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1.
PLoS Negl Trop Dis ; 14(10): e0008797, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33075058

RESUMO

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mannose phosphate isomerase (mpi) gene was applied to 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru for identification of the infecting parasite at the species level, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Although most results (121/134) including 4 hybrids of Leishmania (Viannia) braziliensis and L. (V.) peruviana corresponded to those obtained in the previous study, PCR-RFLP analyses revealed the distribution of putative hybrid strains between L. (V.) peruviana and L. (V.) lainsoni in two samples, which has never been reported. Moreover, parasite strains showing discordance between kinetoplast and nuclear genes (kDNA and nDNA), so-called mito-nuclear discordance, were identified in 11 samples. Of these, six strains had the kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) guyanensis, and three strains had the kDNAs of L. (V.) shawi and nDNAs of L. (V.) braziliensis. The rest were identified as mito-nuclear discordance strains having kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) lainsoni, and kDNAs of L. (V.) lainsoni and nDNAs of L. (V.) braziliensis. The results demonstrate that Leishmania strains in Peru are genetically more complex than previously considered.


Assuntos
Núcleo Celular/parasitologia , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Cruzamentos Genéticos , Citocromos b/genética , Humanos , Leishmania/classificação , Leishmania/isolamento & purificação , Peru , Filogenia , Proteínas de Protozoários/genética
2.
mBio ; 11(2)2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32156805

RESUMO

The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain chronic infection of its host for prolonged periods. Despite this, little is known regarding whether and how T. gondii bradyzoites, a quasi-dormant life stage residing within intracellular cysts, manipulate the host cell to maintain persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro Because MYR1 is known to be involved in the translocation of parasite-derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show, by immunofluorescence assays, that most effectors accumulate in the host nucleus at early but not late time points after infection, during the tachyzoite-to-bradyzoite transition and when parasites further along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon gamma signaling, which was previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications regarding how this highly successful parasite maintains persistent infection of its host.IMPORTANCEToxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life stage successfully establishes and maintains persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different time points after bradyzoite differentiation and found that they accumulated largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediated its function after prolonged infection with bradyzoites, and we provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.


Assuntos
Interações Hospedeiro-Parasita , Estágios do Ciclo de Vida , Transporte Proteico , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Animais , Núcleo Celular/parasitologia , Núcleo Celular/fisiologia , Células Cultivadas , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Proteômica , Transdução de Sinais
3.
EMBO Rep ; 20(12): e48896, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31584242

RESUMO

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. Super-resolution microscopy and real-time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites.


Assuntos
Actinas/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Plasmodium falciparum/fisiologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/fisiologia , Toxoplasma/parasitologia , Toxoplasma/patogenicidade , Actinas/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/parasitologia , Núcleo Celular/fisiologia , Células Cultivadas , Técnicas de Inativação de Genes , Humanos , Merozoítos/genética , Merozoítos/patogenicidade , Merozoítos/fisiologia , Modelos Biológicos , Mutação , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Transdução de Sinais , Toxoplasma/genética , Virulência/fisiologia
4.
Genes (Basel) ; 10(5)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31052454

RESUMO

Identification and characterization of protein complexes and interactomes has been essential to the understanding of fundamental nuclear processes including transcription, replication, recombination, and maintenance of genome stability. Despite significant progress in elucidation of nuclear proteomes and interactomes of organisms such as yeast and mammalian systems, progress in other models has lagged. Protists, including the alveolate ciliate protozoa with Tetrahymena thermophila as one of the most studied members of this group, have a unique nuclear biology, and nuclear dimorphism, with structurally and functionally distinct nuclei in a common cytoplasm. These features have been important in providing important insights about numerous fundamental nuclear processes. Here, we review the proteomic approaches that were historically used as well as those currently employed to take advantage of the unique biology of the ciliates, focusing on Tetrahymena, to address important questions and better understand nuclear processes including chromatin biology of eukaryotes.


Assuntos
Infecções por Cilióforos/genética , Proteínas Nucleares/genética , Proteômica , Tetrahymena thermophila/genética , Núcleo Celular/genética , Núcleo Celular/parasitologia , Cromatina/genética , Cromatina/parasitologia , Infecções por Cilióforos/parasitologia , Citoplasma/genética , Citoplasma/parasitologia , Humanos , Tetrahymena thermophila/patogenicidade
5.
Mol Biochem Parasitol ; 229: 15-23, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30772422

RESUMO

The nuclear import of proteins in eukaryotic cells is a fundamental biological process. While it has been analysed to different extents in model eukaryotic organisms, this event has rarely been studied in the early divergent protozoa of the order Kinetoplastida. The work presented here represents an overview of nuclear import in these important species of human pathogens. Initially, an in silico study of classical nuclear localization signals within the published nuclear proteomes of Trypanosoma brucei and Trypanosoma cruzi was carried out. The basic amino acids that comprise the monopartite and bipartite classical nuclear localization signals (cNLS) in trypanosomal proteins are similar to the consensus sequences observed for the nuclear proteins of yeasts, animals and plants. In addition, a summarized description of published studies that experimentally address the NLS of nuclear proteins in trypanosomatids is presented, and the clear occurrence of non-classical NLS (NLS that lack the consensus motifs of basic amino acids) in the analysed reports indicate a complex scenario for the types of receptors in these species. In general, the information presented here agrees with the hypothetical appearance of mechanisms for the recognition of nuclear proteins in early eukaryotic evolution.


Assuntos
Núcleo Celular/parasitologia , Doença de Chagas/parasitologia , Sinais de Localização Nuclear/química , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/metabolismo , Tripanossomíase Africana/parasitologia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/química , Trypanosoma cruzi/genética
6.
Mem Inst Oswaldo Cruz ; 113(6): e170471, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29846382

RESUMO

Toxoplasma gondii uses epigenetic mechanisms to regulate both endogenous and host cell gene expression. To identify genes with putative epigenetic functions, we developed an in silico pipeline to interrogate the T. gondii proteome of 8313 proteins. Step 1 employs PredictNLS and NucPred to identify genes predicted to target eukaryotic nuclei. Step 2 uses GOLink to identify proteins of epigenetic function based on Gene Ontology terms. This resulted in 611 putative nuclear localised proteins with predicted epigenetic functions. Step 3 filtered for secretory proteins using SignalP, SecretomeP, and experimental data. This identified 57 of the 611 putative epigenetic proteins as likely to be secreted. The pipeline is freely available online, uses open access tools and software with user-friendly Perl scripts to automate and manage the results, and is readily adaptable to undertake any such in silico search for genes contributing to particular functions.


Assuntos
Núcleo Celular/parasitologia , Simulação por Computador , Epigênese Genética/genética , Interações Hospedeiro-Parasita/fisiologia , Proteoma/genética , Toxoplasma/genética , Toxoplasma/fisiologia
8.
Mem. Inst. Oswaldo Cruz ; 113(6): e170471, 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1040597

RESUMO

Toxoplasma gondii uses epigenetic mechanisms to regulate both endogenous and host cell gene expression. To identify genes with putative epigenetic functions, we developed an in silico pipeline to interrogate the T. gondii proteome of 8313 proteins. Step 1 employs PredictNLS and NucPred to identify genes predicted to target eukaryotic nuclei. Step 2 uses GOLink to identify proteins of epigenetic function based on Gene Ontology terms. This resulted in 611 putative nuclear localised proteins with predicted epigenetic functions. Step 3 filtered for secretory proteins using SignalP, SecretomeP, and experimental data. This identified 57 of the 611 putative epigenetic proteins as likely to be secreted. The pipeline is freely available online, uses open access tools and software with user-friendly Perl scripts to automate and manage the results, and is readily adaptable to undertake any such in silico search for genes contributing to particular functions.


Assuntos
Toxoplasma/genética , Simulação por Computador , Núcleo Celular/parasitologia , Proteoma/genética , Epigênese Genética/genética , Interações Hospedeiro-Parasita/fisiologia , Toxoplasma/fisiologia
9.
Infect Immun ; 85(12)2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28923894

RESUMO

Urogenital schistosomiasis, caused by the parasitic trematode Schistosoma haematobium, affects over 112 million people worldwide. As with Schistosoma mansoni infections, the pathology of urogenital schistosomiasis is related mainly to the egg stage, which induces granulomatous inflammation of affected tissues. Schistosoma eggs and their secretions have been studied extensively for the related organism S. mansoni, which is more amenable to laboratory studies. Indeed, we have shown that IPSE/alpha-1 (here M-IPSE), a major protein secreted from S. mansoni eggs, can infiltrate host cells. Although the function of M-IPSE is unknown, its ability to translocate to the nuclei of host cells and bind DNA suggests a possible role in immune modulation of host cell tissues. Whether IPSE homologs are expressed in other schistosome species has not been investigated. Here, we describe the cloning of two paralog genes, H03-IPSE and H06-IPSE, which are orthologs of M-IPSE, from egg cDNA of S. haematobium Using PCR and immunodetection, we confirmed that the expression of these genes is restricted to the egg stage and female adult worms, while the H-IPSE protein is detectable only in mature eggs and not adults. We show that both H03-IPSE and H06-IPSE proteins can infiltrate HTB-9 bladder cells when added exogenously to culture medium. Monopartite C-terminal nuclear localization sequence (NLS) motifs conserved in H03-IPSE, SKRRRKY, and H06-IPSE SKRGRKY, are responsible for targeting the proteins to the nucleus of HTB-9 cells, as demonstrated by site-directed mutagenesis and green fluorescent protein (GFP) tagging. Thus, S. haematobium eggs express IPSE homologs that appear to perform similar functions in infiltrating host cells.


Assuntos
Proteínas de Helminto/metabolismo , Óvulo/metabolismo , Schistosoma haematobium/patogenicidade , Animais , Linhagem Celular Tumoral , Núcleo Celular/parasitologia , Clonagem Molecular , Proteínas de Ligação a DNA , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Proteínas de Helminto/genética , Humanos , Imunomodulação , Inflamação , Proteínas Recombinantes/genética , Esquistossomose Urinária/parasitologia , Bexiga Urinária/citologia , Bexiga Urinária/efeitos dos fármacos
10.
Parasitol Res ; 115(12): 4559-4571, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27640151

RESUMO

Sj16 is a Schistosoma japonicum-derived protein (16 kDa in molecular weight) that has been identified as an immune modulation molecule, but the mechanisms of modulation of immune responses are not known. In this report, we aimed to investigate the host immune regulation mechanism by recombinant Sj16 (rSj16) and thus illuminate the molecular mechanism of immune evasion by S. japonicum. The effect of rSj16 and rSj16 mutants on the biology of dendritic cells (DCs) was assessed by examining DC maturation, cytokine production, and expression of surface markers by flow cytometry and enzyme-linked immunosorbent assay. We found that rSj16 significantly stimulated interleukin (IL)-10 production and inhibited LPS-induced bone marrow-derived dendrite cell (BMDC) maturation in a dose-dependent manner. By using antibody neutralization experiments and IL-10-deficient (knockout) mice, we confirmed that the inhibitory effect of rSj16 on LPS-induced BMDCs is due to its induction of IL-10 production. To understand how rSj16 induces the production of IL-10, we analyzed the protein sequence and revealed two potential nuclear localization signals (NLS) in Sj16. The N-terminal NLS (NLS1) is both necessary and sufficient for translocation of rSj16 to the nucleus of BMDCs and is important for subsequent induction of IL-10 production and the inhibition of BMDC maturation by rSj16. The results of our study concluded that the ability of rSj16 to inhibit DC functions is IL-10 dependent which is operated by IL-10R signal pathway. This study also confirmed that NLS is an important domain associated with increased production of IL-10. Our findings will extend the current understanding on host-schistosome relationship and provide insight about bottleneck of parasitic control.


Assuntos
Núcleo Celular/metabolismo , Células Dendríticas/parasitologia , Proteínas de Helminto/metabolismo , Interleucina-10/metabolismo , Sinais de Localização Nuclear/genética , Schistosoma japonicum/isolamento & purificação , Esquistossomose Japônica/parasitologia , Animais , Núcleo Celular/parasitologia , Células Dendríticas/metabolismo , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Interleucina-12/genética , Interleucina-12/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sinais de Localização Nuclear/metabolismo , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schistosoma japonicum/genética , Schistosoma japonicum/metabolismo , Esquistossomose Japônica/metabolismo
11.
Antimicrob Agents Chemother ; 60(8): 4701-7, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27216059

RESUMO

The current treatment of Chagas disease (CD), based on nifurtimox and benznidazole (Bz), is unsatisfactory. In this context, we performed the phenotypic in vitro screening of novel mono- and diamidines and drug interaction assays with selected compounds. Ten novel amidines were tested for their activities against bloodstream trypomastigote (BT) and amastigote forms of Trypanosoma cruzi (Y and Tulahuen strains) and their toxicities for mammalian host cells (L929 cells and cardiac cells). Seven of 10 molecules were more active than Bz against BT, with the most active compound being the diamidine DB2267 (50% effective concentration [EC50] = 0.23 µM; selectivity index = 417), which was 28-fold more active and about 3 times more selective than the standard drug. Five of the six monoamidines were also more active than Bz. The combination of DB2267 and DB2236 in fixed-ratio proportions showed an additive effect (sum of fractional inhibitory concentrations < 4) on BT. Interestingly, when intracellular forms were exposed to DB2267, its activity was dependent on the parasite strain, being effective (EC50 = 0.87 ± 0.05 µM) against a discrete typing unit (DTU) II strain (strain Y) but not against a representative DTU VI strain (strain Tulahuen) even when different vehicles (ß-cyclodextrin and dimethyl sulfoxide) were used. The intrinsic fluorescence of several diamidines allowed their uptake to be studied. Testing of the uptake of DB2236 (inactive) and DB2267 (active) by amastigotes of the Y strain showed that the two compounds were localized intracellularly in different compartments: DB2236 in the cytoplasm and DB2267 in the nucleus. Our present data encourage further studies regarding the activities of amidines and provide information which will help with the identification of novel agents for the treatment of CD.


Assuntos
Amidinas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/parasitologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Citoplasma/efeitos dos fármacos , Citoplasma/parasitologia , Mamíferos/parasitologia , Testes de Sensibilidade Parasitária/métodos , Fenótipo
12.
J Proteome Res ; 14(11): 4805-14, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26465659

RESUMO

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 Tandem
13.
Turkiye Parazitol Derg ; 39(2): 131-4, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26081887

RESUMO

OBJECTIVE: The aim of this study was to provide a description of a new haemogregarine, Haemogregarina sundarbanensis n. sp., from mud turtles collected from Sundarban regions, West Bengal, India. METHODS: The turtles were collected from fish markets near Canning, South 24-Parganas District, West Bengal. Thin blood films were prepared using the blood drawn from the head and feet. The blood films were then air-dried, fixed in absolute methanol, and stained with Giemsa. RESULTS: The erythrocytic stages could be differentiated into small and large forms. The young gamonts measured 7.896×3.469 µm, microgamonts measured 8.876×4.425 µm, and macrogamonts measured 8.919×4.272 µm. The cytoplasm of the macrogamonts was stained deep blue with Giemsa, and many metachromatic granules are found uniformly distributed. The oval or rounded nucleus was central in position and stained deep red with Giemsa stain, whereas in microgamonts, the nucleus was not at all compact but with a few dispersed chromatin granules. CONCLUSION: The prevalence of infection was found to be 33.3% (5/15). The infected erythrocytes reveal shape alteration, marginal and atrophic nucleus, and were larger than non-parasitized erythrocytes. The cytoplasm of the infected erythrocytes stained darker than that of the uninfected erythrocytes.


Assuntos
Coccidiose/veterinária , Eritrócitos/parasitologia , Eucoccidiida/ultraestrutura , Tartarugas/parasitologia , Animais , Corantes Azur , Núcleo Celular/parasitologia , Núcleo Celular/ultraestrutura , Coccidiose/sangue , Coccidiose/epidemiologia , Coccidiose/parasitologia , Eritrócitos/patologia , Eucoccidiida/classificação , Eucoccidiida/isolamento & purificação , Humanos , Índia/epidemiologia , Prevalência , Esquizontes/ultraestrutura
14.
Eukaryot Cell ; 13(8): 965-76, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24951442

RESUMO

Intracellular pathogens can replicate efficiently only after they manipulate and modify their host cells to create an environment conducive to replication. While diverse cellular pathways are targeted by different pathogens, metabolism, membrane and cytoskeletal architecture formation, and cell death are the three primary cellular processes that are modified by infections. Toxoplasma gondii is an obligate intracellular protozoan that infects ∼30% of the world's population and causes severe and life-threatening disease in developing fetuses, in immune-comprised patients, and in certain otherwise healthy individuals who are primarily found in South America. The high prevalence of Toxoplasma in humans is in large part a result of its ability to modulate these three host cell processes. Here, we highlight recent work defining the mechanisms by which Toxoplasma interacts with these processes. In addition, we hypothesize why some processes are modified not only in the infected host cell but also in neighboring uninfected cells.


Assuntos
Toxoplasma/fisiologia , Toxoplasmose/imunologia , Animais , Apoptose , Metabolismo dos Carboidratos , Membrana Celular/metabolismo , Membrana Celular/parasitologia , Núcleo Celular/parasitologia , Núcleo Celular/fisiologia , Metabolismo Energético , Interações Hospedeiro-Parasita , Humanos , Inflamassomos/fisiologia , Metabolismo dos Lipídeos , Toxoplasmose/parasitologia
15.
J Eukaryot Microbiol ; 61(2): 173-81, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24612333

RESUMO

Members of Amoebophrya ceratii complex are known to infect a number of free-living dinoflagellates including harmful algal bloom species. In August and October 2012, Amoebophrya infections during two bloom events of the dinoflagellate Cochlodinium polykrikoides were observed along southern coastal waters of Korea. Microscopic observations and molecular data revealed that two different Amoebophrya parasites infected the same host species. In addition, while one developed in the host's nucleus, the other in the host's cytoplasm. Phylogenetic analyses showed that the two parasites were not nested in the previously recognized "Amoebophrya ceratii complex clade", which contained sequences of parasites infecting numerous dinoflagellate species. Instead, they branched as sister taxa to the isolate (possibly Amoebophrya) from radiolarians Hexacontium gigantheum. Our result indicates that the two Amoebophrya parasites infecting C. polykrikoides may be different species from those inside the "complex."


Assuntos
Alveolados/classificação , Alveolados/isolamento & purificação , Dinoflagellida/parasitologia , Água do Mar/parasitologia , Alveolados/citologia , Alveolados/genética , Núcleo Celular/parasitologia , Análise por Conglomerados , Citoplasma/parasitologia , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes de RNAr , Proliferação Nociva de Algas , Microscopia , Dados de Sequência Molecular , Filogenia , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , República da Coreia , Análise de Sequência de DNA
16.
PLoS One ; 9(2): e88548, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24551116

RESUMO

BACKGROUND: Several clinical forms of malaria such as chronic carriage, gestational malaria or hyper-reactive malarial splenomegaly may follow a cryptic evolution with afebrile chronic fatigue sometimes accompanied by anemia and/or splenomegaly. Conventional parasitological tests are often negative or not performed, and severe complications may occur. Extensive explorations of these conditions often include the search for antinuclear autoantibodies (ANA). METHODS: We analysed fluorescence patterns in the ANA test in patients with either chronic cryptic or acute symptomatic malaria, then conducted a one-year prospective study at a single hospital on all available sera drawn for ANA detections. We then identified autoantibodies differentially expressed in malaria patients and in controls using human protein microarray. RESULTS: We uncovered and defined a new, malaria-related, nucleo-cytoplasmic ANA pattern displaying the specific association of a nuclear speckled pattern with diffuse cytoplasmic perinuclearly-enhanced fluorescence. In the one-year prospective analysis, 79% of sera displaying this new nucleo-cytoplasmic fluorescence were from patients with malaria. This specific pattern, not seen in other parasitic diseases, allowed a timely reorientation of the diagnosis toward malaria. To assess if the autoantibody immune response was due to autoreactivity or molecular mimicry we isolated 42 autoantigens, targets of malarial autoantibodies. BLAST analysis indicated that 23 of recognized autoantigens were homologous to plasmodial proteins suggesting autoimmune responses directly driven by the plasmodial infection. CONCLUSION: In patients with malaria in whom parasitological tests have not been performed recognition of this new, malaria-related fluorescence pattern on the ANA test is highly suggestive of the diagnosis and triggers immediate, easy confirmation and adapted therapy.


Assuntos
Anticorpos Antinucleares/sangue , Antígenos de Protozoários/sangue , Malária Falciparum/sangue , Malária Falciparum/diagnóstico , Plasmodium falciparum/imunologia , Anticorpos Antinucleares/imunologia , Antígenos de Protozoários/imunologia , Núcleo Celular/imunologia , Núcleo Celular/parasitologia , Doença Crônica , Citoplasma/imunologia , Citoplasma/parasitologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Microscopia de Fluorescência , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/parasitologia , Plasmodium falciparum/isolamento & purificação , Análise Serial de Proteínas
17.
PLoS One ; 8(9): e74701, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24040327

RESUMO

The mechanisms of stage-specific gene regulation in the malaria parasite Plasmodium falciparum are largely unclear, with only a small number of specific regulatory transcription factors (AP2 family) having been identified. In particular, the transcription factors that function in the intraerythrocytic stage remain to be elucidated. Previously, as a model case for stage-specific transcription in the P. falciparum intraerythrocytic stage, we analyzed the transcriptional regulation of pf1-cys-prx, a trophozoite/schizont-specific gene, and suggested that some nuclear factors bind specifically to the cis-element of pf1-cys-prx and enhance transcription. In the present study, we purified nuclear factors from parasite nuclear extract by 5 steps of chromatography, and identified a factor termed PREBP. PREBP is not included in the AP2 family, and is a novel protein with four K-homology (KH) domains. The KH domain is known to be found in RNA-binding or single-stranded DNA-binding proteins. PREBP is well conserved in Plasmodium species and partially conserved in phylum Apicomplexa. To evaluate the effects of PREBP overexpression, we used a transient overexpression and luciferase assay combined approach. Overexpression of PREBP markedly enhanced luciferase expression under the control of the pf1-cys-prx cis-element. These results provide the first evidence of a novel transcription factor that activates the gene expression in the malaria parasite intraerythrocytic stage. These findings enhance our understanding of the evolution of specific transcription machinery in Plasmodium and other eukaryotes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Fatores de Transcrição/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/parasitologia , Biologia Computacional , Citoplasma/metabolismo , Eritrócitos/parasitologia , Regulação da Expressão Gênica , Genes Reporter , Humanos , Espectrometria de Massas , Plasmodium falciparum/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Transgenes
18.
Cell Host Microbe ; 13(4): 489-500, 2013 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-23601110

RESUMO

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/parasitologia , Endopeptidases/genética , Endopeptidases/metabolismo , Feminino , Expressão Gênica , Células HEK293 , Interações Hospedeiro-Parasita , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Alinhamento de Sequência , Toxoplasma/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Virulência
19.
PLoS One ; 7(12): e50772, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23227206

RESUMO

The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.


Assuntos
Hepatócitos/parasitologia , Membranas Intracelulares/parasitologia , Mutação/genética , Parasitos/crescimento & desenvolvimento , Plasmodium berghei/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Vacúolos/parasitologia , Animais , Núcleo Celular/parasitologia , Núcleo Celular/ultraestrutura , Feminino , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Humanos , Membranas Intracelulares/ultraestrutura , Malária/parasitologia , Malária/patologia , Merozoítos/crescimento & desenvolvimento , Merozoítos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Parasitos/ultraestrutura , Plasmodium berghei/ultraestrutura , Vacúolos/ultraestrutura
20.
Artigo em Inglês | MEDLINE | ID: mdl-23211925

RESUMO

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria additional to their more characterized role of suppressing plant defense. Recent studies suggest that effectors may manipulate host transcription or other nuclear regulatory components for the benefit of pathogen development. However, the specific mechanisms by which these effectors promote susceptibility remain unclear. Of two recent screenings, we identified 15 nuclear-localized Hpa effectors (HaRxLs) that interact directly or indirectly with host nuclear components. When stably expressed in planta, nuclear HaRxLs cause diverse developmental phenotypes highlighting that nuclear effectors might interfere with fundamental plant regulatory mechanisms. Here, we report recent advances in understanding how a pathogen can manipulate nuclear processes in order to cause disease.


Assuntos
Arabidopsis/parasitologia , Núcleo Celular/parasitologia , Interações Hospedeiro-Parasita/imunologia , Peronospora/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal/imunologia , Proteínas/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/imunologia , Fenótipo , Doenças das Plantas/parasitologia , Ligação Proteica , Fatores de Transcrição/metabolismo
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