Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 59
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Trends Parasitol ; 35(12): 996-1008, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615721

RESUMO

Phosphoinositides (or phosphatidylinositol phosphates, PIPs) are low-abundance membrane phospholipids that act, in conjunction with their binding partners, as important constitutive signals defining biochemical organelle identity as well as membrane trafficking and signal transduction at eukaryotic cellular membranes. In this review, we present roles for PIP residues and PIP-binding proteins in endocytosis and autophagy in protist parasites such as Trypanosoma brucei, Toxoplasma gondii, Plasmodium falciparum, Entamoeba histolytica, and Giardia lamblia. Molecular parasitologists with an interest in comparative cell and molecular biology of membrane trafficking in protist lineages beyond the phylum Apicomplexa, along with cell and molecular biologists generally interested in the diversification of membrane trafficking in eukaryotes, will hopefully find this review to be a useful resource.

2.
Adv Parasitol ; 106: 105-127, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31630756

RESUMO

Over the past years, the subcellular organization of the Excavata member Giardia lamblia (syn. duodenalis, intestinalis) has been investigated in considerable detail. There are several reasons for this endeavour which go beyond this parasite's medical importance and are mostly concerned with its reduced subcellular complexity and debated evolutionary status. One may say that simplification has emerged as a paradigm for the evolution of Giardia's subcellular architecture. However, a complete appreciation of the evolutionary and ecological significance of this phenomenon is far from complete. In this chapter, we present and discuss the most recent data on the main trafficking pathways in G. lamblia which include endo- and exo-cytosis, organellar import and function. We provide perspectives on open questions concerning organelle replication and inheritance and include a technical outlook on methods and approaches to genetic manipulations in G. lamblia. A better understanding of G. lamblia subcellular organization at the morphological and molecular level complements any effort aimed at elucidating this parasitic species' evolutionary status and could provide us with the basis for novel strategies to interfere with parasite transmission and/or pathogenesis.


Assuntos
Giardia lamblia/metabolismo , Giardíase/parasitologia , Proteínas de Protozoários/metabolismo , Giardíase/transmissão , Transporte Proteico
3.
Sci Rep ; 9(1): 1474, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30728393

RESUMO

Almost any warm-blooded creature can be an intermediate host for Toxoplasma gondii. However, sexual reproduction of T. gondii occurs only in felids, wherein fertilisation of haploid macrogametes by haploid microgametes, results in diploid zygotes, around which a protective wall develops, forming unsporulated oocysts. Unsporulated oocysts are shed in the faeces of cats and meiosis gives rise to haploid sporozoites within the oocysts. These, now infectious, sporulated oocysts contaminate the environment as a source of infection for people and their livestock. RNA-Seq analysis of cat enteric stages of T. gondii uncovered genes expressed uniquely in microgametes and macrogametes. A CRISPR/Cas9 strategy was used to create a T. gondii strain that exhibits defective fertilisation, decreased fecundity and generates oocysts that fail to produce sporozoites. Inoculation of cats with this engineered parasite strain totally prevented oocyst excretion following infection with wild-type T. gondii, demonstrating that this mutant is an attenuated, live, transmission-blocking vaccine.

5.
Genome Biol Evol ; 10(9): 2310-2325, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30060189

RESUMO

The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.


Assuntos
Blastocystis/metabolismo , Diatomáceas/metabolismo , Glicólise , Mitocôndrias/metabolismo , Evolução Biológica , Blastocystis/citologia , Blastocystis/enzimologia , Blastocystis/genética , Diatomáceas/citologia , Diatomáceas/enzimologia , Diatomáceas/genética , Metabolismo Energético , Genoma Mitocondrial , Mitocôndrias/genética , Simbiose , Transformação Genética
6.
Int J Parasitol ; 48(7): 519-530, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29530647

RESUMO

Understanding the complex Entamoeba communities in the mammalian intestine has been, to date, complicated by the lack of a suitable approach for molecular detection of multiple variants co-occurring in mixed infections. Here, we report on the application of a high throughput sequencing approach based on partial 18S rDNA using the Illumina MiSeq platform. We describe, to our knowledge, for the first time, the Entamoeba communities in humans, free-ranging western lowland gorillas and central chimpanzees living in the Dja Faunal Reserve in Cameroon. We detected 36 Entamoeba haplotypes belonging to six haplotype clusters, containing haplotypes possessing high and low host specificity. Most of the detected haplotypes belonged to commensal Entamoeba, however, the pathogenic species (Entamoeba histolytica and Entamoeba nuttalli) were also detected. We observed that some Entamoeba haplotypes are shared between humans and other hosts, indicating their zoonotic potential. The findings are important not only for understanding the epidemiology of amoebiasis in humans in rural African localities, but also in the context of wild great ape conservation.


Assuntos
Doenças dos Símios Antropoides/parasitologia , Entamoeba , Entamebíase/veterinária , Gorilla gorilla/parasitologia , Sequenciamento de Nucleotídeos em Larga Escala , Pan troglodytes/parasitologia , África/epidemiologia , Animais , Doenças dos Símios Antropoides/epidemiologia , Conservação dos Recursos Naturais , Entamebíase/epidemiologia , Entamebíase/parasitologia , Humanos , Enteropatias Parasitárias/epidemiologia , Enteropatias Parasitárias/parasitologia , Enteropatias Parasitárias/veterinária
7.
Int J Parasitol ; 48(6): 413-422, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29432770

RESUMO

Cryptosporidium parvum is a major cause of diarrhoea in humans and animals. There are no vaccines and few drugs available to control C. parvum. In this study, we used RNA-Seq to compare gene expression in sporozoites and intracellular stages of C. parvum to identify genes likely to be important for successful completion of the parasite's life cycle and, thereby, possible targets for drugs or vaccines. We identified 3774 protein-encoding transcripts in C. parvum. Applying a stringent cut-off of eight fold for determination of differential expression, we identified 173 genes (26 coding for predicted secreted proteins) upregulated in sporozoites. On the other hand, expression of 1259 genes was upregulated in intestinal stages (merozoites/gamonts) with a gene ontology enrichment for 63 biological processes and upregulation of 117 genes in 23 metabolic pathways. There was no clear stage specificity of expression of AP2-domain containing transcription factors, although sporozoites had a relatively small repertoire of these important regulators. Our RNA-Seq analysis revealed a new calcium-dependent protein kinase, bringing the total number of known calcium-dependent protein kinases (CDPKs) in C. parvum to 11. One of these, CDPK1, was expressed in all stages, strengthening the notion that it is a valid drug target. By comparing parasites grown in vivo (which produce bona fide thick-walled oocysts) and in vitro (which are arrested in sexual development prior to oocyst generation) we were able to confirm that genes encoding oocyst wall proteins are expressed in gametocytes and that the proteins are stockpiled rather than generated de novo in zygotes. RNA-Seq analysis of C. parvum revealed genes expressed in a stage-specific manner and others whose expression is required at all stages of development. The functional significance of these can now be addressed through recent advances in transgenics for C. parvum, and may lead to the identification of viable drug and vaccine targets.


Assuntos
Cryptosporidium parvum/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Protozoários/metabolismo , Animais , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Meiose/fisiologia , Camundongos , Mucinas/genética , Mucinas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Protozoários/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
8.
Elife ; 62017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28898199

RESUMO

Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.


Assuntos
Ácido Aspártico Proteases/farmacologia , Organelas/metabolismo , Proteínas de Protozoários/farmacologia , Toxoplasma/enzimologia , Toxoplasma/metabolismo , Anticorpos , Antimaláricos/farmacologia , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/imunologia , Moléculas de Adesão Celular/genética , Linhagem Celular , DNA de Protozoário , Escherichia coli/genética , Fibroblastos , Técnicas de Silenciamento de Genes , Genes de Protozoários , Humanos , Proteínas de Protozoários/genética , Proteínas Recombinantes , Toxoplasma/genética
9.
Int J Parasitol ; 47(12): 811-821, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28899692

RESUMO

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/isolamento & purificação , Sarcocystidae/efeitos dos fármacos , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Fibroblastos/citologia , Fibroblastos/parasitologia , Imunofluorescência , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Proteínas Quinases/química , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/genética , Reação em Cadeia da Polimerase em Tempo Real , Sarcocystidae/genética , Sarcocystidae/crescimento & desenvolvimento , Sarcocystidae/ultraestrutura , Inoculações Seriadas
10.
Sci Rep ; 7(1): 3357, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28611446

RESUMO

Infections of dogs with virulent strains of Babesia canis are characterized by rapid onset and high mortality, comparable to complicated human malaria. As in other apicomplexan parasites, most Babesia virulence factors responsible for survival and pathogenicity are secreted to the host cell surface and beyond where they remodel and biochemically modify the infected cell interacting with host proteins in a very specific manner. Here, we investigated factors secreted by B. canis during acute infections in dogs and report on in silico predictions and experimental analysis of the parasite's exportome. As a backdrop, we generated a fully annotated B. canis genome sequence of a virulent Hungarian field isolate (strain BcH-CHIPZ) underpinned by extensive genome-wide RNA-seq analysis. We find evidence for conserved factors in apicomplexan hemoparasites involved in immune-evasion (e.g. VESA-protein family), proteins secreted across the iRBC membrane into the host bloodstream (e.g. SA- and Bc28 protein families), potential moonlighting proteins (e.g. profilin and histones), and uncharacterized antigens present during acute crisis in dogs. The combined data provides a first predicted and partially validated set of potential virulence factors exported during fatal infections, which can be exploited for urgently needed innovative intervention strategies aimed at facilitating diagnosis and management of canine babesiosis.


Assuntos
Babesia/genética , Babesiose/genética , Doenças do Cão/parasitologia , Regulação da Expressão Gênica , Genoma de Protozoário , Proteínas de Protozoários/metabolismo , Fatores de Virulência/metabolismo , Animais , Babesia/isolamento & purificação , Babesia/metabolismo , Babesiose/parasitologia , Babesiose/transmissão , Cães , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Filogenia , Proteômica , Proteínas de Protozoários/genética , Transcriptoma , Fatores de Virulência/genética
11.
Int J Parasitol ; 47(10-11): 597-600, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28526607

RESUMO

The apicomplexan, Toxoplasma gondii, infects all warm-blooded animals as intermediate hosts but only felids as definitive hosts. Dense granule proteins are critical for the survival of Toxoplasma within host cells but, whilst these proteins have been studied intensively in tachyzoites, little is known about their expression in the coccidian stages in the cat intestine. Transcriptomic profiling indicates that two putative dense granule proteins, TgGRA11A and TgGRA11B, are expressed uniquely in merozoites. Immunofluorescent microscopy of Toxoplasma-infected cat intestine and tachyzoites engineered to express TgGRA11B, reveals that it is a dense granule protein that traffics into the parasitophorous vacuole and its membrane.


Assuntos
Regulação da Expressão Gênica/fisiologia , Merozoítos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Vacúolos/fisiologia , Animais , Doenças do Gato/parasitologia , Gatos , Intestinos , Conformação Proteica , Toxoplasmose Animal/parasitologia
12.
Trends Parasitol ; 33(2): 76, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28081986
13.
Nat Commun ; 7: 13859, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27976675

RESUMO

The genome of the protozoan parasite Giardia lamblia is organized in two diploid nuclei, which has so far precluded complete analysis of gene function. Here we use a previously developed Cre/loxP-based knock-out and selection marker salvage strategy in the human-derived isolate WB-C6 to eliminate all four copies of the Cyst-Wall-Protein-1 locus (CWP1). Because these loci are silenced in proliferating trophozoites and highly expressed only in encysting cells, CWP1 ablation allows functional characterization of a conditional phenotype in parasites induced to encyst. We show that encysting Δcwp1 cells are unable to establish the stage-regulated trafficking machinery with Golgi-like encystation-specific vesicles required for cyst-wall formation but show morphological hallmarks of cyst development and karyokinesis. This 'pseudocyst' phenotype is rescued by transfection of Δcwp1 cells with an episomally maintained CWP1 expression vector. Genome editing in genera Giardia and Trypanosoma are the only reported examples addressing questions on pathogen transmission within the Excavata supergroup.


Assuntos
Vias Biossintéticas , Giardia lamblia/metabolismo , Complexo de Golgi/metabolismo , Biogênese de Organelas , Proteínas de Protozoários/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Sobrevivência Celular , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Vesículas Citoplasmáticas/metabolismo , Genes Reporter , Teste de Complementação Genética , Giardia lamblia/citologia , Humanos , Mutação/genética , Solubilidade
14.
PLoS Pathog ; 12(12): e1006036, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27926928

RESUMO

Protozoan parasites of the genus Giardia are highly prevalent globally, and infect a wide range of vertebrate hosts including humans, with proliferation and pathology restricted to the small intestine. This narrow ecological specialization entailed extensive structural and functional adaptations during host-parasite co-evolution. An example is the streamlined mitosomal proteome with iron-sulphur protein maturation as the only biochemical pathway clearly associated with this organelle. Here, we applied techniques in microscopy and protein biochemistry to investigate the mitosomal membrane proteome in association to mitosome homeostasis. Live cell imaging revealed a highly immobilized array of 30-40 physically distinct mitosome organelles in trophozoites. We provide direct evidence for the single giardial dynamin-related protein as a contributor to mitosomal morphogenesis and homeostasis. To overcome inherent limitations that have hitherto severely hampered the characterization of these unique organelles we applied a novel interaction-based proteome discovery strategy using forward and reverse protein co-immunoprecipitation. This allowed generation of organelle proteome data strictly in a protein-protein interaction context. We built an initial Tom40-centered outer membrane interactome by co-immunoprecipitation experiments, identifying small GTPases, factors with dual mitosome and endoplasmic reticulum (ER) distribution, as well as novel matrix proteins. Through iterative expansion of this protein-protein interaction network, we were able to i) significantly extend this interaction-based mitosomal proteome to include other membrane-associated proteins with possible roles in mitosome morphogenesis and connection to other subcellular compartments, and ii) identify novel matrix proteins which may shed light on mitosome-associated metabolic functions other than Fe-S cluster biogenesis. Functional analysis also revealed conceptual conservation of protein translocation despite the massive divergence and reduction of protein import machinery in Giardia mitosomes.


Assuntos
Giardia lamblia/fisiologia , Giardia lamblia/ultraestrutura , Homeostase/fisiologia , Proteínas de Protozoários/metabolismo , Imunofluorescência , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Organelas , Organismos Geneticamente Modificados , Reação em Cadeia da Polimerase , Trofozoítos/fisiologia , Trofozoítos/ultraestrutura
15.
mBio ; 7(4)2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27555307

RESUMO

UNLABELLED: Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia's sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-Rac(CA)) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-Rac(CA)-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis. IMPORTANCE: The encystation process is crucial for the transmission of giardiasis and the life cycle of many protists. Encystation for Giardia lamblia involves the assembly of a protective cyst wall via sequential production, trafficking, and secretion of cyst wall material. However, the regulatory pathways that coordinate cargo maturation and secretion remain unknown. Here, we asked whether the signaling activities of G. lamblia's single Rho family GTPase, GlRac, might have a regulatory role in the encystation process. We show that GlRac localizes to endomembranes and its signaling activities regulate the production of cyst wall protein 1 (CWP1), the maturation of encystation-specific vesicles (ESVs), and secretion of CWP1. We also show that secreted CWP1 is available for the development of cysts at the population level, a finding that in part could explain why Giardia encystation proceeds more efficiently at high cell densities.


Assuntos
Regulação da Expressão Gênica , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/genética , Proteínas de Protozoários/metabolismo , Esporos de Protozoários/crescimento & desenvolvimento , Esporos de Protozoários/genética , Proteínas rac de Ligação ao GTP/metabolismo , Transporte Proteico
16.
PLoS Pathog ; 12(7): e1005756, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27438602

RESUMO

Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs), which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM). Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain (GlCHC), dynamin-related protein (GlDRP), and assembly polypeptide complex 2 (AP2) subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular remodeling of endocytosis in this species.


Assuntos
Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Giardia lamblia/metabolismo , Vacúolos/metabolismo , Membrana Celular/ultraestrutura , Imunofluorescência , Giardia lamblia/ultraestrutura , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Microscopia Confocal , Microscopia Eletrônica , Vacúolos/ultraestrutura
17.
Parasit Vectors ; 9: 124, 2016 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-26935317

RESUMO

BACKGROUND: Eimeria is an important genus of apicomplexan parasites. A defining feature of these parasites is the oocyst, which is transmitted into the environment via the faeces of definitive hosts. The oocyst wall contains cross-linked, tyrosine-rich proteins and protects eight infectious sporozoites, housed in pairs within a second walled structure, the sporocyst. The biochemical basis for sporocyst wall formation is not known. FINDINGS: Here, we report the discovery of a novel tyrosine-rich protein, EtSWP1, in Eimeria tenella. Like the tyrosine-rich proteins of the oocyst wall, EtSWP1 is an intrinsically disordered protein with the tyrosine residues concentrated in a specific region of the protein, located immediately following the region of intrinsic disorder. We engineered E. tenella to express mCherry-tagged EtSWP1 and showed that the tagged protein localises specifically to sporocyst walls, indicating that the biochemistry of sporocyst wall assembly is analagous to that of oocyst walls. CONCLUSIONS: Tyrosine-rich proteins are known to be key components of the oocyst wall and we now demonstrate, using gene and protein analyses combined with genetic manipulation, that a novel tyrosine-rich protein is specific for the sporocyst wall. This finding is important because it shows that the biochemistry of these two distinct walls is similar and, hence, brings targeted disruption of sporulation and, therefore, potential neutralisation of oocysts in the environment, a step closer.


Assuntos
Parede Celular/química , Eimeria tenella/química , Oocistos/química , Proteínas de Protozoários/isolamento & purificação , Parede Celular/genética , Eimeria tenella/genética , Proteínas de Protozoários/genética , Tirosina/análise , Tirosina/genética
18.
J Pathol ; 237(4): 495-507, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26235267

RESUMO

The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes and delayed the formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in progenitor-based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells.


Assuntos
Células Acinares/citologia , Desdiferenciação Celular/fisiologia , Pâncreas Exócrino/fisiologia , Serotonina/metabolismo , Envelhecimento , Animais , Modelos Animais de Doenças , Immunoblotting , Imuno-Histoquímica , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Pancreatite/patologia , Regeneração
19.
BMC Genomics ; 16: 66, 2015 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-25757795

RESUMO

BACKGROUND: The apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into. RESULTS: To determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites. CONCLUSIONS: The distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.


Assuntos
Enterócitos/parasitologia , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Protozoário , Toxoplasma/genética , Animais , Gatos , Hibridização Genômica Comparativa , Estágios do Ciclo de Vida/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Elementos Reguladores de Transcrição/genética , Análise de Sequência de RNA , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologia , Toxoplasmose Animal/patologia
20.
BMC Genomics ; 16: 94, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25765081

RESUMO

BACKGROUND: The protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite's sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission. RESULTS: Comparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered. CONCLUSIONS: The need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.


Assuntos
Eimeria tenella/genética , Transcriptoma , Sequência de Aminoácidos , Animais , Galinhas/parasitologia , Coccidiose/parasitologia , Coccidiose/patologia , Eimeria tenella/crescimento & desenvolvimento , Genoma de Protozoário , Merozoítos/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Oocistos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA/química , RNA/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Esporozoítos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA