell. The parasite-derived membranous structures called Maurer's clefts (MC) play an important role in protein trafficking from the parasite to the red blood cell membrane. However, their specific function has yet to be determined. We identified and characterized a new MC membrane protein, termed small exported membrane protein 1 (SEMP1). Upon invasion it is exported into the RBC cytosol where it inserts into the MCs before it is partly translocated to the RBC membrane. Using conventional and conditional loss-of-function approaches we showed that SEMP1 is not essential for parasite survival, gametocytogenesis, or PfEMP1 export under culture conditions. Co-IP experiments identified several potential interaction partners, including REX1 and other membrane-associated proteins that were confirmed to co-localize with SEMP1 at MCs. Transcriptome analysis further showed that expression of a number of exported parasite proteins was up-regulated in SEMP1-depleted parasites. By using Co-IP and transcriptome analysis for functional characterization of an exported parasite protein we provide a new starting point for further detailed dissection and characterisation of MC-associated protein complexes.
Assuntos
Interações Hospedeiro-Parasita , Malária/genética , Proteínas de Membrana/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Animais , Citosol/parasitologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Gametogênese , Humanos , Malária/parasitologia , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , VirulênciaRESUMO
The protozoan parasite Toxoplasma gondii resides within a nonfusogenic vacuole during intracellular replication. Although the limiting membrane of this vacuole provides a protective barrier to acidification and degradation by lysosomal hydrolases, it also physically segregates the parasite from the host cytosol. Accordingly, it has been suggested that T. gondii acquires material from the host via membrane channels or transporters. The ability of the parasite to internalize macromolecules via endocytosis during intracellular replication has not been tested. Here, we show that Toxoplasma ingests host cytosolic proteins and digests them using cathepsin L and other proteases within its endolysosomal system. Ingestion was reduced in mutant parasites lacking an intravacuolar network of tubular membranes, implicating this apparatus as a possible conduit for trafficking to the parasite. Genetic ablation of proteins involved in the pathway is associated with diminished parasite replication and virulence attenuation. We show that both virulent type I and avirulent type II strain parasites ingest and digest host-derived protein, indicating that the pathway is not restricted to highly virulent strains. The findings provide the first definitive evidence that T. gondii internalizes proteins from the host during intracellular residence and suggest that protein digestion within the endolysosomal system of the parasite contributes to toxoplasmosis. Importance: Toxoplasma gondii causes significant disease in individuals with weak immune systems. Treatment options for this infection have drawbacks, creating a need to understand how this parasite survives within the cells it infects as a prelude to interrupting its survival strategies. This study reveals that T. gondii internalizes proteins from the cytoplasm of the cells it infects and degrades such proteins within a digestive compartment within the parasite. Disruption of proteins involved in the pathway reduced parasite replication and lessened disease severity. The identification of a novel parasite ingestion pathway opens opportunities to interfere with this process and improve the outcome of infection.
Assuntos
Citosol/metabolismo , Proteínas/metabolismo , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/metabolismo , Animais , Citosol/parasitologia , Feminino , CamundongosRESUMO
Protein export into the host red blood cell is one of the key processes in the pathobiology of the malaria parasite Plasmodiumtrl falciparum, which extensively remodels the red blood cell to ensure its virulence and survival. In this study, we aimed to shed further light on the protein export mechanisms in the rodent malaria parasite P. berghei and provide further proof of the conserved nature of host cell remodeling in Plasmodium spp. Based on the presence of an export motif (R/KxLxE/Q/D) termed PEXEL (Plasmodium export element), we have generated transgenic P. berghei parasite lines expressing GFP chimera of putatively exported proteins and analysed one of the newly identified exported proteins in detail. This essential protein, termed PbCP1 (P. berghei Cleft-like Protein 1), harbours an atypical PEXEL motif (RxLxY) and is further characterised by two predicted transmembrane domains (2TMD) in the C-terminal end of the protein. We have functionally validated the unusual PEXEL motif in PbCP1 and analysed the role of the 2TMD region, which is required to recruit PbCP1 to discrete membranous structures in the red blood cell cytosol that have a convoluted, vesico-tubular morphology by electron microscopy. Importantly, this study reveals that rodent malaria species also induce modifications to their host red blood cell.
Assuntos
Citosol/parasitologia , Eritrócitos/parasitologia , Plasmodium berghei/metabolismo , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Citosol/ultraestrutura , Eritrócitos/ultraestrutura , Expressão Gênica , Proteínas de Fluorescência Verde , Estágios do Ciclo de Vida/genética , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmodium berghei/genética , Plasmodium berghei/ultraestrutura , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
During the intraerythrocytic development of Plasmodium falciparum, the malaria parasite remodels the host cell cytosol by inducing membranous structures termed Maurer's clefts and inserting parasite proteins into the red blood cell cytoskeleton and plasma membrane. Pf332 is the largest known asexual malaria antigen that is exported into the red blood cell cytosol where it associates with Maurer's clefts. In the current work, we have utilized a set of different biochemical assays to analyze the solubility of the endogenous Pf332 molecule during its trafficking from the endoplasmic reticulum within the parasite to the host cell cytosol. Solubilization studies demonstrate that Pf332 is synthesized and trafficked within the parasite as a peripheral membrane protein, which after export into the host cell cytosol associates with the cytoplasmic side of Maurer's clefts in a peripheral manner. By immunofluorescence microscopy and flow cytometry, we show that Pf332 persists in close association with Maurer's clefts throughout trophozoite maturation and schizogony, and does not become exposed at the host cell surface. Our data also indicate that Pf332 interacts with the host cell cytoskeleton, but only in very mature parasite stages. Thus, the present study describes Pf332 as a resident peripheral membrane protein of Maurer's clefts and suggests that the antigen participates in host cytoskeleton modifications at completion of the intraerythrocytic developmental cycle.
Assuntos
Eritrócitos/citologia , Eritrócitos/metabolismo , Proteínas de Membrana/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Carbonatos , Citosol/parasitologia , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/parasitologia , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Modelos Biológicos , Octoxinol , Parasitos/citologia , Parasitos/metabolismo , Biossíntese de Proteínas , Transporte Proteico , Proteínas de Protozoários/isolamento & purificação , Esquizontes/metabolismo , Solubilidade , Trofozoítos/metabolismo , UreiaRESUMO
Cell stress and infection promote the formation of ubiquitinated aggregates in both non-immune and immune cells. These structures are recognised by the autophagy receptor p62/sequestosome 1 and are substrates for selective autophagy. The intracellular growth of Salmonella enterica occurs in a membranous compartment, the Salmonella-containing vacuole (SCV), and is dependent on effectors translocated to the host cytoplasm by the Salmonella pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS). Here, we show that bacterial replication is accompanied by the formation of ubiquitinated structures in infected cells. Analysis of bacterial strains carrying mutations in genes encoding SPI-2 T3SS effectors revealed that in epithelial cells, formation of these ubiquitinated structures is dependent on SPI-2 T3SS effector translocation, but is counteracted by the SPI-2 T3SS deubiquitinase SseL. In macrophages, both SPI-2 T3SS-dependent aggregates and aggresome-like induced structures (ALIS) are deubiquitinated by SseL. In the absence of SseL activity, ubiquitinated structures are recognized by the autophagy receptor p62, which recruits LC3 and targets them for autophagic degradation. We found that SseL activity lowers autophagic flux and favours intracellular Salmonella replication. Our data therefore show that there is a host selective autophagy response to intracellular Salmonella infection, which is counteracted by the deubiquitinase SseL.
Assuntos
Autofagia , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Células Epiteliais/parasitologia , Infecções por Salmonella/metabolismo , Salmonella enterica/enzimologia , Animais , Linhagem Celular , Citosol/metabolismo , Citosol/parasitologia , Humanos , Immunoblotting , Imunoprecipitação , Macrófagos/parasitologia , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Ubiquitina/metabolismo , Vacúolos/metabolismo , Vacúolos/parasitologiaRESUMO
Plasmodium falciparum is predicted to transport over 300 proteins to the cytosol of its chosen host cell, the mature human erythrocyte, including 19 members of the Hsp40 family. Here, we have generated transfectant lines expressing GFP- or HA-Strep-tagged versions of these proteins, and used these to investigate both localization and other properties of these Hsp40 co-chaperones. These fusion proteins labelled punctate structures within the infected erythrocyte, initially suggestive of a Maurer's clefts localization. Further experiments demonstrated that these structures were distinct from the Maurer's clefts in protein composition. Transmission electron microscopy verifies a non-cleft localization for HA-Strep-tagged versions of these proteins. We were not able to label these structures with BODIPY-ceramide, suggesting a lower size and/or different lipid composition compared with the Maurer's clefts. Solubility studies revealed that the Hsp40-GFP fusion proteins appear to be tightly associated with membranes, but could be released from the bilayer under conditions affecting membrane cholesterol content or organization, suggesting interaction with a binding partner localized to cholesterol-rich domains. These novel structures are highly mobile in the infected erythrocyte, but based on velocity calculations, can be distinguished from the 'highly mobile vesicles' previously described. Our study identifies a further extra-parasitic structure in the P. falciparum-infected erythrocyte, which we name 'J-dots' (as their defining characteristic so far is the content of J-proteins). We suggest that these J-dots are involved in trafficking of parasite-encoded proteins through the cytosol of the infected erythrocyte.