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1.
Nature ; 625(7994): 366-376, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38093015

RESUMO

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.


Assuntos
Gatos , Técnicas In Vitro , Estágios do Ciclo de Vida , Toxoplasma , Animais , Gatos/parasitologia , Humanos , Cromatina/genética , Cromatina/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Técnicas In Vitro/métodos , Estágios do Ciclo de Vida/genética , Merozoítos/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/fisiologia , Toxoplasmose/genética , Toxoplasmose/parasitologia , Toxoplasmose/transmissão , Transcrição Gênica
2.
J Cell Sci ; 136(11)2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37288670

RESUMO

Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The '9+2' axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella.


Assuntos
Parasitos , Plasmodium , Masculino , Animais , Axonema/metabolismo , Parasitos/metabolismo , Microtúbulos/metabolismo , Sementes , Proteínas/metabolismo , Flagelos/metabolismo , Eucariotos/metabolismo , Plasmodium/metabolismo , Dineínas/metabolismo
3.
Plant J ; 2023 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-37665331

RESUMO

Centromeres are the chromosomal domains, where the kinetochore protein complex is formed, mediating proper segregation of chromosomes during cell division. Although the function of centromeres has remained conserved during evolution, centromeric DNA is highly variable, even in closely related species. In addition, the composition of the kinetochore complexes varies among organisms. Therefore, it is assumed that the centromeric position is determined epigenetically, and the centromeric histone H3 (CENH3) serves as an epigenetic marker. The loading of CENH3 onto centromeres depends on centromere-licensing factors, chaperones, and transcription of centromeric repeats. Several proteins that regulate CENH3 loading and kinetochore assembly interact with the centromeric transcripts and DNA in a sequence-independent manner. However, the functional aspects of these interactions are not fully understood. This review discusses the variability of centromeric sequences in different organisms and the regulation of their transcription through the RNA Pol II and RNAi machinery. The data suggest that the interaction of proteins involved in CENH3 loading and kinetochore assembly with centromeric DNA and transcripts plays a role in centromere, and possibly neocentromere, formation in a sequence-independent manner.

4.
PLoS Pathog ; 18(11): e1010955, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36395346

RESUMO

Cyst-forming Apicomplexa (CFA) of the Sarcocystidae have a ubiquitous presence as pathogens of humans and farm animals transmitted through the food chain between hosts with few notable exceptions. The defining hallmark of this family of obligate intracellular protists consists of their ability to remain for very long periods as infectious tissue cysts in chronically infected intermediate hosts. Nevertheless, each closely related species has evolved unique strategies to maintain distinct reservoirs on global scales and ensuring efficient transmission to definitive hosts as well as between intermediate hosts. Here, we present an in-depth comparative mRNA expression analysis of the tachyzoite and bradyzoite stages of Besnoitia besnoiti strain Lisbon14 isolated from an infected farm animal based on its annotated genome sequence. The B. besnoiti genome is highly syntenic with that of other CFA and also retains the capacity to encode a large majority of known and inferred factors essential for completing a sexual cycle in a yet unknown definitive host. This work introduces Besnoitia besnoiti as a new model for comparative biology of coccidian tissue cysts which can be readily obtained in high purity. This model provides a framework for addressing fundamental questions about the evolution of tissue cysts and the biology of this pharmacologically intractable infectious parasite stage.


Assuntos
Besnoitia , Estágios do Ciclo de Vida , Animais , Humanos , Estágios do Ciclo de Vida/genética , Cadeia Alimentar , Expressão Gênica
5.
Cell Microbiol ; 17(2): 191-206, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25154861

RESUMO

Gametocytes are the sole Plasmodium parasite stages that infect mosquitoes; therefore development of functional gametes is required for malaria transmission. Flagellum assembly of the Plasmodium male gamete differs from that of most other eukaryotes in that it is intracytoplasmic but retains a key conserved feature: axonemes assemble from basal bodies. The centriole/basal body protein SAS-6 normally regulates assembly and duplication of these organelles and its depletion causes severe flagellar/ciliary abnormalities in a diverse array of eukaryotes. Since basal body and flagellum assembly are intimately coupled to male gamete development in Plasmodium, we hypothesized that SAS-6 disruption may cause gametogenesis defects and perturb transmission. We show that Plasmodium berghei sas6 knockouts display severely abnormal male gametogenesis presenting reduced basal body numbers, axonemal assembly defects and abnormal nuclear allocation. The defects in gametogenesis reduce fertilization and render Pbsas6 knockouts less infectious to mosquitoes. Additionally, we show that lack of Pbsas6 blocks transmission from mosquito to vertebrate host, revealing an additional yet undefined role in ookinete to sporulating oocysts transition. These findings underscore the vulnerability of the basal body/SAS-6 to malaria transmission blocking interventions.


Assuntos
Corpos Basais/fisiologia , Malária/transmissão , Plasmodium berghei/fisiologia , Proteínas de Protozoários/metabolismo , Animais , Culicidae/parasitologia , Técnicas de Inativação de Genes , Camundongos , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Proteínas de Protozoários/genética
6.
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
7.
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
8.
PLoS Pathog ; 8(9): e1002948, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23028336

RESUMO

Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl(-) mutants, with global phosphorylation studies of ookinete differentiation from 1.5-24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.


Assuntos
Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Plasmodium berghei/enzimologia , Plasmodium berghei/crescimento & desenvolvimento , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Alveolados/química , Alveolados/genética , Motivos de Aminoácidos , Animais , Anopheles/parasitologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sequência de Bases , Diferenciação Celular , Genes de Protozoários , Malária/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfoproteínas Fosfatases/genética , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Proteínas de Protozoários/genética , Análise de Sequência de DNA , Viridiplantae/química
9.
Microorganisms ; 11(3)2023 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-36985278

RESUMO

Microtubules and specialized microtubule-containing structures are assembled from tubulins, an ancient superfamily of essential eukaryotic proteins. Here, we use bioinformatic approaches to analyze features of tubulins in organisms from the phylum Apicomplexa. Apicomplexans are protozoan parasites that cause a variety of human and animal infectious diseases. Individual species harbor one to four genes each for α- and ß-tubulin isotypes. These may specify highly similar proteins, suggesting functional redundancy, or exhibit key differences, consistent with specialized roles. Some, but not all apicomplexans harbor genes for δ- and ε-tubulins, which are found in organisms that construct appendage-containing basal bodies. Critical roles for apicomplexan δ- and ε-tubulin are likely to be limited to microgametes, consistent with a restricted requirement for flagella in a single developmental stage. Sequence divergence or the loss of δ- and ε-tubulin genes in other apicomplexans appears to be associated with diminished requirements for centrioles, basal bodies, and axonemes. Finally, because spindle microtubules and flagellar structures have been proposed as targets for anti-parasitic therapies and transmission-blocking strategies, we discuss these ideas in the context of tubulin-based structures and tubulin superfamily properties.

10.
bioRxiv ; 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36711883

RESUMO

Sexual reproduction of Toxoplasma gondii , which is restricted to the small intestine of felids, is sparsely documented, due to ethical concerns surrounding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described 1 . In this study, we found that transcription factors AP2XII-1 and AP2XI-2, expressed in tachyzoite stage that causes acute toxoplasmosis, can silence genes necessary for merozoites, a developmental stage critical for sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a drastic change in the transcriptional program, promoting a complete transition from tachyzoites to merozoites. Pre-gametes produced in vitro under these conditions are characterized by specific protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit the epigenitors MORC and HDAC3 1 , which in turn restrict the accessibility of chromatin to the transcriptional machinery. Thus, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. This effective in vitro culture of merozoites paves the way to explore Toxoplasma sexual reproduction without the need to infect kittens and has potential for the development of therapeutics to block parasite transmission.

11.
Proc Natl Acad Sci U S A ; 106(50): 21270-5, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19940242

RESUMO

Insect hemocytes mediate important cellular immune responses including phagocytosis and encapsulation and also secrete immune factors such as opsonins, melanization factors, and antimicrobial peptides. However, the molecular composition of these important immune cells has not been elucidated in depth, because of their scarcity in the circulating hemolymph, their adhesion to multiple tissues and the lack of primary culture methods to produce sufficient material for a genome-wide analysis. In this study, we report a genome-wide molecular characterization of circulating hemocytes collected from the hemolymph of adult female Anopheles gambiae mosquitoes--the major mosquito vector of human malaria in subSaharan Africa. Their molecular profile identified 1,485 transcripts with enriched expression in these cells, and many of these genes belong to innate immune gene families. This hemocyte-specific transcriptome is compared to those of Drosophila melanogaster and two other mosquitoes, Aedes aegypti and Armigeres subalbatus. We report the identification of two genes as ubiquitous hemocyte markers and several others as hemocyte subpopulation markers. We assess, via an RNAi screen, the roles in development of Plasmodium berghei of 63 genes expressed in hemocytes and provide a molecular comparison of the transcriptome of these cells during malaria infection.


Assuntos
Anopheles/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Inseto/genética , Hemócitos/metabolismo , Aedes/genética , Animais , Drosophila melanogaster/genética , Feminino , Marcadores Genéticos , Malária , Plasmodium , RNA Mensageiro/análise
12.
Mol Biochem Parasitol ; 243: 111373, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33961917

RESUMO

The Coccidia is the largest group of parasites within the Apicomplexa, a phylum of unicellular, obligate parasites characterized by the possession of an apical complex of organelles and structures in the asexual stages of their life cycles, as well as by a sexual reproductive phase that occurs enterically in host animals. Coccidian sexual reproduction involves morphologically distinct microgametes and macrogametes that combine to form a diploid zygote and, ultimately, following meiosis and mitosis, haploid, infectious sporozoites, inside sporocysts within an oocyst. Recent transcriptomic analyses have identified genes involved in coccidian sexual stage development and reproduction, including genes encoding for microgamete- and macrogamete-specific proteins with roles in gamete motility, fusion and fertilization, and in the formation of the resilient oocyst wall that allows coccidians to persist for long periods in the environment. Transcriptomics has also provided important clues about the regulation of gene expression in the transformation of parasites from one developmental stage to the next, a complex sequence of events that may involve transcription factors such as the apicomplexan Apetala2 (ApiAP2) family, alternative splicing, regulatory RNAs and MORC (a microrchida homologue and regulator of sexual stage development in Toxoplasma gondii). The molecular dissection of coccidian sexual development and reproduction by transcriptomic analyses may lead to the development of novel transmission-blocking strategies.


Assuntos
Coccídios/fisiologia , Regulação da Expressão Gênica , Proteínas de Protozoários/genética , Processamento Alternativo , Coccídios/isolamento & purificação , Coccídios/patogenicidade , Perfilação da Expressão Gênica , Estágios do Ciclo de Vida , Merozoítos/genética , MicroRNAs , Oocistos/genética , RNA Longo não Codificante , RNA de Protozoário , Análise de Célula Única/métodos
13.
Nat Commun ; 12(1): 3788, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145271

RESUMO

Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.


Assuntos
Membrana Celular/parasitologia , Proteínas de Protozoários/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/patologia , Transporte Biológico/fisiologia , Células Cultivadas , Interações Hospedeiro-Parasita , Humanos , Via Secretória/fisiologia , Fatores de Virulência
14.
Artigo em Inglês | MEDLINE | ID: mdl-32500038

RESUMO

The pathogenesis of bovine besnoitiosis and the molecular bases that govern disease progression remain to be elucidated. Thus, we have employed an in vitro model of infection based on primary bovine aortic endothelial cells (BAEC), target cells during the acute infection. Host-parasite interactions were investigated by RNA-Seq at two post-infection (pi) time points: 12 hpi, when tachyzoites have already invaded host cells, and 32 hpi, when tachyzoites have replicated for at least two generations. Additionally, the gene expression profile of B. besnoiti tachyzoites was studied at both pi time points. Up to 446 differentially expressed B. taurus genes (DEGs) were found in BAEC between both pi time points: 249 DEGs were up-regulated and 197 DEGs were down-regulated at 32 hpi. Upregulation of different genes encoding cytokines, chemokines, leukocyte adhesion molecules predominantly at 12 hpi implies an activation of endothelial cells, whilst upregulation of genes involved in angiogenesis and extracellular matrix organization was detected at both time points. NF-κB and TNF-α signaling pathways appeared to be mainly modulated upon infection, coordinating the expression of several effector proteins with proinflammatory and pro-fibrotic phenotypes. These mediators are thought to be responsible for macrophage recruitment setting the basis for chronic inflammation and fibrosis characteristic of chronic besnoitiosis. Angiogenesis regulation also predominated, and this multistep process was evidenced by the upregulation of markers involved in both early (e.g., growth factors and matrix metalloproteinases) and late steps (e.g., integrins and vasohibin). Besnoitia besnoiti ortholog genes present in other Toxoplasmatinae members and involved in the lytic cycle have shown to be differentially expressed among the two time points studied, with a higher expression at 32 hpi (e.g., ROP40, ROP5B, MIC1, MIC10). This study gives molecular clues on B. besnoiti- BAECs interaction and shows the progression of type II endothelial cell activation upon parasite invasion and proliferation.


Assuntos
Doenças dos Bovinos , Sarcocystidae , Animais , Bovinos , Proliferação de Células , Células Endoteliais , Fibrose , RNA-Seq , Sarcocystidae/genética
15.
Sci Rep ; 9(1): 1474, 2019 02 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.


Assuntos
Proteínas de Protozoários/genética , Vacinas Protozoárias/administração & dosagem , Toxoplasma/genética , Toxoplasmose Animal/prevenção & controle , Vacinas Atenuadas/administração & dosagem , Animais , Sistemas CRISPR-Cas , Gatos , Fezes/parasitologia , Feminino , Fertilização/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Inativação Gênica , Masculino , Vacinas Protozoárias/farmacologia , Análise de Sequência de RNA , Toxoplasma/efeitos dos fármacos , Toxoplasmose Animal/transmissão , Vacinas Atenuadas/farmacologia
16.
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
17.
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
18.
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
19.
Artigo em Inglês | MEDLINE | ID: mdl-16682771

RESUMO

Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ(gamma) subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5-15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000), Protein Sci. 9, 755-757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 A resolution. The crystals belong to space group P2(1), with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 angstroms, beta = 104.5 degrees. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.


Assuntos
Corismato Mutase/química , Mycobacterium tuberculosis/enzimologia , Corismato Mutase/antagonistas & inibidores , Corismato Mutase/isolamento & purificação , Cristalização/métodos , Cristalografia por Raios X
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